( A - C ) Cells from the thyroid gland of 5 mpf Tg ( tg : nls - GFP ) ; pax2amKO2 animals were labelled with calcein ( live cell marker ) and analysed using FACS . ( A ) A FACS plot showing calcein on Y - axis and GFP on X - axis . The box encompassing the GFP + cells represents the thyrocyte population , which was gated for further analysis . ( B ) Histogram showing the distribution of GFP intensity in thyrocytes . ( C ) Histogram showing the distribution of mKO2 intensity in thyrocytes . Thyrocytes were selected by gating for GFP + population . Horizontal lines indicate the mKO2 - Low and mKO2 - High expression level , with percentage values representing proportion of thyrocytes with mKO2 - Low and mKO2 - High expression .

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(A - C) Cells from the thyroid gland of 5 mpf Tg(tg:nls-GFP); pax2amKO2 animals were labelled with calcein (live cell marker) and analysed using FACS. (A) A FACS plot showing calcein on Y-axis and GFP on X-axis. The box encompassing the GFP+ cells represents the thyrocyte population, which was gated for further analysis. (B) Histogram showing the distribution of GFP intensity in thyrocytes. (C) Histogram showing the distribution of mKO2 intensity in thyrocytes. Thyrocytes were selected by gating for GFP+ population. Horizontal lines indicate the mKO2-Low and mKO2-High expression level, with percentage values representing proportion of thyrocytes with mKO2-Low and mKO2-High expression.


( D , E ) RNA - Seq . analysis of transcriptome isolated from pax2amKO2 - High and pax2amKO2 - Low subpopulations . ( D ) Volcano plot representing the differential expression of genes between the two thyrocyte subpopulations . Dots for pax2a , tg , mKO2 and EGFP are marked . Dots for genes that display significant differential expression ( DESeq2 , adjusted p - value < 0 . 05 ) are coloured . ( E ) Heatmap displaying the expression of selected genes in the two subpopulations . Biological replicates are represented by individual columns .

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(D , E) RNA-Seq. analysis of transcriptome isolated from pax2amKO2-High and pax2amKO2-Low subpopulations. (D) Volcano plot representing the differential expression of genes between the two thyrocyte subpopulations. Dots for pax2a, tg, mKO2 and EGFP are marked. Dots for genes that display significant differential expression (DESeq2, adjusted p-value < 0.05) are coloured. (E) Heatmap displaying the expression of selected genes in the two subpopulations. Biological replicates are represented by individual columns.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . A ) Transcription in fft2Δ relative to WT over chromosome 1 . The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands , respectively . Coordinates are shown in the middle panel ; genes more than 2 - fold upregulated are marked : Tf2 retrotransposons ( black ) ; antisense to Tf2 elements ( gray ) ; coding genes ( green ) ; noncoding RNAs ( blue )

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). A) Transcription in fft2Δ relative to WT over chromosome 1. The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands, respectively. Coordinates are shown in the middle panel; genes more than 2-fold upregulated are marked: Tf2 retrotransposons (black); antisense to Tf2 elements (gray); coding genes (green); noncoding RNAs (blue)


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . B ) Northern blot for the Tf2 ORF ; rRNA staining of the same membrane by methylene blue is shown below . Two different exposures of the molecular size marker are shown ( left ) .

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). B) Northern blot for the Tf2 ORF; rRNA staining of the same membrane by methylene blue is shown below. Two different exposures of the molecular size marker are shown (left).


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . C ) qPCR scheme . Amplicon 1 spans the end of the LTR and into the ORF . Amplicon 2 does not include the LTR but covers the translation start site . Amplicon 3 is entirely within the ORF . D ) Bar graph representing RNA levels for the amplicons depicted in ( C ) , relative to a control locus ( SPAC1F8 . 07c ) and to WT levels , as measured by qPCR . Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates .

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Retrotransposon transcription in WT (Hu303), fft2Δ(Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). C) qPCR scheme. Amplicon 1 spans the end of the LTR and into the ORF. Amplicon 2 does not include the LTR but covers the translation start site. Amplicon 3 is entirely within the ORF. D) Bar graph representing RNA levels for the amplicons depicted in (C), relative to a control locus (SPAC1F8.07c) and to WT levels, as measured by qPCR. Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . E ) 5 ' RACE products run on a 1 . 5 % agarose gel .

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). E) 5'RACE products run on a 1.5% agarose gel.


Top panel : Y - axis shows the CAGE signal intensity ( tag - per - million normalized number of mapped CAGE tags on plus strand ) . X axis shows LTR and 5 ' of Tf2 ORF . Three biological replicates are shown for each strain : fft2Δfft3 and WT . Bottom : LTR and tf2 ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer and primer binding sequence ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of all Tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above the sequence elements . The black square indicates the 5 ' end of the Tf2 mRNA in WT and the green square indicates the 5 ' end in fft2Δ fft3Δ detected by 5 ' RACE .

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Top panel: Y-axis shows the CAGE signal intensity (tag-per-million normalized number of mapped CAGE tags on plus strand). X axis shows LTR and 5' of Tf2 ORF. Three biological replicates are shown for each strain: fft2Δfft3 and WT. Bottom: LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all Tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements. The black square indicates the 5' end of the Tf2 mRNA in WT and the green square indicates the 5' end in fft2Δ fft3Δ detected by 5'RACE.


A ) Fft2 and Fft3 are both enriched at LTRs , while Fft2 is also enriched at the tf2 coding region . Fft2 LTR enrichment increases in fft3Δ cells , while Fft3 LTR enrichment decreases slightly in fft2Δ cells . Top : Fft2 - myc enrichment in WT ( solid line ) and in fft3Δ ( dashed line ) . Bottom : Fft3 - myc enrichment in WT ( solid line ) and in fft2Δ ( dashed line ) . ChIP - chip signal is normalized against no - epitope control arrays .

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A) Fft2 and Fft3 are both enriched at LTRs, while Fft2 is also enriched at the tf2 coding region. Fft2 LTR enrichment increases in fft3Δ cells, while Fft3 LTR enrichment decreases slightly in fft2Δ cells. Top: Fft2 -myc enrichment in WT (solid line) and in fft3Δ (dashed line). Bottom: Fft3 -myc enrichment in WT (solid line) and in fft2Δ (dashed line). ChIP-chip signal is normalized against no-epitope control arrays.


B ) Nucleosome occupancy over the LTR U3 is significantly reduced ( average reduction ~ 50 % ; average p - values at each position < 0 . 001 , Poisson test ) in fft2Δ fft3Δ cells . MNase - seq data aligned at the start of tf25 ' LTRs is shown for WT ( black line ) , and fft2Δ fft3Δ ( dark gray line ) . Light gray line indicates significance of difference between WT and mutant signals : amplitude reflects significance ( p - value ) and sign reflects the sign of occupancy change in mutant vs . WT cells . Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements . LTR and tf2ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer and primer binding sequence ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of all tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above the sequence elements , while the green dot indicates the TSS in fft2Δ fft3Δ .

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B) Nucleosome occupancy over the LTR U3 is significantly reduced (average reduction ~50%; average p-values at each position <0.001, Poisson test) in fft2Δ fft3Δ cells. MNase-seq data aligned at the start of tf25'LTRs is shown for WT (black line), and fft2Δ fft3Δ (dark gray line). Light gray line indicates significance of difference between WT and mutant signals: amplitude reflects significance (p-value) and sign reflects the sign of occupancy change in mutant vs. WT cells. Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements. LTR and tf2ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements, while the green dot indicates the TSS in fft2Δ fft3Δ.


A ) Mean nucleosome occupancy over solo LTRs ( n = 236 ) and tf2 - associated LTRs ( n = 25 ) , in WT and in fft2Δ fft3Δ .

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A) Mean nucleosome occupancy over solo LTRs (n=236) and tf2-associated LTRs (n=25), in WT and in fft2Δ fft3Δ.


B ) Mean ratio of expression ( fft2Δ fft3Δ / WT ) for each of the following categories : non - coding with LTR ( n = 69 ) / without LTR ( n = 1665 ) ( p = 0 . 008 ) ; protein coding with LTR ( n = 246 ) / without LTR ( n = 4887 ) ( p < 0 . 001 ) ; Tf2s ( n = 13 ) . A and B ) Data shown as box plots , significance of difference between categories was assessed by Wilcoxon - Mann - Whitney test ( * * p < 0 . 001 ) .

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B) Mean ratio of expression (fft2Δ fft3Δ /WT) for each of the following categories: non-coding with LTR (n=69) / without LTR (n=1665) (p=0.008); protein coding with LTR (n=246) / without LTR (n=4887) (p<0.001); Tf2s (n=13). A and B) Data shown as box plots, significance of difference between categories was assessed by Wilcoxon-Mann-Whitney test (** p<0.001).


A ) Heat and oxidative stress cause a Tf2 TSS shift in WT cells , resulting in the production of reverse transcription competent Tf2 transcripts . Transcription fold change as in Figure 1D , but relative to act1 transcription and to transcription in the same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological duplicates .

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A) Heat and oxidative stress cause a Tf2 TSS shift in WT cells, resulting in the production of reverse transcription competent Tf2 transcripts. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates.


B ) Heat shock - associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy . The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells , where nucleosome occupancy is already very low ( Fig 2B , Fig S3 ) . LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF + 1 nucleosome , measured by MNase - qPCR .

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B) Heat shock-associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy. The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells, where nucleosome occupancy is already very low (Fig 2B, Fig S3). LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF +1 nucleosome, measured by MNase-qPCR.


C ) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells . Transcription fold change as in Figure 1D , but relative to act1 transcription and to transcription in the same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological

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C) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates


B ) Bar graph representing RNA levels as in Fig . 1D , relative to a control locus ( act1 ) and to WT levels , as measured by qPCR . Error bars represent standard deviation of nucleosome occupancy of biological duplicates .

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B) Bar graph representing RNA levels as in Fig. 1D, relative to a control locus (act1) and to WT levels, as measured by qPCR. Error bars represent standard deviation of nucleosome occupancy of biological duplicates.


C ) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy , measured by MNase - qPCR as in Fig . 5B .

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C) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy, measured by MNase-qPCR as in Fig. 5B.


A ) Fluorescencein situ hybridization with a probe against tf2 elements ( green ) , and DAPI staining ( blue ) in WT and fft2Δ fft3Δ cells . B ) The number of tf2 element signal clusters is significantly elevated ( p = 9 . 8x10 - 18 , χ2 - test ) in fft2Δ fft3Δ cells , indicating a loss of retrotransposon clustering . Tf2 clustering was counted in wt cells ( n = 663 ) , fft2 ∆ ( n = 556 ) , fft3 ∆ ( n = 493 ) , fft2 ∆ fft3 ∆ ( n = 636 ) . Significance of difference between strains was assessed by Chi - square test .

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A) Fluorescencein situ hybridization with a probe against tf2 elements (green), and DAPI staining (blue) in WT and fft2Δ fft3Δ cells. B) The number of tf2 element signal clusters is significantly elevated (p=9.8x10-18, χ2-test) in fft2Δ fft3Δ cells, indicating a loss of retrotransposon clustering. Tf2 clustering was counted in wt cells (n=663), fft2∆ (n=556), fft3∆ (n=493), fft2∆ fft3∆ (n=636). Significance of difference between strains was assessed by Chi-square test.


A ) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D ( PP3 ) and fft2Dfft3D ( PP4 ) mutant strains with new integrations of Tf2 - 12 - neoAI . After 3 days at 32o C .

A) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D (PP3) and fft2Dfft3D (PP4) mutant strains with new integrations of Tf2-12-neoAI. After 3 days at 32o C.


C ) Southern blot probed for neoA fft3D ( PP3 ) and fft2Dfft3D mutant strains ( PP4 ) and derived G418 resistant subclones .

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C) Southern blot probed for neoA fft3D (PP3) and fft2Dfft3D mutant strains (PP4) and derived G418 resistant subclones.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) in response to different levels of KCl stress . Points reflect the measured values , and lines correspond to best model fit .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) at 0 . 4M KCl .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) at 0.4M KCl.


Representation of the experiments shown in ( B , C ) in parameter space . Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter ( Y axis ) vs . KCl concentration ( X axis ) of the KCl gradient experiment The inferred total open time - toff − ton ( Y axis ) vs . the inferred production rate ( X axis ) of the KCl gradient

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Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment


Summary of Hsp12 - GFP dynamics single mutant screen . 68 mutants were divided into three groups : increased / decreased / did not change the total GFP produced . Each group was independently hierarchically clustered . The values shown are the Z - Scores of each parameter : ton - response onset time ; production rate ; total open time - length of transcriptional window ( toff − ton ) ; Basal GFP - GFP levels measured before exposure to stress ; Delta GFP - amount of GFP produced throughout the experiment ( Max GFP - Basal GFP ) .

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Summary of Hsp12-GFP dynamics single mutant screen. 68 mutants were divided into three groups: increased/decreased/did not change the total GFP produced. Each group was independently hierarchically clustered. The values shown are the Z-Scores of each parameter: ton- response onset time; production rate; total open time - length of transcriptional window (toff − ton); Basal GFP - GFP levels measured before exposure to stress; Delta GFP - amount of GFP produced throughout the experiment (Max GFP - Basal GFP).


mRNA levels of HSP12 ( Y axis ) as a function of time ( X axis ) in six representative strains .

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mRNA levels of HSP12 (Y axis) as a function of time (X axis) in six representative strains.


Comparison of RNA levels to protein levels . X axis is the sum over the HSP12 mRNA counts ( see D ) . Y axis is the maximum over the Hsp12 - GFP values . The red line indicates the best linear fit .

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Comparison of RNA levels to protein levels. X axis is the sum over the HSP12 mRNA counts (see D). Y axis is the maximum over the Hsp12-GFP values. The red line indicates the best linear fit.


Comparing the total open time parameter inferred from mRNA data ( X axis ) to the one inferred from protein data ( Y axis ) .

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Comparing the total open time parameter inferred from mRNA data (X axis) to the one inferred from protein data (Y axis).


Time - lapse microscopy of Hsp12 - GFP in response to stress ( 0 . 4M KCl ) . Examples of raw images ( top ) and traces of individual cells ( bottom ) . Traces are colored according to their basal GFP level .

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Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.


Scatter plot showing the production rate estimated from single traces vs . the production rate estimated from the population average . Each point represents a different yeast strain .

Scatter plot showing the production rate estimated from single traces vs. the production rate estimated from the population average. Each point represents a different yeast strain.


Estimated parameters ( median over repeats ) of all ~ 1600 double mutant strains . The color represents the total Hsp12 - GFP produced . The same amount of GFP can be produced with multiple parameter combinations . The ellipses illustrate iso - expression areas in the graph . Three examples of individual strains ( marked in B ) with nearly the same total expression but dramatically different dynamics .

Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph. Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics.


Illustration of the genetic interaction trends of individual mutants . For each single mutant we plot the median of all the double mutants that contain it ( purple dots ) . The gray dots are all individual double mutant strains ( B ) .

Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B).


Illustration of the interactions of Δhog1 in the production rate parameter . Shown the production rate of the single mutant ( X axis ) versus the production rate of the double mutant with Δhog1 ( Y axis ) . The gray line marks the Hog1 single mutant levels . Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 ( pink dots ) .

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Illustration of the interactions of Δhog1 in the production rate parameter. Shown the production rate of the single mutant (X axis) versus the production rate of the double mutant with Δhog1 (Y axis). The gray line marks the Hog1 single mutant levels. Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 (pink dots).


Illustration of the interactions of Δhog1 for the total open time parameter .

Illustration of the interactions of Δhog1 for the total open time parameter.


Epistatic interactions of Δmck1 in the production rate parameter


Epistatic interactions of Δmck1 in the total open time parameter


Upper : HSP12 mRNA in response to osmotic stress ( 0 . 4M KCl ) . Lower : Estimate of Msn2 / 4 dependent production of HSP12 mRNA over time ( difference between WT and Δmsn2Δmsn4 production profiles , Figure EV4A , Methods ) .

Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).


Δmck1 effect on the response of all stress genes . The integral over mRNA values in WT strain ( X axis ) vs . the integral in the Δmck1 strain ( Y values ) . The inset shows the integral for the HSP12 gene .

Δmck1 effect on the response of all stress genes. The integral over mRNA values in WT strain (X axis) vs. the integral in the Δmck1 strain (Y values). The inset shows the integral for the HSP12 gene.


Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints ( See 2C ) . Each gene is presented relative to its pre - stress level and normalized by the median over WT values to visualize the dynamic range in all genes . The genes are separated into MSN dependent and independent genes ( Figure EV4B ) . The MSN - dependent genes are sorted based on the onset time to three groups ( Early , Average and Late onset ) .

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Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints (See 2C). Each gene is presented relative to its pre-stress level and normalized by the median over WT values to visualize the dynamic range in all genes. The genes are separated into MSN dependent and independent genes (Figure EV4B). The MSN-dependent genes are sorted based on the onset time to three groups (Early, Average and Late onset).


The behaviour of early and late onset genes . Similar to ( C ) but showing the average over the genes in each group .

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The behaviour of early and late onset genes. Similar to (C) but showing the average over the genes in each group.


Msn2 - GFP localization following stress ( 0 . 4M KCl ) in selected strains . Top : The percentage of nuclear cells ( Y axis ) over time ( X axis ) . Bottom : count of the number of nuclear import events in live - cell time - lapse microscopy at the two relevant periods .

Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods.


A . Heatmap summary of Aft1 induction experiment showing all genes that change with | log2 ( fold - change ) | > 1 . 5 .

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A. Heatmap summary of Aft1 induction experiment showing all genes that change with |log2(fold-change)| > 1.5.


A ) Correlations of mean fold - changes of the proteins changing PD - dependently in the Columbia and LCC cohorts . Only proteins quantified in both cohorts are shown ( n = 330 ) . Proteins overlapping between the two cohorts are labeled with their name .

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A) Correlations of mean fold-changes of the proteins changing PD-dependently in the Columbia and LCC cohorts. Only proteins quantified in both cohorts are shown (n=330). Proteins overlapping between the two cohorts are labeled with their name.


B ) Mean fold - changes for each of the 33 proteins that were LRRK2 - dependently regulated in both cohorts using a pairwise t - test comparing the four subgroups ( HC , NMC , iPD and LRRK2 PD ) .

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B) Mean fold-changes for each of the 33 proteins that were LRRK2-dependently regulated in both cohorts using a pairwise t-test comparing the four subgroups (HC, NMC, iPD and LRRK2 PD).


A ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the MoCA total score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included in the analysis . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in red .

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A) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the MoCA total score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included in the analysis. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


B ) Pearson correlation scores and associated p - values [ - log10 ] of all protein intensities with the UPDRS - III score . Either all PD patients ( left ) , iPD patients ( middle ) or LRRK2 PD patients ( right ) were included . Significantly correlated proteins with an FDR of 5 % after Benjamini - Hochberg correction are highlighted in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Pearson correlation scores and associated p-values [-log10] of all protein intensities with the UPDRS-III score. Either all PD patients (left), iPD patients (middle) or LRRK2 PD patients (right) were included. Significantly correlated proteins with an FDR of 5% after Benjamini-Hochberg correction are highlighted in red.


( A ) GFP - PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation ( GS ) for 4 h . The nuclei were stained with Hoechst 33342 . Scale bar : 10 μm .

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(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.


( B ) Immunoblotting ( IB ) analysis of wild type ( WT ) and PAQR3 - deficient MEFs after GS for different time as indicated .

(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.


( C ) WT and PAQR3 - deficient MEFs were treated with 80 mM chloroquine ( CQ ) for 2 h to induce LC3 - II accumulation . Whole - cell lysates were harvested for IB analysis of LC3 - II .

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(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.


( D ) Autophagosome - like structures in WT and PAQR3 - deleted MEFs were detected after GS for 4 h by transmission electron microscopy . * indicates autophagosome - like structures . N , nucleus . ( E ) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images . Thirty cells were quantified from each independent experiment ， which was repeated for three times with similar results . Values are presented as mean ± SD , * * * p < 0 . 001 .

(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.


( F ) Immunofluorescence staining of LC3 in WT or PAQR3 - deficient MEFs after GS for 4 h . The arrowhead indicates representative LC3 - positive puncta . Scale bar : 10 μm .

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(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.


( G , H ) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus . After GS for 1 h , the cell lysates were harvested for IB .

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(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.


( A ~ C ) HeLa cells stably expressing GFP - DFCP1 were transfected with or without PAQR3 - specific shRNA . GFP signals were observed in these cells before or after glucose starvation ( GS , shown in A ) . The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP - positive puncta per cell is illustrated in C . Values are presented as mean ± SD , * * * P < 0 . 001 . Scale bar : 10 μm .

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(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.


( D ) GFP - WIPI1 was transfected into WT or PAQR3 - deficient MEFs . 24 h later , the cells were incubated under GS for 4 h , followed by fluorescence observation . Scale bar : 10 μm .

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(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.


( E ) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium ( NM ) or GS . Then PI ( 3 ) P level was determined by a quantitative ELISA assay . The PI ( 3 ) P level was normalized to the amount of ATG14L or UVRAG used in the assay ( n = 5 ; * * * p < 0 . 001 ; ns , not significant ) .

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(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).


( F ) HeLa cells infected with control or PAQR3 - overexpressed lentivirus were treated with GS for 4 h . PI ( 3 ) P levels were determined as in Figure 2E ( n = 5 ; * * p < 0 . 01 ; ns , not significant ) .

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(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).


( G , H ) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants . At 24 h after the transfection , the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody , followed by immunoblotting . The activity of ATG14L - linked VPS34 activity was detected by a quantitative ELISA assay . The PI ( 3 ) P level was normalized to the amount of ATG14L ( n = 5 ; * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).


( A ) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells . The cell lysates were used for immunoprecipitation ( IP ) and immunoblotting ( IB ) with the antibodies as indicated .

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(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) Cytosol and Golgi fractionations were isolated from the livers of PAQR3 - deleted mice and their littermate controls . Equal protein amounts of cytosol and Golgi fractions were subjected to IB .

(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.


( C ) HEK293T cells were transiently transfected with Myc - tagged PAQR3 . At 24 h after transfection , the cells were treated with normal medium ( NM ) ， amino acid starvation ( AS ) or glucose starvation ( GS ) , HBSS solution , or rapamycin ( Rapa , 50 nM ) for 4 h respectively . The cell lysates were then used in IP and IB .

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(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.


( D ) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM , AS or GS conditions . The relative abundance of VPS34 - binding partners was determined by IB . Each IP was normalized to the amount of VPS34 .

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(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.


( E ) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB .

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(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.


( F ) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB .

(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.


( G ) MEFs were infected with control or PAQR3 - expressing lentivirus . Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies . Supernatants of MEF lysates after immunodepletion ( Post - IP ) were examined to determine the level of VPS34 complex proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.


( A ) A two - step co - IP assay in HEK293T cells to determine the ternary complex containing PAQR3 , Beclin1 and ATG14L . The procedures of the two - step co - IP are outlined in the top . The samples were subjected to immunoprecipitation ( IP ) and immunoblotting ( IB ) with the antibodies as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) HeLa cells were transiently transfected with the plasmids as indicated . 24 h after transfection , the cells were treated with or without glucose starvation ( GS ) for 4 h . The cell lysates were used in IP and IB .

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(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.


( C , D ) Different truncations of ATG14L ( C ) were co - transfected with Myc - tagged PAQR3 into HEK293T cells as indicated , followed by IP and IB .

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(C, D) Different truncations of ATG14L (C) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( C , D ) Different truncations of Beclin1 ( D ) were co - transfected with Myc - tagged PAQR3 into HEK293T cells as indicated , followed by IP and IB .

(C, D) Different truncations of Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( E ) Purified GST or GST - fused PAQR3 ( 1 - 71 ) ( GST fused NH2 - terminal 71aa of PAQR3 ) on glutathione agarose beads was mixed with purified His tagged proteins as indicated . After incubation at 4 ° C for 3 h , the samples were washed extensively and subjected to IB .

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(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with Flag - tagged Beclin1 ( F ) into HEK293T cells . The cells were then subjected to IP and IB .

(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) into HEK293T cells. The cells were then subjected to IP and IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with GFP - fused ATG14L ( G ) into HEK293T cells . The cells were then subjected to IP and IB .

(F, G) Different PAQR3 deletion constructs were co-transfected with GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.


( A ) HeLa cells were cultured in normal medium ( NM ) or glucose starvation ( GS ) for 4 h . The starved lysates were treated with calf intestinal phosphatase ( CIP ) to remove phosphate groups and analyzed by immunoblotting ( IB ) with Phos - tag gel or regular SDS - PAGE .

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(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.


( B ) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time ( lane 1 ~ 5 ) , or GS for 2 h followed by 2 h chase in NM ( lane 6 ) . The cell lysates were subjected to Phos - tag gel and regular SDS PAGE respectively .

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(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.


( C ) WT or AMPKa1 / 2 double knockout MEFs were incubated under NM or GS for 4 h , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(C) WT or AMPKa1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.


( D ) HeLa cells were incubated with compound C ( 20 mM , 30 min ) with or without GS , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.


( E ) Bacterial purified His - tagged NH2 - terminal 71 amino acid of PAQR3 was incubated with or without purified Flag - tagged ATG14L . Then the complexes were treated with WT AMPK or DN - AMPK ( dominant negative AMPK ) for the indicated time in vitro . PAQR3 phosphorylation was examined by Phos - tag gel .

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(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.


( F ) HeLa cells infected with control or ATG14L - expressing lentivirus were incubated under GS for different time as indicated . Whole cell lysates were analyzed by both Phos - tag gel and regular SDS - PAGE .

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(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( G ) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L - specific shRNA . Then the ATG14L knockdown cells were stably re - expressed with shRNA - resistant ATG14L ( rATG14L ) or Beclin1 and incubated under NM or GS for 4 h . Whole cell lysates were analyzed by both Phos - tag gel and regular SDS - PAGE .

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(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( A ) Bacterial purified His - tagged WT or T32A NH2 - terminal 71aa of PAQR3 was incubated with or without Flag - tagged ATG14L purified from HEK293T cells . Then the complexes were incubated with AMPK for the indicated time and subjected to Phos - tag gel or regular SDS - PAGE .

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(A) Bacterial purified His-tagged WT or T32A NH2-terminal 71aa of PAQR3 was incubated with or without Flag-tagged ATG14L purified from HEK293T cells. Then the complexes were incubated with AMPK for the indicated time and subjected to Phos-tag gel or regular SDS-PAGE.


( B ) PAQR3 - deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids . 24 h later , the cells were incubated with normal medium ( NM ) or glucose starvation ( GS ) for 4 h , followed by immunoblotting ( IB ) in Phos - tag gel or regular SDS - PAGE .

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(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.


( C ) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C ( C . C , 20 mM , 30 min ) prior to GS for 1 h . In parallel , 25 mM 2 - DG or 1 mM AICAR was added in NM for 1 h . Cell lysates were analyzed by IB .

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(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.


( D ) HEK293T cells were transfected with Myc - tagged PAQR3 . 24 h after transfection , the cells were treated with GS for 4 h . Then the cell lysates were used in IB and immunoprecipitation ( IP ) with the antibodies as indicated . The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos - tag gel or regular SDS - PAGE .

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(D) HEK293T cells were transfected with Myc-tagged PAQR3. 24 h after transfection, the cells were treated with GS for 4 h. Then the cell lysates were used in IB and immunoprecipitation (IP) with the antibodies as indicated. The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos-tag gel or regular SDS-PAGE.


( E ) WT , phosphorylation defective ( T32A ) , or phospho - mimetic ( T32E ) PAQR3 were transfected in the presence or absence of constitutively active AMPK ( CA - AMPK ) into PAQR3 - deficient HeLa cells as indicated . 24 h later , the cells without AMPK transfection were treated with GS for 4 h . 10 % of the cell lysates were subjected to IB to detect PAQR3 expression level ( shown in Appendix Fig S4A ) , the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody , followed by VPS34 activity measurement by a quantitative PI ( 3 ) P ELISA assay . The PI ( 3 ) P level was normalized to the amount of ATG14L ( n = 5 ; * p < 0 . 05 ; * * p < 0 . 01 as compared to the first group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).


( F ) PAQR3 - deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3 . Then PAQR3 expression levels were examined by IB .

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(F) PAQR3-deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.


( G ) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively . Cell lysates were analyzed by IB .

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(G) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively. Cell lysates were analyzed by IB.


( A , B ) Representative immunohistochemical images of LC3 staining in skeletal muscle ( A ) and liver ( B ) after 80 min exercise in WT and PAQR3 - deleted mice . Scale bar in ( A ) : 20 μm . Scale bar in ( B ) : 50 μm .

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(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm.


( C , D ) Immunoblotting analysis of skeletal muscle ( C ) and liver ( D ) from WT or PAQR3 knockout mice at rest or after 80 min exercise .

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(C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise.


( E ) Abnormal limb - clasping of PAQR3 knockout mice when suspended by its tail . Quantification scoring of the limb clasping phenotype is shown on the right ( n = 7 for each group ; * * p < 0 . 01 ) .

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(E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01).


( F ) Motor performance was measured using an accelerating rotarod apparatus , and the time of each mouse spent on the rotating rod until it fell off was recorded . Average time on the rod for each group is shown ( n = 7 for each group ; * * p < 0 . 01 ) .

(F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01).


( G ) Co - immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype . Immunoprecipitation ( IP ) and immunoblotting ( IB ) were performed using the antibodies as indicated .

(G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated.


( H ) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis , followed by IB .

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(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.


( I ) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI ( 3 ) P ELISA assay ( n = 5 ; * * p < 0 . 01 ) .

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(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).


( Ci ) The levels of WDR6 were determined and normalized against GAPDH in three groups . Normal , n = 21 ; Noninvasive , n = 15 ; Invasive , n = 8 . ( Cii ) The levels of WDR6 in 10 pairs of patients ' tissues .

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(Ci) The levels of WDR6 were determined and normalized against GAPDH in three groups. Normal, n=21; Noninvasive, n=15; Invasive, n=8. (Cii) The levels of WDR6 in 10 pairs of patients' tissues.


( E ) The DFS and OS assays of HCC patients ( n = 201 ) stratified by WDR6 expression .

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(E) The DFS and OS assays of HCC patients (n=201) stratified by WDR6 expression.


( F ) Analysis of WDR6 using TCGA LIHC data . Fi and Fiii were plotted with GEPIA analyzing tool , and Fii were analyzed by UALCAN analyzing tool .

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(F) Analysis of WDR6 using TCGA LIHC data. Fi and Fiii were plotted with GEPIA analyzing tool, and Fii were analyzed by UALCAN analyzing tool.


( A ) Western blots confirmed WDR6 knockdown in HCC cells transduced with shWDR6 or shcontrol lentiviral vector .

(A) Western blots confirmed WDR6 knockdown in HCC cells transduced with shWDR6 or shcontrol lentiviral vector.


( B ) The effects of WDR6 silencing on the invasive and proliferative ability of HCC cells , respectively . Left panel : HCC - LM3 : P = 0 . 912 shControl vs shWDR6 - # 1 , P = 0 . 889 shControl vs shWDR6 - # 2 ; HepG2 : P = 0 . 826 shControl vs shWDR6 - # 1 , P = 0 . 796 shControl vs shWDR6 - # 2 ; right panel : HCC - LM3 : P = 0 . 856 shControl vs shWDR6 - # 1 , P = 0 . 814 shControl vs shWDR6 - # 2 ; HepG2 : P = 0 . 726 shControl vs shWDR6 - # 1 , P = 0 . 745 shControl vs shWDR6 - # 2 .

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(B) The effects of WDR6 silencing on the invasive and proliferative ability of HCC cells, respectively. Left panel: HCC-LM3: P=0.912 shControl vs shWDR6-#1, P=0.889 shControl vs shWDR6-#2; HepG2: P=0.826 shControl vs shWDR6-#1, P=0.796 shControl vs shWDR6-#2; right panel: HCC-LM3: P=0.856 shControl vs shWDR6-#1, P=0.814 shControl vs shWDR6-#2; HepG2: P=0.726 shControl vs shWDR6-#1, P=0.745 shControl vs shWDR6-#2.


( C ) The survival plot of Albumin - Cre / + ; Wdr6f / f mice with DEN - induced HCC .

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(C) The survival plot of Albumin-Cre/+; Wdr6f/f mice with DEN-induced HCC.


( F ) Flow cytometric analysis of MDSC and T cells in tumors from BALB / c mice with H22 - WDR6 or empty vector cells orthotopically implantation ( n = 6 / group ) .

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(F) Flow cytometric analysis of MDSC and T cells in tumors from BALB/c mice with H22-WDR6 or empty vector cells orthotopically implantation (n=6/group).


( G ) α - Gr1 antibody injection markedly decreased the tumor volume and lung metastatic nodules ' number in BALB / c mice with H22 - WDR6 cells orthotopically implantation when compared to IgG control ( n = 5 / group ) .

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(G) α-Gr1 antibody injection markedly decreased the tumor volume and lung metastatic nodules' number in BALB/c mice with H22-WDR6 cells orthotopically implantation when compared to IgG control (n=5/group).


( Ai ) ATAC - sequencing showed the effects of WDR6 knockdown on the chromatin accessibility of IL10 , IL1β , IL6 , and TNFα loci . ( Aii ) quantitative real - time polymerase chain reaction ( qRT - PCR ) analyzing cytokines ' mRNA levels in Hepa1 - 6 and H22 cells treated with or without shWDR6 or WDR6 , respectively .

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(Ai) ATAC-sequencing showed the effects of WDR6 knockdown on the chromatin accessibility of IL10, IL1β, IL6, and TNFα loci. (Aii) quantitative real-time polymerase chain reaction (qRT-PCR) analyzing cytokines' mRNA levels in Hepa1-6 and H22 cells treated with or without shWDR6 or WDR6, respectively.


( B ) TNFα ELISA assays were performed in the conditioned medium from Hepa1 - 6 and H22 cells with WDR6 knockdown or overexpression , respectively .

(B) TNFα ELISA assays were performed in the conditioned medium from Hepa1-6 and H22 cells with WDR6 knockdown or overexpression, respectively.


( D ) The effects of TNFα knockdown in H22 - WDR6 cells on MDSC recruitment ( Dii ) , tumor size ( Diii ) , and the ratio of IFN - γ + CD8 + T cells ( Div ) in tumors from syngeneic mice ( n = 5 / group ) .

(D) The effects of TNFα knockdown in H22-WDR6 cells on MDSC recruitment (Dii), tumor size (Diii), and the ratio of IFN-γ+ CD8+ T cells (Div) in tumors from syngeneic mice (n=5/group).


( E ) TNFα supplementation decreased MDSC infiltration induced by WDR6 knockdown in tumor tissues from syngeneic mice ( n = 6 / group ) .

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(E) TNFα supplementation decreased MDSC infiltration induced by WDR6 knockdown in tumor tissues from syngeneic mice (n=6/group).


( A ) The effects of NF ‐ κB inhibitor IMD0354 or IκBα ‐ SR on TNFα expression in MHCC - 97L cells with or without WDR6 overexpression .

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(A) The effects of NF‐κB inhibitor IMD0354 or IκBα‐SR on TNFα expression in MHCC-97L cells with or without WDR6 overexpression.


( B ) The effects of WDR6 knockdown on the protein levels of p65 , RelB , c - Rel , p52 , and p50 in HCC cells .

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(B) The effects of WDR6 knockdown on the protein levels of p65, RelB, c-Rel, p52, and p50 in HCC cells.


( Di - ii ) The effects of autophagic deficiency using 3 - MA on the levels of p65 protein and TNFα secretion in HCC cells with WDR6 knockdown .

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(Di-ii) The effects of autophagic deficiency using 3-MA on the levels of p65 protein and TNFα secretion in HCC cells with WDR6 knockdown.


( Ei ) WDR6 mRNA levels in Hep3B and MHCC - 97L cells treated with or without TNFα in culture media . ( Eii ) Schematic diagram showing three candidate NF ‐ κB - binding sites in WDR6 promoter . ( Eiii ) The impact of IMD0354 on the luciferase reporter of WDR6 proximal promoter in HCC cells . ( Eiv - v ) ChIP assays using HCC cells or HCC tissues from patients ( P # 36 and P # 54 ) .

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(Ei) WDR6 mRNA levels in Hep3B and MHCC-97L cells treated with or without TNFα in culture media. (Eii) Schematic diagram showing three candidate NF‐κB-binding sites in WDR6 promoter. (Eiii) The impact of IMD0354 on the luciferase reporter of WDR6 proximal promoter in HCC cells. (Eiv-v) ChIP assays using HCC cells or HCC tissues from patients (P#36 and P#54). Da


( C ) Co - IP analysis indicated the effect of WDR6 mutant ( R123A ) or ( R203A ) on the binding of CUL4A to endogenous UVRAG in MHCC - 97L cells .

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(C) Co-IP analysis indicated the effect of WDR6 mutant (R123A) or (R203A) on the binding of CUL4A to endogenous UVRAG in MHCC-97L cells.


( Di ) UVRAG ubiquitination assay in MHCC - 97L cells expressing WT - WDR6 or its mutant WDR6 - R123A . ( Dii ) UVRAG ubiquitination in HCC - LM3 cells treated as shown .

(Di) UVRAG ubiquitination assay in MHCC-97L cells expressing WT-WDR6 or its mutant WDR6-R123A. (Dii) UVRAG ubiquitination in HCC-LM3 cells treated as shown.


( Ei ) MS assay identified K176 of UVRAG as major ubiquitination site . ( Eii ) K176R mutant abolished UVRAG ubiquitination promoted by WDR6 .

(Ei) MS assay identified K176 of UVRAG as major ubiquitination site. (Eii) K176R mutant abolished UVRAG ubiquitination promoted by WDR6.


( A ) WDR6 or its mutant ( Ai ) or WDR6 shRNAs ( Aii ) or WDR6 with CUL4A siRNA ( Aiii ) affected the levels of endogenous UVRAG in HCC cell lines .

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(A) WDR6 or its mutant (Ai) or WDR6 shRNAs (Aii) or WDR6 with CUL4A siRNA (Aiii) affected the levels of endogenous UVRAG in HCC cell lines.


( B ) CHX assays of UVRAG stability in HCC - LM3 cells transfected with WDR6 shRNA .

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(B) CHX assays of UVRAG stability in HCC-LM3 cells transfected with WDR6 shRNA.


( C ) The effects of WT - WDR6 or WDR6 - R123A mutant on UVRAG levels in MHCC - 97L cells in the presence of MG132 .

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(C) The effects of WT-WDR6 or WDR6-R123A mutant on UVRAG levels in MHCC-97L cells in the presence of MG132.


( Di - iii ) UVRAG knockdown in Hepa1 - 6 cells reverted WDR6 - deficient - induced chromatin accessibility of TNFα locus and TNFα secretion in culture medium . The cells in ( Di ) were orthotopically implanted into C57BL / 6J mice .

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(Di-iii) UVRAG knockdown in Hepa1-6 cells reverted WDR6-deficient-induced chromatin accessibility of TNFα locus and TNFα secretion in culture medium. The cells in (Di) were orthotopically implanted into C57BL/6J mice.


( F ) Heat - map showing the expression of altered genes related to functional signatures in MDSCs after WDR6 alone or with UVRAG knockdown .

(F) Heat-map showing the expression of altered genes related to functional signatures in MDSCs after WDR6 alone or with UVRAG knockdown.


( A ) HEK293T cells with indicated constructs were treated with 5 μM Pep2 - WDxR or Pep2 - con or Pep2 - WDxA mutant for 16 hours , respectively . Then co - IP assay was used to assess the impacts of these peptides on the WDR6 - UVRAG binding .

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(A) HEK293T cells with indicated constructs were treated with 5 μM Pep2-WDxR or Pep2-con or Pep2-WDxA mutant for 16 hours, respectively. Then co-IP assay was used to assess the impacts of these peptides on the WDR6-UVRAG binding.


( C ) BALB / c mice introduced with H22 - WDR6 overexpression cells were administered with Pep2 - WDxR or Pep2 - con with or without anti - PD - L1 antibody . The images and sizes of HCC ( Ci ) , hematoxylin - eosin ( H & E ) staining and metastasis nodules of lung were presented ( Cii ) . The survival analysis of the mouse after treatment was performed ( n = 9 / group ) ( Ciii ) . In the BALB / c mice from ( C ) , Flow cytometry was performed to analyze PMN - MDSC , M - MDSC , and IFN - γ + CD8 + T cells in tumors ( Civ ) .

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(C) BALB/c mice introduced with H22-WDR6 overexpression cells were administered with Pep2-WDxR or Pep2-con with or without anti-PD-L1 antibody. The images and sizes of HCC (Ci), hematoxylin-eosin (H&E) staining and metastasis nodules of lung were presented (Cii). The survival analysis of the mouse after treatment was performed (n = 9/group) (Ciii). In the BALB/c mice from (C), Flow cytometry was performed to analyze PMN-MDSC, M-MDSC, and IFN-γ+ CD8+ T cells in tumors (Civ).


( D ) The survival test of DEN - and CCl4 - induced HCC using WDR6 - knockout and WT mice . At twenty - six weeks , mice were administered with or without anti - PD - L1 antibody ( n = 6 / group ) .

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(D) The survival test of DEN- and CCl4-induced HCC using WDR6-knockout and WT mice. At twenty-six weeks, mice were administered with or without anti-PD-L1 antibody (n = 6/group).


( C ) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures . The synthetic growth defects of vps4 ∆ tul1 ∆ or vps4 ∆ ubx3 ∆ double mutants at 26 ° C and 37 ° C were complemented with plasmids encoding VPS4 or TUL1 or UBX3 . The dashed boxes indicate the non - complemented double mutants .

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(C) Equal amounts of WT cells and indicated single or double mutants in serial dilutions were incubated on agar plates at the indicated temperatures. The synthetic growth defects of vps4∆ tul1∆ or vps4∆ ubx3∆ double mutants at 26°C and 37°C were complemented with plasmids encoding VPS4 or TUL1 or UBX3. The dashed boxes indicate the non-complemented double mutants.


( A ) Vacuolar proteolysis - deficient pep4 ∆ and tul1 ∆ pep4 ∆ cells were labeled to saturation with heavy 13C6 , 15C2 - L - lysine and then chased for 3 hours in presence of light L - lysine , following quantitative proteome analysis by LC - MS ( two biological replicates ) . To analyze Dsc complex - dependent protein turnover , the ratio of H / L ratios of tul1 ∆ pep4 ∆ over pep4 ∆ cells of 2511 proteins quantified in both strains was plotted , and proteins with a ratio of H / L ratios > 2 were selected . Red squares : regulated proteins with at least one predicted transmembrane ( TM ) domain ; black triangles : regulated proteins without TM domains .

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(A) Vacuolar proteolysis-deficient pep4∆ and tul1∆ pep4∆ cells were labeled to saturation with heavy 13C6,15C2-L-lysine and then chased for 3 hours in presence of light L-lysine, following quantitative proteome analysis by LC-MS (two biological replicates). To analyze Dsc complex-dependent protein turnover, the ratio of H/L ratios of tul1∆ pep4∆ over pep4∆ cells of 2511 proteins quantified in both strains was plotted, and proteins with a ratio of H/L ratios >2 were selected. Red squares: regulated proteins with at least one predicted transmembrane (TM) domain; black triangles: regulated proteins without TM domains.


SDS - PAGE and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated

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SDS-PAGE and Western blot analysis with the indicated antibodies of total yeast lysates from WT cells and the indicated mutants


SDS - PAGE and Western blot analysis with the indicated antibodies ( E ) of input and elution ( with FLAG peptide ) from denaturing FLAG - Orm2 immunoprecipitations ( IP ) from WT cells and the indicated mutants . Control cells expressed untagged

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SDS-PAGE and Western blot analysis with the indicated antibodies (E) of input and elution (with FLAG peptide) from denaturing FLAG-Orm2 immunoprecipitations (IP) from WT cells and the indicated mutants. Control cells expressed untagged Orm2


SDS - PAGE and Western blot analysis with the indicated antibodies ( F ) of input and elution ( with FLAG peptide ) from native FLAG - Orm2 co - immunoprecipitations from WT cells and the indicated mutants . The control strain expresses untagged Orm2 .

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SDS-PAGE and Western blot analysis with the indicated antibodies (F) of input and elution (with FLAG peptide) from native FLAG-Orm2 co-immunoprecipitations from WT cells and the indicated mutants. The control strain expresses untagged Orm2.


( A ) Epifluorescence and phase contrast microscopy of living WT yeast cells and the indicated mutants expressing GFP - Orm2 or GFP - Orm2 - K25 , 33R ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside the endoplasmic reticulum ( ER ) . Data information : Scale bars = 5 µm .

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(A) Epifluorescence and phase contrast microscopy of living WT yeast cells and the indicated mutants expressing GFP-Orm2 or GFP-Orm2-K25,33R (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Data information: Scale bars = 5 µm.


( B ) Epifluorescence and phase contrast microscopy of tul1 ∆ mutants expressing GFP - Orm2 ( green ) with Vps4 - mCherry ( red , endosomes ) or with Sec7 - mCherry ( red , Golgi ) or Vps4 - eGFP ( green ) with Sec7 - mCherry . Data information : Scale bars = 5 µm .

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(B) Epifluorescence and phase contrast microscopy of tul1∆ mutants expressing GFP-Orm2 (green) with Vps4-mCherry (red, endosomes) or with Sec7-mCherry (red, Golgi) or Vps4-eGFP (green) with Sec7-mCherry. Data information: Scale bars = 5 µm.


( C ) Left panel : Epifluorescence and phase contrast microscopy of the indicated mutants expressing GFP - Orm2 ( green ) with mCherry - Cps1 ( red , MVB cargo ) . E indicates class E compartments ; Right panel : Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures . Data information : Scale bars = 5 µm .

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(C) Left panel: Epifluorescence and phase contrast microscopy of the indicated mutants expressing GFP-Orm2 (green) with mCherry-Cps1 (red, MVB cargo). E indicates class E compartments; Right panel: Equal amounts of the indicated mutants in serial dilutions were incubated on agar plates at the indicated temperatures. Data information: Scale bars = 5 µm.


WT cells and the indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block protein synthesis for 90 min and 180 min at 26 ° C or at non - permissive temperature ( 37 ° C ) where indicated . Total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies . ( A ) Graphs display the FLAG - Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0 , 90 and 180 min after the addition of CHX . Each experiment was repeated at least three times , FLAG - Orm2 levels were normalized to Pgk1 loading controls , and each time - point was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation .

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WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (A) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots from cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.


WT cells and the indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block protein synthesis for 90 min and 180 min at 26 ° C or at non - permissive temperature ( 37 ° C ) where indicated . Total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies . Each experiment was repeated at least three times , FLAG - Orm2 levels were normalized to Pgk1 loading controls , and each time - point was related to t = 0 min ( set to 1 ) .

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WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. Each experiment was repeated at least three times, FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each time-point was related to t=0 min (set to 1).


WT cells and the indicated mutants were left untreated ( 0 min ) or were treated with cycloheximide ( CHX ) to block protein synthesis for 90 min and 180 min at 26 ° C or at non - permissive temperature ( 37 ° C ) where indicated . Total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies . ( C ) SEC13 WT and sec13 - 4 cells were preincubated for 30 minutes at 37 ° C before CHX was added .

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WT cells and the indicated mutants were left untreated (0 min) or were treated with cycloheximide (CHX) to block protein synthesis for 90 min and 180 min at 26°C or at non-permissive temperature (37°C) where indicated. Total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. (C) SEC13 WT and sec13-4 cells were preincubated for 30 minutes at 37°C before CHX was added.


( D ) Epifluorescence and phase contrast microscopy of living WT cells and cdc48 - 3 mutants expressing GFP - Orm2 incubated for 5h at the non - permissive temperature ( 37 ° C ) . Data information : Scale bars 5µm .

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(D) Epifluorescence and phase contrast microscopy of living WT cells and cdc48-3 mutants expressing GFP-Orm2 incubated for 5h at the non-permissive temperature (37°C). Data information: Scale bars 5µm.


( E ) Epifluorescence and phase contrast microscopy of living tul1 ∆ and tul1 ∆ sec13 - 4 mutants expressing GFP - Orm2 ( green ) and dsRed - HDEL ( red ) incubated at 26 ° C or shifted for 90 minutes to the non - permissive temperature ( 37 ° C ) . Data information : Scale bars 5µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Epifluorescence and phase contrast microscopy of living tul1∆ and tul1∆ sec13-4 mutants expressing GFP-Orm2 (green) and dsRed-HDEL (red) incubated at 26°C or shifted for 90 minutes to the non-permissive temperature (37°C). Data information: Scale bars 5µm.


( A ) WT ( pdr5 ∆ ) cells expressing FLAG - Orm2 and Sec61 - GFP ( integral ER membrane protein ) treated with MG - 132 were fractionated into insoluble membrane ( P100 ) and soluble cytoplasmic ( S100 ) fractions and analyzed by SDS - PAGE and Western blot with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP (integral ER membrane protein) treated with MG-132 were fractionated into insoluble membrane (P100) and soluble cytoplasmic (S100) fractions and analyzed by SDS-PAGE and Western blot with the indicated antibodies.


WT cells , tul1 ∆ mutants ( all in a pdr5 ∆ strain background ) were left untreated or treated with the proteasome inhibitor ( MG - 132 ) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG - Orm2 immunoprecipitations ( IP ) . Input and eluted fractions ( with FLAG peptide ) were analyzed by SDS - PAGE and Western blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT cells, tul1∆ mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


WT cells and cdc48 - 3 mutants ( all in a pdr5 ∆ strain background ) were left untreated or treated with the proteasome inhibitor ( MG - 132 ) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG - Orm2 immunoprecipitations ( IP ) . Input and eluted fractions ( with FLAG peptide ) were analyzed by SDS - PAGE and Western blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT cells and cdc48-3 mutants (all in a pdr5∆ strain background) were left untreated or treated with the proteasome inhibitor (MG-132) as indicated and subsequently subjected to subcellular fractionation S100 and P100 fractions were subjected to denaturing FLAG-Orm2 immunoprecipitations (IP). Input and eluted fractions (with FLAG peptide) were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


( D ) P100 fractions from MG - 132 - treated WT ( pdr5 ∆ ) cells expressing FLAG - Orm2 and Sec61 - GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100 . 000 x g centrifugation into soluble ( S ) and insoluble membrane ( M ) fractions , subjected to denaturing FLAG - immunoprecipitations and analyzed by Western blotting with the indicated antibodies . The asterisk indicates reactivity of the secondary antibody with IgG heavy chains .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) P100 fractions from MG-132-treated WT (pdr5∆) cells expressing FLAG-Orm2 and Sec61-GFP were extracted with Na2CO3 solution at the indicated pH and subsequently fractionated by 100.000 x g centrifugation into soluble (S) and insoluble membrane (M) fractions, subjected to denaturing FLAG-immunoprecipitations and analyzed by Western blotting with the indicated antibodies. The asterisk indicates reactivity of the secondary antibody with IgG heavy chains.


( A ) The levels of two major ceramide ( Cer ) species in lipid extracts from WT ( black ) , tul1Δ ( dark gray ) or Orm2 - K25 , 33R mutants ( light grey ) were measured using LC - MS and quantified using spiked non - yeast ceramides as internal standard . Data are normalized to Cer44 : 0 ; 4 levels of WT cells ( set to 1 ) and presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - test . p values are given above the respective bars .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) The levels of two major ceramide (Cer) species in lipid extracts from WT (black), tul1Δ (dark gray) or Orm2-K25,33R mutants (light grey) were measured using LC-MS and quantified using spiked non-yeast ceramides as internal standard. Data are normalized to Cer44:0;4 levels of WT cells (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( B ) Autoradiogram of sphingolipid extracts from [ 3H ] serine radiolabelled WT cells and tul1 ∆ mutants were separated by thin layer chromatography . IPC , Inositolphosphorylceramide ; MIPC , mannosylinositolphosphorylceramide ; MIP2C , mannosyldiinositolphosphorylceramide . X indicates an unknown lipid species that is insensitive to myriocin treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O

(B) Autoradiogram of sphingolipid extracts from [3H]serine radiolabelled WT cells and tul1∆ mutants were separated by thin layer chromatography. IPC, Inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; MIP2C, mannosyldiinositolphosphorylceramide. X indicates an unknown lipid species that is insensitive to myriocin treatment.


( C ) SDS - PAGE and Western blot analysis with the indicated antibodies of input and elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from cells expressing FLAG - Orm2 - WT or FLAG - Orm2 - K25 , 33R . Control cells expressed untagged Orm2 WT . The asterisks label unspecific cross - reactions of the respective antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-WT or FLAG-Orm2-K25,33R. Control cells expressed untagged Orm2 WT. The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE and Western blot analysis with the indicated antibodies of total cell lysates ( D ) from WT cells expressing FLAG - Orm2 or FLAG - Orm2 - K25 , 33R The asterisks label unspecific cross - reactions of the respective antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (D) from WT cells expressing FLAG-Orm2 or FLAG-Orm2-K25,33R The asterisks label unspecific cross-reactions of the respective antibodies.


SDS - PAGE or Phos - tag - PAGE and Western blot analysis with the indicated antibodies of total cell lysates ( E ) from WT cells and tul1 ∆ mutants expressing FLAG - Orm2 , FLAG - Orm2 - 3A or FLAG - Orm2 - 3D with or without Myriocin treatment ( 1 . 5 µM ) for one hour .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O

SDS-PAGE or Phos-tag-PAGE and Western blot analysis with the indicated antibodies of total cell lysates (E) from WT cells and tul1∆ mutants expressing FLAG-Orm2, FLAG-Orm2-3A or FLAG-Orm2-3D with or without Myriocin treatment (1.5 µM) for one hour.


( A ) Left panel : In tor1 - 1 avo3 ∆ 1274 - 1430 cells rapamycin specifically inhibits TORC2 but not TORC1 ( Gaubitz et al . , 2015 ) . tor1 - 1 avo3 ∆ 1274 - 1430 cells were treated with cycloheximide ( CHX ) and rapamycin or vehicle ( methanol ) for the indicated times . Right panel : WT or Orm2 mutant cells were treated with CHX for the indicated times . The protein levels of FLAG - Orm2 or of the indicated FLAG - Orm2 mutants from total cell lysates were analyzed by SDS - PAGE and Western blotting with the indicated antibodies .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Left panel: In tor1-1 avo3∆1274-1430 cells rapamycin specifically inhibits TORC2 but not TORC1 (Gaubitz et al., 2015). tor1-1 avo3∆1274-1430 cells were treated with cycloheximide (CHX) and rapamycin or vehicle (methanol) for the indicated times. Right panel: WT or Orm2 mutant cells were treated with CHX for the indicated times. The protein levels of FLAG-Orm2 or of the indicated FLAG-Orm2 mutants from total cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies.


( B ) Graphs display the FLAG - Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0 , 90 and 180 min after the addition of CHX . Each experiment was repeated at least three times . FLAG - Orm2 levels were normalized to Pgk1 loading controls , and each timepoint was related to t = 0 min ( set to 1 ) . Data are presented as mean ± standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Graphs display the FLAG-Orm2 protein levels determined by densitometric quantification of Western blots in cell lysates of WT cells and the indicated mutants 0, 90 and 180 min after the addition of CHX. Each experiment was repeated at least three times. FLAG-Orm2 levels were normalized to Pgk1 loading controls, and each timepoint was related to t=0 min (set to 1). Data are presented as mean ±standard deviation.


( C ) Denaturing immunoprecipitation ( IP ) of FLAG - Orm2 , FLAG - Orm2 - 3A and FLAG - Orm2 - 3D . The control strain expresses untagged Orm2 . Input and elutions ( with FLAG peptides ) were analyzed by SDS - PAGE and Western blot with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Denaturing immunoprecipitation (IP) of FLAG-Orm2, FLAG-Orm2-3A and FLAG-Orm2-3D. The control strain expresses untagged Orm2. Input and elutions (with FLAG peptides) were analyzed by SDS-PAGE and Western blot with the indicated antibodies.


( D ) Epifluorescence and phase contrast microscopy of living tul1 ∆ mutants expressing GFP - Orm2 , GFP - Orm2 - 3A or GFP - Orm2 - 3D ( green ) with dsRed - HDEL ( red ) . Arrowheads point to GFP - Orm2 accumulations outside the endoplasmic reticulum ( ER ) . Scale bars = 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Epifluorescence and phase contrast microscopy of living tul1∆ mutants expressing GFP-Orm2, GFP-Orm2-3A or GFP-Orm2-3D (green) with dsRed-HDEL (red). Arrowheads point to GFP-Orm2 accumulations outside the endoplasmic reticulum (ER). Scale bars = 5 µm.


( E ) SDS - PAGE and Western blot analysis with the indicated antibodies of input and elution ( with FLAG peptide ) from native anti - FLAG co - immunoprecipitations from cells expressing FLAG - Orm2 - K25 , 33R , FLAG - Orm2 - K25 , 33R - 3A or FLAG - Orm2 - K25 , 33R - 3D . The control strain expresses untagged Orm2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) SDS-PAGE and Western blot analysis with the indicated antibodies of input and elution (with FLAG peptide) from native anti-FLAG co-immunoprecipitations from cells expressing FLAG-Orm2-K25,33R, FLAG-Orm2-K25,33R-3A or FLAG-Orm2-K25,33R-3D. The control strain expresses untagged Orm2.


( F ) The levels of the long chain bases ( LCB ) C18 - dihydrosphingosine ( C18 - DHS ) and C18 - phytosphingosine ( C18 - PHS ) and of two major ceramide ( Cer ) species in lipid extracts from WT , tul1 ∆ , Orm2 - K25 , 33R , Orm2 - 3A and Orm2 - 3D mutants were measured using LC - MS and quantified using spiked non - yeast LCBs / ceramides as internal standard . Data are normalized to WT levels of the most abundant LCB / ceramide species ( set to 1 ) and presented as mean ± standard deviation from three independent experiments . Pairwise statistical significance was assessed by Student ' s t - test . p values are given above the respective bars .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The levels of the long chain bases (LCB) C18-dihydrosphingosine (C18-DHS) and C18-phytosphingosine (C18-PHS) and of two major ceramide (Cer) species in lipid extracts from WT, tul1∆, Orm2-K25,33R, Orm2-3A and Orm2-3D mutants were measured using LC-MS and quantified using spiked non-yeast LCBs/ceramides as internal standard. Data are normalized to WT levels of the most abundant LCB/ceramide species (set to 1) and presented as mean ±standard deviation from three independent experiments. Pairwise statistical significance was assessed by Student's t-test. p values are given above the respective bars.


( B ) When transposon insertion libraries subjected to ampicillin resistance selection , a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor ( pBR322 bla : : Im7 L53A I54A ) relative to a strain containing just pBR322 . Transposon insertion frequency increased 21 - , 24 - , and 8 - fold in the pstS , pstA , and pstC genes , respectively , compared to the insertion frequency found with no ampicillin selection .

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(B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.


( A ) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla : : Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect , pBR322 and pBR322 bla : : Im7 L53A I54A were transformed into strains containing in - frame deletion mutants of each of the pst genes ( pstS , pstC , and pstA , respectively ) . The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla : : Im7 L53A I54A , but did not shower higher ampicillin resistance in the presence of pBR322 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322.


( B ) Levels of β - lactamase ( Bla ) and β - lactamase - Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β - lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes ( CL248 ( pstS : : Tn5 ) , CL249 ( pstC : : Tn5 ) , CL250 ( pstA : : Tn5 ) ) . Maltose binding protein ( MBP ) was used as a control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.