pex35 is a regulator of peroxisome...

RESEARCH ARTICLE

Pex35 is a regulator of peroxisome abundanceIdo Yofe1 Kareem Soliman2 Silvia G Chuartzman1 Bruce Morgan34 Uri Weill1 Eden Yifrach1 Tobias P Dick4Sara J Cooper5 Christer S Ejsing6 Maya Schuldiner1 Einat Zalckvar1 and Sven Thoms2

ABSTRACTPeroxisomes are cellular organelles with vital functions in lipid aminoacid and redox metabolism The cellular formation and dynamics ofperoxisomes are governed by PEX genes however the regulation ofperoxisome abundance is still poorly understood Here we use ahigh-content microscopy screen in Saccharomyces cerevisiae toidentify new regulators of peroxisome size and abundance Ourscreen led to the identification of a previously uncharacterized genewhich we term PEX35 which affects peroxisome abundance PEX35encodes a peroxisomal membrane protein a remote homolog toseveral curvature-generating human proteins We systematicallycharacterized the genetic and physical interactome as well as themetabolome of mutants in PEX35 and we found that Pex35functionally interacts with the vesicle-budding-inducer Arf1 Ourresults highlight the functional interaction between peroxisomesand the secretory pathway

KEY WORDS Peroxisomes Pex35 Ygr168c Arf1 High-contentscreen Yeast

INTRODUCTIONOf the cellular organelles peroxisomes are among the most poorlyunderstood Peroxisomes are equipped with only a modest numberof proteins (sim100) and have a diameter in the range of the resolutionof light microscopes (sim200 nm) but they are extremely dynamic insize number and protein content (Smith and Aitchison 2013) Inaddition peroxisomes are intriguing and central organelles due totheir involvement in various cellular metabolic pathways such asthose of lipids reactive oxygen species (ROS) carbohydratespolyamines and amino acids (Wanders and Waterham 2006) aswell as their contribution to an increasing number of non-metabolicfunctions such as innate immunity (Dixit et al 2010 Magalhatildeeset al 2016 Odendall et al 2014)It is not surprising therefore that malfunction of peroxisomes

leads to peroxisome-specific diseases (Braverman et al 2013Weller et al 2003) and contributes to the pathology of Alzheimerrsquosand Parkinsonrsquos diseases aging cancer type 2 diabetes and heartfailure (Beach et al 2012 Colasante et al 2015 Fransen et al

2013 Islinger et al 2012 Trompier et al 2014) A usefuldistinction divides peroxisomal diseases into two groups those inwhich single enzymatic functions are defective and those whereperoxisome biogenesis is defective per se In peroxisome biogenesisdisorders (PBD) genes involved in peroxisome formation ormaintenance are mutated Such genes are termed PEX genes andthe proteins they encode are termed peroxins In humans 16 PEXgenes are known (Thoms and Gaumlrtner 2012 Wanders andWaterham 2006) Nearly all of the human PEX genes haveorthologs in yeasts however yeasts have additional PEX geneswithout obvious homologues in mammals PEX34 from the yeastSaccharomyces cerevisiae a recent addition to the growing list ofPEX genes encodes a peroxisomal membrane protein that functionstogether with the Pex11 family to control peroxisome number(Tower et al 2011) The Pex11 family in yeast encompasses theorthologous proteins Pex11 Pex25 and Pex27 and is involved inperoxisome division and proliferation (Thoms and Erdmann 2005)Therefore the Pex11 family is also referred to as PEX11-type ofperoxisome proliferators (PPP) (Kettelhut and Thoms 2014 Thomsand Gaumlrtner 2012) Despite the identification of new genesinvolved in division and proliferation our understanding ofperoxisome division is still partial and hence in-depthcharacterization of the functions of known effectors as well asdiscovery of more players in this process is forthcoming

Classically PEX genes have been identified by complementationscreens using mammalian cells or yeast cells (Just and Kunau 2014)The earlier yeast screens forPEX genes were based on the finding thatβ-oxidation in yeast is exclusively located in the peroxisome so thatonly peroxisome-competent cells were supposed to be able to utilizeoleic acid as a sole carbon source (Just and Kunau 2014) Morerecently it was shown that oleic acid becomes toxic when someperoxisomal functions are compromised (Lockshon et al 2007) andgenes with these functions can therefore not be identified intraditional screen settings Also owing to the experimentallimitations inherent in classical forward genetic screens yeastscreens for peroxisomal functions have identified mainly non-essential genes with strong phenotypes Other non-PEX genes thatare required for peroxisome biogenesis or function have beenidentified bymore dedicated approaches These include the Sec61 ERtranslocon complex as an entry point into the endomembrane systemfor peroxisomal membrane proteins (Thoms et al 2011a) or theADP-ribosylation factors (ARFs) small GTPases involved in thecontrol of many vesicular processes that are also required forperoxisome formation (Anthonio et al 2009 Lay et al 2006)

So how can we identify more proteins required for peroxisomalfunction In recent years this has been achieved by both genome-wide screens and dedicated proteomic analyses For exampleproteomics screens of peroxisomes were able to identify newperoxisomal proteins in fungi (Marelli et al 2004 Schaumlfer et al2001 Thoms et al 2008 Yi et al 2002) plants (Eubel et al 2008Fukao et al 2002 Reumann et al 2007 2009) and mammaliantissues such as rat liver (Islinger et al 2007 2010 Kikuchi et alReceived 10 February 2016 Accepted 24 November 2016

1Department of Molecular Genetics Weizmann Institute of Science Rehovot7610001 Israel 2Department of Child and Adolescent Health University MedicalCenter Gottingen 37075 Germany 3Department of Cellular BiochemistryUniversity of Kaiserslautern Kaiserslautern 67653 Germany 4Division of RedoxRegulation ZMBH-DKFZ Alliance German Cancer Research Center (DKFZ)Heidelberg 69121 Germany 5HudsonAlpha Institute for Biotechnology HuntsvilleAL 35806 USA 6Department of Biochemistry and Molecular Biology VILLUMCenter for Bioanalytical Sciences University of Southern Denmark Odense 5230Denmark

Authors for correspondence (mayaschuldinerweizmannacil einatzalckvarweizmannacil sventhomsmeduni-goettingende)

ST 0000-0003-3018-6363

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2004) mouse liver and kidney (Mi et al 2007 Ofman et al 2006Wiese et al 2007) and human liver (Gronemeyer et al 2013) Inaddition several genome-wide genetic screens in yeast have shedlight on new proteins and their potential functions For examplefitness and transcriptome responses to oleic and myristic acid havebeen used to define the networks underlying the metabolicresponses to these fatty acids and to identify new proteinsinvolved in peroxisome function (Lockshon et al 2007 Smithet al 2002 2006) A collection of kinase and phosphatase genedeletion strains has also been evaluated for the expression of theperoxisomal matrix protein enzyme 3-ketoacyl-CoA thiolase (Pot1)(Saleem et al 2008) Following a similar approach a genome-widescreen for alterations in the expression of a GFP-labeledperoxisomal matrix protein led to the identification of 169 genesrequired for all the processes that lead to faithful biogenesisand propagation of peroxisomes (Saleem et al 2010) Othersystematic approaches including high-throughput experiments withquantitative analysis and modeling have been used to complete theperoxisomal proteome and to improve the understanding ofperoxisome metabolism and biogenesis (Cohen et al 2014Wolinski et al 2009) More recently systematic high-contentscreens and bioinformatics analyses led to the identification of anew peroxisomal targeting protein Pex9 and two new peroxisomematrix proteins Pxp1 and Pxp2 (Effelsberg et al 2016 Noumltzelet al 2016 Yifrach et al 2016 Yofe et al 2016) With each newstudy revealing additional new peroxisomal proteins it is clear thatwe have not yet saturated our understanding of the peroxisomalproteome and new peroxisomal proteins await discoveryIn this work we report the results of a high-content screen in

the yeast Saccharomyces cerevisiae tailored to uncover mutantsaffecting peroxisome abundance By creating a systematic libraryof gene mutants in yeast cells expressing a fluorescent markerfor peroxisomes followed by automated microscopy andcomputational image analysis we identified a previouslyuncharacterized protein affecting peroxisome abundance Weshow that this protein which we name Pex35 is a peroxisomalmembrane protein that controls peroxisome abundance

RESULTSA high-content screen uncovers regulators of peroxisomesize and numberIn order to identify genes that regulate peroxisome number or sizewe wanted to quantify these parameters in mutants of every yeastgene To this end we created a query strain that enables visualizationof peroxisomes and is compatible with the Synthetic Genetic Array(SGA) automated mating approach in yeast (Cohen and Schuldiner2011 Tong and Boone 2006) The strain expressed the peroxisomalmembrane protein Pex3 tagged with the red fluorescent proteinmCherry (Pex3ndashmCherry) and a fluorescent marker for theendoplasmic reticulum (ER) Spf1ndashGFP to enable automated cellsegmentation The query strain was then crossed into nearly 5000strains of the deletion library (Giaever et al 2002) and more than1000 strains of the DAmP (Decreased Abundance by mRNAPerturbation) hypomorphic allele library for essential genes(Breslow et al 2008) Following sporulation and selection ofhaploids we obtained about 6000 unique haploid strains eachmutated in one yeast gene and marked with Pex3ndashmCherry andSpf1ndashGFP This screening library was subjected to automatedimage acquisition and analysis during growth in glucose Using anautomated high-content microscopy setup specifically configuredfor this purpose (Breker et al 2013) we recorded three imagescovering hundreds of cells per strain (Fig 1A) We then developed a

computational pipeline using the green fluorescent ER marker tofind individual cells and counted the mean size of peroxisomes aswell as their number per cell (Fig S1) When analyzing peroxisomesize (Fig 1B for complete data see Table S1) we found that meancell peroxisome size varied through a range of approximately six-fold Although some peroxisomal proteins and proteins associatedwith peroxisome formation were enriched at the edges of thedistribution there was no strong enrichment or near-completecoverage of mutants in peroxisomal proteins or peroxisomebiogenesis factors in the strains with decreased or increasedperoxisome size Rather genes controlling mitochondrial functionor localization were dramatically enriched Among the top 130mutants with enlarged peroxisomes 80 were mitochondrial proteins(five-fold enrichment P-value=2times10minus31) (Eden et al 2009)confirming a tight connection between peroxisomes andmitochondria (Cohen et al 2014 Mohanty and McBride 2013Schrader et al 2013 Thoms et al 2009) Indeed it is known thatwhen mitochondrial metabolism is compromised cells cancompensate by upregulating peroxisome metabolism and size(Eisenberg-Bord and Schuldiner 2016) Interestingly a mutantcausing some of the smallest peroxisomes was defective incarbamoyl phosphate synthase (Cpa1) which is important for thebiosynthesis of the amino acid citrulline These findings indicatethat peroxisome size is not the best parameter to identify newperoxisomal proteins However the information regarding size maybe used in the future to further understand the factors that create thefunctional link between mitochondria and peroxisomes

When focusing on peroxisome number we noticed that mutantswith increased peroxisome number were not enriched forperoxisomal proteins and seemed to have defects in centralcellular processes like translation chromatin control orcytoskeletal dynamics (Fig 1C Table S1) However the strainswith the strongest reduction in peroxisome number were strikinglyenriched in known peroxisomal membrane proteins (105-foldenrichment P-value=10minus26) (Eden et al 2009) (Fig 1C) Amongthose are strains known to affect peroxisome morphology (Δpex19Δpex27 Δinp1 and Δvps1) At the other end of the spectrum loss ofPex17 a protein that is associated with the docking complex forperoxisome matrix protein import (Huhse et al 1998) caused adrastic increase in peroxisome number Importantly severalunknown proteins affected peroxisome number to a similar extentas mutations in known peroxisomal proteins [Fig 1C (inset) andTable 1] Of those the most highly ranking uncharacterized openreading frame was YGR168C

Deletion or overexpression of YGR168C affects peroxisomenumber and functionWe decided to focus on YGR168C since a deletion of this previouslyuncharacterized gene caused a decrease in peroxisome abundance thatis as dramatic as defects in the known peroxisomal biogenesis andinheritance proteins Pex12 and Vps1 (Fig 2A) To ensure that thisphenotype is independent of the peroxisomal marker (Pex3ndashmCherry)that we chose for the initial screen we deleted YGR168C in fourstrains with GFP-tagged peroxisomal markers including both matrixand membrane proteins We found that peroxisome recognition of ourimage analysis program depended on the intensity of the marker usedand the analysis settings Therefore peroxisome abundance valuesfrommeasurement of fluorescent markers should be stated in arbitraryunits that may only be used to compare between strains with the samemarker However regardless of the marker used loss of YGR168Ccaused a significant reduction in peroxisome number per cell whencompared to a control strain (Fig 2B)

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Next we manipulated Ygr168c protein expression by puttingit under control of the strong TEF1 promoter (TEF1prom)Overexpression of YGR168C also led to a decrease inperoxisomal fluorescent puncta either in glucose (Fig 2C) oroleic acid as a sole carbon source (Fig 2D) The fact that bothdeletion and overexpression caused similar phenotypes waspuzzling to us and we decided to follow peroxisomal architecturein more detail

Super-resolution microscopy demonstrates that YGR168Coverexpression leads to multi-lobular peroxisomemorphologyTo better characterize the effect on peroxisomes produced byeither deletion or overexpression of YGR168C we analyzedperoxisome size and morphology by stimulated emissiondepletion (STED) microscopy Strains were transformed with aplasmid expressing a peroxisome-targetedGFP variant (EYFPndashSKL)

Fig 1 A high-content screen uncovers regulators of peroxisome size and number (A) Schematic representation of the high-content screen workflow Aquery strain containing the peroxisomal marker Pex3ndashmCherry and the ER marker Spf1ndashGFP was crossed with strain collections of gene deletions andhypomorphic alleles Following selection the resulting haploid yeast each contained both a single mutation and the markers The libraries were imaged using ahigh-throughput automatedmicroscope Imageswere analyzed by software-assisted single-cell and object (peroxisome) recognition allowing the identification ofmutants that show aberrant peroxisome size or number (B) Distribution of the Z score of the mean peroxisome size for successfully analyzed mutant strainsMutants of peroxisomal membrane proteins are marked in red and other peroxisomal proteins marked in black (C) Distribution of the Z score of the meanperoxisome number per cell for successfully analyzed mutant strains A high enrichment of peroxisomal membrane protein mutants and Δpexmutants is found inthe group of decreased peroxisome number as illustrated by the inset pie chart presenting functional classes of the 30 mutants in this group YGR168C is apreviously uncharacterized gene found within this group Mutants of peroxisomal membrane proteins are marked in red and other peroxisomal proteins aremarked in black

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and were analyzed by fluorescence nanoscopy using nanobodiesdirected against GFPFirst using STED microscopy we determined the size of

peroxisomes with unprecedented accuracy We used images of71 peroxisomes to determine that the diameter of wild-typeperoxisomes is 174 nm (plusmn8 nm sem) which is below theresolution of conventional light microscopy (Fig 3ADFig S2A) Second these images as opposed to regulardiffraction-limited analysis revealed a clue as to the basis of thefunction of YGR168C While the number of peroxisomes wasreduced in Δygr168c their size was not (Fig 3BD Fig S2A)However peroxisomes in the overexpressing strain which appearedto be enlarged in confocal microscopy analysis showed a strikingmulti-lobular morphology when analyzed by STED microscopy(Fig 3CD Fig S2A) This phenotype may be due to hyper-fissionof the organelles into tiny peroxisomes which clump togethergiving the false impression of a single big peroxisome whenvisualizing cells by fluorescence microscopy alternatively themulti-lobular phenotype may be due to the initiation of aproliferation process that is aborted at an early stage Bothscenarios are compatible with a role of Ygr168c in controllingperoxisome fission Loss of YGR168C may therefore cause theopposite effect ndash reduction in fission ndash causing the observedreduction in peroxisome number Taken together our resultsdemonstrate that Ygr168c is a bona fide regulator of peroxisomenumber and size in yeast

YGR168C is a new PEX gene which we denote PEX35Since Ygr168c has such a strong effect on peroxisome numberand size we wanted to better characterize this protein TheC-terminally tagged strain could not be visualized abovebackground autofluorescence when expressed under its ownpromoter in cells growing in glucose medium indicating thatexpression of the protein in glucose medium is very low When weexpressed the protein with either a C- or N-terminal fluorescent tagunder control of the medium strength TEF1 promoter Ygr168ctagged on either terminus was localized to punctate structures thatcompletely colocalized with the peroxisomal markers Pex3ndash

mCherry or Pex3ndashGFP (Fig 4A) The Ygr168c protein ispredicted by TMHMM (Krogh et al 2001) and TOPCONS(Bernsel et al 2008) to be a membrane protein with fivetransmembrane domains and a large C-terminal domain facing thecytosol (Fig 4B) We examined the expression and localization ofGFPndashYgr168c in the absence of Pex19 a farnesylated membraneprotein biogenesis factor (Rucktaumlschel et al 2009) In Δpex19cells the residual GFP signal is distributed in the cytosol and theER (Fig 4C) These findings further support that Ygr168c is aperoxisomal protein that is withheld in or mislocalized to the ER inthe absence of Pex19 Similar results were obtained in the absenceof PEX3 GFPndashYgr168c expression was reduced and the residualGFP signal was distributed in the cytosol and the ER (Fig S2B) inagreement with Ygr168c being a peroxisomal protein To confirmthat Ygr168c is a bona fide peroxisomal protein we purifiedperoxisomes from oleate-induced wild-type cells with a GFP-marked Ygr168c Ygr168c was recovered from the peroxisomefraction of the Nycodenz density gradient (24ndash35 interface) andwas absent in the cytosolic and the mitochondrial fractions(Fig 4D)

The N-terminal domain of Ygr168c is predicted to contain amitochondrial-targeting signal (MTS) (Emanuelsson et al 2007)As some peroxisomal membrane proteins can be dually targeted tomitochondria we analyzed the intracellular localization of anN-terminal fragment of Ygr168c comprising the first and secondpredicted transmembrane domain (TMD amino acids 1 to 107) thatcontain the predicted MTS as a fusion protein with mCherryMitochondrial targeting of the truncated protein was not observedsuggesting that the N-terminal domain does not function as an MTSin this context or under these conditions Interestingly this fusionprotein now localized to both the ER and peroxisomes (Fig 4E) inagreement with the proposed passage of peroxisomal membraneproteins through the ER (van der Zand et al 2010) Our finding alsosuggests that peroxisome-specific targeting information is in theC-terminal domain of Ygr168c

The hallmark of many strains with a defect in maintaining normalperoxisome abundance is a growth defect when grown on oleic acidas the sole carbon source Indeed the deletion of YGR168C did notaffect growth on glucose (Fig S2C) yet showed reduced growthand reduced formation of halos on oleic acid mediumdemonstrating the lack of oleic acid consumption (Fig S2D)Interestingly the overexpression did not give a similar phenotype(Fig S2E) However overexpression of a GFP-tagged isoform(TEF1promGFP-YGR168C) did cause a phenotype suggesting thatit somehow aggravates the effect of overexpression or creates adominant negative (Fig S2E) Growth curves of these strains onoleate as a sole carbon source confirm this finding the growth ofΔygr168 and TEF1promGFP-YGR168C is slower than a controlstrain although is better than Δpex3 (Fig 4F) Quantitativeassessment of oleate consumption at the end point of the growthassay showed that mutant cells indeed utilized less of the availableoleic acid (Fig 4G)

Finally we assayed the accumulation of peroxisomes in real timeThe GAL1 promoter was introduced to regulate GFPndashYgr168c in astrain containing Pex3ndashRFP as a peroxisomal marker Time-lapsemicroscopy following a transfer of this strain from glucose- togalactose-containing medium showed that GFPndashYgr168caccumulated in Pex3-marked peroxisomes Furthermore whilecells that did not show GFPndashYgr168c expression had a normalperoxisome abundance cells with GFPndashYgr168c expressiondisplayed an abnormal phenotype of one enlarged peroxisome percell over time (Fig 4H)

Table 1 Top hits of the microscopic screen for proteins involved inperoxisome formation

Rank Gene Protein Abundance

1 YDL065C Pex19 minus762 YKR001C Vps1 minus603 YOL044W Pex15 minus554 YNL329C Pex6 minus555 YOR193W Pex27 minus536 YKL197C Pex1 minus527 YDR265W Pex10 minus498 YMR204C Inp1 minus479 YGR077C Pex8 minus4710 YJL210W Pex2 minus4611 YCL056C Pex34 minus4412 YPL112C Pex25 minus4413 YMR026C Pex12 minus4114 YMR205C Pfk2 minus4015 YMR163C Inp2 minus4016 YLR191W Pex13 minus3517 YGR168C minus3218 YDR233C Rtn1 minus2919 YLR126C minus2820 YPR128C Ant1 minus2721 YOR084W Lpx1 minus24

Abundance peroxisome number per cell (Z score)

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Taken together these data show that Ygr168c is a newperoxisomal membrane protein in yeast Manipulation of theexpression level of Ygr168c led to changes in peroxisomenumber and size as well as to growth defects on oleate AsYgr168c bears all signs of a peroxin we named it Pex35

Characterizing the molecular function of Pex35Having identified a new peroxisomal membrane protein we wereeager to functionally characterize it and understand how it exerts itseffect on peroxisome number and size Since peroxisomes have animportant role in lipid metabolism we first performed a detailedlipidomic analysis but could find no significant changes in whole-cell lipid composition compared to a control strain (Table S2) Thisresult suggests that Pex35 does not influence gross membrane lipidcomposition Coupled with the relatively small growth defect ofΔpex35 strains grown on oleic acid our results suggest that Pex35 isnot directly involved in lipid metabolism

We then performed a synthetic growth screen to try and uncoverproteins or pathways that become important when cells are lackingPex35 In this screen a Δpex35 strain was crossed with the librariescomprising thesim6000 deletion and hypomorphic strains that we hadpreviously used for the visual screen After mating sporulation andselection of haploids growth of the double mutants was comparedto growth of the individual mutant strain (Table 2 Table S3)Focusing on synthetic lethal interactions where the double mutantsare dead while each single mutant is alive we saw that redoxhomeostasis becomes important in the absence of Pex35 Forexample deletion of the glutathione (GSH) synthetase (GSH2)required for the synthesis of GSH from γ-glutamyl cysteine andglycine was synthetic lethal with Δpex35 This suggests that themutant strongly depends on a reducing environment for survival Totest this we measured the glutathione redox potential in theperoxisomal matrix using a newly described peroxisomal reporter(Elbaz-Alon et al 2014) Notably overexpression of Pex35 had

Fig 2 Deletion or overexpression of YGR168C affects peroxisome number and function (A) Examples of mutants from the high-content screenshowing aberrant peroxisome content Images of strains from the high-content screen containing Pex3ndashRFP and Spf1ndashGFP Peroxisome size is increased inΔvps1 and peroxisome number is decreased inΔvps1Δpex12 and Δygr168c compared to awild-type (WT) strain Scale bar 5 micromGraph shows quantification ofthese parameters (see also Fig S1) ngt72 Plt001 compared with WT (t-test) (B) Reduction in peroxisome number in Δygr168c is independent of theperoxisomalmarker used The numberof peroxisomes per cell was analyzed by using several peroxisomalmembrane ormatrix proteins taggedwithGFP either ina WT or Δygr168c background The decrease in peroxisome number in Δygr168c is seen with all peroxisome markers used n=6 Plt001 (Δygr168c versusWT t-test foreachmarker) (C)OverexpressionofYGR168C reduces thenumberof peroxisomepuncta Imagesof strainswithPex3ndashGFPand that areWTΔygr168cor overexpress YGR168C (OE-YGR168C) Scale bar 5 microm (D) Peroxisome number is affected in ygr168cmutants grown in glucose and oleate Quantification ofmean cell peroxisome number per cell in WT Δygr168c or OE-YGR168C cells visualized after growth in either glucose (gray) or oleate (red) medium While anincrease in peroxisome number is observed in oleate compared to glucose for each strain peroxisome number is decreased in the mutants compared to WT inglucose medium and in OE-YGR168C in oleate ngt731 Plt001 compared with the same medium for WT (t-test) All quantitative data are meanplusmnsem

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already caused a substantial pro-oxidative shift under steady stateconditions Moreover following treatment with a bolus of hydrogenperoxide the probe remained more reduced in the deletion strainwhereas it became more oxidized upon overexpression of Pex35(Fig S2F)

Other pathways that were over-represented were those ofpolyamine cysteine lysine and arginine biosynthesis (Table 2)To test how amino acid levels are altered in the absence of Pex35we extracted metabolites from wild-type Δpex35 TEFpromGFP-PEX35 and Δpex3 strains We used high-performance liquidchromatography (HPLC)-based metabolomics to characterizeamino acid levels in each strain and found that amino acid levelswere indeed significantly affected (Fig S2G) There was aremarkable 70 reduction in the level of citrulline in Δpex35which was matched by a more than two-fold increase of citrulline inTEFpromGFP-PEX35 This was in agreement with the stronglethality of this strain in combination with mutants in the argininebiosynthetic pathway These findings reinforce the notion thatperoxisomes in yeast are important in regulating or executingaspects of amino acid metabolism (Natarajan et al 2001) andhighlights how defects in mitochondrial function (such as inarginine biosynthesis) can activate the retrograde response toenhance peroxisomal functions (Chelstowska and Butow 1995)

Pex35 is located in the proximity of Pex11 family proteinsand ArfsSo far our data suggested that Pex35 is a peroxisome membraneprotein affecting peroxisome number and size as well asredox and amino acid homeostasis To try and understand themechanism by which Pex35 functions we performed a systematiccomplementation assay using the two parts of a specially adapteddihydrofolate reductase (DHFR) enzyme (Tarassov et al 2008) Inthis assay one half of the DHFR enzyme is fused to Pex35 (lsquobaitrsquo)which is expressed insim5700 yeast strains expressing fusions of eachindividual proteins with the other half of DHFR (lsquopreyrsquo) When thePex35 bait is in proximity to one of the library prey proteinsmethotrexate-resistant DHFR activity is reconstituted and cells cangrow in the presence of methotrexate an inhibitor of theendogenous essential DHFR activity (Fig 5A)

When seeking for growth with an N-terminally tagged Pex35 wecould not find any high-confidence complementing proteinssuggesting that the N-terminus is essential for the interaction ofPex35 with effector proteins localized at the peroxisome (data notshown) and reinforcing the previous notion that the overexpressionof an N-terminally tagged GFPndashPex35 is more toxic than regular

Fig 3 STED microscopy reveals characteristics of wild-type and ygr168cperoxisomes (AndashC) Examples of STED super-resolution microscopy of wild-type (WT A) Δygr168c (B) and strains overexpressing YGR168C (OE-YGR168C C) expressing EYFPndashSKL as a peroxisomalmarker and labeled withanti-GFP nanobodies Scale bar 200 nm The size of peroxisomes wasdetermined as the FWHM of a Gaussian fit Dashed lines positions of size andcluster measurements Peroxisome diameter in Δygr168c is not alteredOverexpression of YGR168C causes an increase in the apparent size ofperoxisomes In conventional light microscopy these aggregates appear asenlarged single peroxisomes Sub-diffraction imaging reveals that enlargedperoxisomes consist of multi-lobular structures whereby the individualsubstructures appear as peroxisomes of wild-type-like morphology Thedistances between the maxima varies between 137 and 174 nm (D) The meandiameter of WT peroxisomes is 174plusmn8 nm (sem) The size difference betweenwild-type and knockout peroxisomes 96plusmn7 nm (sem) was not significant(P=01) The mean diameter of OE-YGR168C peroxisome clusters is 285 nm(plusmn16 nm sem) (P=6times10minus8 compared with WT) ngt50 in all cases

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Fig 4 See next page for legend

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overexpression However when we performed the assay with aC-terminally tagged strain either under the native Pex35 promoteror the strong TEF1 promoter we identified 11 proteins previouslyknown to be involved in peroxisomal functions among the top 16hits (Fig 5BC Table S4) These included Pex19 the peroxisomalmembrane protein chaperone supporting the notion that Pex35 is amembrane protein Other proteins that were in the vicinity of Pex35were peroxisomal membrane proteins or membrane-associatedproteins (enrichment factor 70 P=16times10minus17) (Eden et al 2009)Especially intriguing was the identification of the Pex11 familyproteins Pex11 and Pex25 and Arf1 because these proteins havepreviously been shown to interact with each other and all of themhave a role in peroxisome fission a process that would affectperoxisome number and size (Fig 5BC)

Pex35 modulates the effect of Arf1To examine whether the physical proximity with Arf1 can explainthe effect of Pex35 on peroxisome abundance we first testedwhether the abundance of Arf1 or the localization of Arf1ndashmCherrywere affected by deleting or overexpressing Pex35 We found thatboth deletion and overexpression of PEX35 dramatically increasedArf1 levels (Fig S3A) Moreover PEX35 also affected Arf1ndashmCherry distribution In control strains Arf1 localizes mainly inpunctate structures in agreement with the Golgi localization of Arf1(Stearns et al 1990) However when PEX35 was deleted or thetagged form was overexpressed Arf1 redistributed to variousinternal membranes (Fig 6A) It is well known that while theC-terminal tag of Arf1 creates a dysfunctional form it does reportaccurately on its subcellular localization (Huh et al 2003 Jianet al 2010)

Arf1 has previously been implicated in peroxisome formation(Anthonio et al 2009 Lay et al 2005 Passreiter et al 1998) Wetherefore investigated whether Pex35 was mediating its effect onperoxisome abundance through Arf1 by looking at the effect ofΔpex35 deletion or overexpression on the background of loss ofArf1 (Δarf1) Deletion of ARF1 in the Δpex35 knockout lead to amarked increase in peroxisome size and aggravated the effect ofΔpex35 on peroxisome number (Fig 6BC) Even more striking thedeletion of ARF1 in the Pex35-overexpressing strain rescued theperoxisome-enlarging effect of Pex35 overexpression andnormalized the number of peroxisomes to control levels (Fig 6BC)These findings suggest that Pex35 exerts its function through amechanism that requires Arf1

Next we wanted to analyze Arf1 at the organellar level Wepurified peroxisomes by cell fractionation from strains thatcontained both GFP-tagged Pex3 as a peroxisome marker andRFP-tagged Arf1 either in a wild-type or in a Δpex35 background(Fig S3B) The absence of the peroxisome marker in the cytosol orthe mitochondria fraction showed that other compartments were freefrom peroxisomes Arf1ndashRFP was clearly enriched in theperoxisome and mitochondria fractions in agreement with theresults of the quantitative whole-cell analysis (Fig S3A) Thedistinction of peroxisomal and mitochondrial pools of Arf1however was not possible in this experiment because the lsquolight-mitochondrialrsquo faction co-fractionates with peroxisomes in the stepgradient

Our findings on the effects of Pex35 on Arf1 made us wonderwhether altering PEX35 could exert an effect on the secretorypathway functions of Arf1 (Fig 6A) In order to test whether Arf1-dependent anterograde traffic was affected we tested secretion of

Fig 4 YGR168C (PEX35) is a new PEX gene (A) Ygr168c colocalizes with aperoxisomal marker Overexpressed (OE) N-terminally GFP-tagged Ygr168ccolocalizes with Pex3ndashmCherry with a notable phenotype of about one largeperoxisome per cell (top) Overexpression of Ygr168c with a C-terminalmCherry tag displays colocalization with Pex3ndashGFP (bottom) with wild-type(WT) peroxisome abundance Scale bars 5 microm (B) Predicted transmembranetopology of Ygr168c (Pex35) Transmembrane helices were predicted by theTMHMM algorithm (v20) The schematic representation demonstrates the fivetransmembrane domains as well as a long cytoplasmic C-terminus for theprotein (C) Deletion of PEX19 leads to the cytosolic localization and ERaccumulation of Ygr168c sfGFP superfolder GFP Scale bar 5 microm(D) Identification of GFPndashYgr168c on purified wild-type peroxisomes isolatedfrom oleate-induced cells Western blot analysis of the post-nuclearsupernatant (PNS) and cytosol (Cyt) peroxisomal (Px) and mitochondrial(Mito) fractions from the Nycodenz step gradient Anti-Por1 antibody is used asa mitochondrial marker (E) Overexpression of amino acids 1ndash107 of Ygr168c(Ygr168c1-107 including the N-terminal region and the first two predictedtransmembrane domains) tagged with mCherry displays colocalization withPex3ndashGFP and also shows localization to the ER Scale bar 5 microm(F)YGR168Cmutants growmore slowly inmedium containing oleic acid as thesole carbon source WT Δygr168c OE-YGR168C and Δpex3 strains weregrown in oleic acid for 128 h and the OD600 was measured to assess growthYGR168Cmutants display a significant growth defect at an intermediate levelbetween WT and the Δpex3 Error bars indicate sd n=4 (G) YGR168Cmutants consume less oleic acid Media from the end-point of the experimentdescribed in D (128 h) were used to assess the relative oleic acid consumptionof each strain Error bars indicate sd n=4 Plt005 (t-test) (H) Induction ofGFPndashYgr168c leads to accumulation of the protein in peroxisomes and toaberrant peroxisome content A strain expressing both Pex3ndashRFP and GFPndashYgr168c under the control of the inducibleGAL1 promoter was transferred fromglucose to galactose medium Time-lapse imaging was carried out startingfrom 90 min (t=0) GFPndashYgr168c becomes highly induced under theseconditions and colocalizes with Pex3ndashRFP over time Notably cells in whichGFPndashYgr168c is not expressed (arrows) display normal peroxisome contentwhile cells in which expression is induced display one large peroxisome ineach cell Scale bar 5 microm Dashed lines in images highlight cell outlines

Table 2 Top hits of the PEX35 synthetic genetic interactions screen

Rank Gene Protein SM DM DMSM

1 YOR130C Ort1 086 024 0272 YKL184W Spe1 067 019 0293 YNL268W Lyp1 013 004 0344 YCR028C-A Rim1 012 005 0395 YDR234W Lys4 081 032 0406 YJL101C Gsh1 022 011 0477 YBR081C Spt7 020 010 0488 YJL071W Arg2 021 011 0519 YNL270C Alp1 130 069 05310 YER116C Slx8 025 014 05411 YGL143C Mrf1 049 028 05712 YBR251W Mrps5 013 007 05713 YNL005C Mrp7 030 017 05814 YHR147C Mrpl6 019 011 06115 YER185W Pug1 151 094 06216 YGL076C Rpl7a 049 031 06317 YMR142C Rpl13b 049 032 06518 YPR099C Ypr099c 030 020 06619 YDR028C Reg1 037 024 06620 YCL058C Fyv5 067 044 06621 YJL088W Arg3 037 025 06822 YDR448W Ada2 036 024 06823 YOL108C Ino4 047 032 06924 YOL095C Hmi1 122 084 06925 YDL077C Vam6 106 073 06926 YBR196C-A Ybr196c-a 040 028 06927 YDR017C Kcs1 082 057 06928 YBR282W Mrpl27 045 031 06929 YJR118C Ilm1 074 051 06930 YNL252C Mrpl17 028 020 069

SM normalized colony size of single mutant DM normalized colony size ofdouble mutant DMSM colony size ratio of the double to single mutant

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the intra-ER chaperone Kar2 (a BiP homolog) The slow leak ofKar2 from the ER through the Golgi and its secretion into themedium is a good way to observe dysregulated secretion (Copicet al 2009) Indeed overexpressing GFPndashPex35 (which led to adrastic increase in Arf1 levels) decreased secretion to 50 or lesssimilar to what is seen upon directly overexpressing Arf1 (Fig 6D)Interestingly the deletion of PEX35 (although also leading to Arf1overexpression) could compensate for the effect of Arf1overexpression suggesting that in normal physiology PEX35restrains Arf1 function (Fig 6D) Equally striking was the findingthat the deletion of PEX3 alone exerted a secretion defect in thesame range like strong activation of the unfolded protein response(UPR seen by deletion of the ER protein SPF1) or theoverexpression of Pex35 or Arf1 (Fig 6D) To follow up thisfinding we tested the localization of Arf1ndashRFP in a Δpex3background The deletion of PEX3 led to a surprising loss offocal Arf1 localization (Fig 6E) At this point it is not clear whetherthis is due to a direct association of Arf1 with Pex3 or Pex35 or if itis due to the upregulation of Arf1 or the large proportion of Arf1associated with peroxisomes that is altered upon Pex3 loss but in allthese cases these data point to a strong role for peroxisomes in thefunction of the secretory pathway

DISCUSSIONOne of the current challenges in molecular cell biology is tointegrate multiple levels of understanding spanning holistic omicsapproaches and detailed analysis of single molecular entities In thisstudy we go through such an analysis starting from a large-scalegenetic screen with the complete deletion and DAmP collectionand end up with the identification of a new peroxisomal protein andinsights into the mechanism by which it may regulate peroxisomeabundance

The initial screen relied on three automated stages generation of ascreening library comprising deletions and functional knockdownsof nearly all yeast genes automated image acquisition and acomputational pipeline for image segregation and quantificationThis approach complements the classical forward genetic screenbased on growth on oleate with the advantage of increasedsensitivity gained by statistical analysis of quantitative data Ourscreen was stringent as well as exhaustive ndash most other genespreviously implicated in many aspects of peroxisome formationinheritance and maintenance clustered on the top of our scoring listWe found that although some strains defective in peroxisomeproliferation have a strongly altered (reduced) peroxisome sizereduction in peroxisome number is a more exhaustive criterion tocover genes required for peroxisome formation and maintenance

Of the genes with the strongest effect on peroxisome number wedecided to focus on YGR168C which we termed PEX35 We showthat Pex35 is a peroxisomal membrane protein that had not beenidentified previously probably due to its low expression level incells grown on glucose and its moderate yet quantifiable effect onperoxisome number and size Deletion of PEX35 leads to areduction in peroxisome number and overexpression of PEX35 toan increase in hyper-fissioned aggregated tiny peroxisomes or to anearly block in the proliferation of peroxisomes These resultstogether with the oleate growth phenotypes classify Pex35 as abona fide peroxin

We found that C-terminally tagged Pex35 localizes in the vicinityof the Pex11-type of peroxisome proliferators Pex11 Pex25 andPex34 and the small GTPase Arf1 All of these proteins are knownto influence peroxisome abundance (Lay et al 2006 Thoms andErdmann 2005 Tower et al 2011) so it is attractive to speculate

Fig 5 Pex35 is found in proximity to peroxisomal membrane proteins aswell as to Arf proteins (A) A protein complementation assay for Pex35interactions reveals a physical proximity to the Pex11 Pex25 and Arf1 Querystrains with Pex35 tagged with either the C- or N-terminal fragment of theDHFR enzyme were mated with collections of strains expressing the other halfof DHFR in the opposing mating type Interaction between the Pex35 (bait) anda screened protein (prey) results in complementation of the DHFR enzymewhich in turn enables growth on the methotrexate medium as shown for Ant1and Pex19 (B) Pex35 is in proximity to Pex11 Pex25 and Arf1 Each of thethree indicated Pex35ndashprey combinations shown in the center of each imagedisplay large colonies in contrast to the neighboring colonies with otherPex35ndashprey combinations (C) Summary of proteins in proximity to Pex35 Foldincrease colony size the maximum colony size of four combinations of theDHFR screen (overexpression or native promoter for PEX35 bait and twoDHFR fragment collections)

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the Pex35 is part of a regulatory unit that controls peroxisomefission and therefore numbers in the cell The effect on Arf1 wasparticularly intriguing because ARFs have previously beenimplicated in peroxisome formation but their contribution hasbeen poorly established The mammalian ARF proteins Arf1 andArf6 have been shown to be present on rat liver peroxisomes and tobind to peroxisomes in a GTP-specific manner Interference witheither Arf1 or Arf6 changes peroxisome morphology (Anthonioet al 2009 Lay et al 2005 Passreiter et al 1998) In yeast theARF proteins Arf1 and Arf3 are required for peroxisome fissionalbeit in opposing manners (Anthonio et al 2009 Lay et al 2005)

Additional support of the involvement of ARFs in peroxisomefunction comes from the finding that the expression of a dominant-negative Arf1 mutant Arf1Q71L blocks protein transport from theER to the peroxisome in plant cells (McCartney et al 2005)However whether ARFs are actively recruited to peroxisomes howARFs affect peroxisomes and how this affects general cellulartrafficking fidelity is still a mystery

Initially Pex11 was suggested to be the receptor of ARFs andcoatomer on peroxisomes (Passreiter et al 1998) This notion wasquestioned when it was found that mutations that prevent coatomerbinding to Pex11 had no effect on peroxisome function in yeast or

Fig 6 Pex35 and peroxisomes control Arf1 distribution and function (A) Arf1 distribution is impaired in pex35 mutants While Arf1ndashRFP is localized inpunctate structures (potentially including Golgi and mitochondria) and partially colocalizes to peroxisomes (white arrowheads) in wild-type (WT) strains deletionor overexpression (OE) of PEX35 results in re-distribution to other internal membranes and loss of punctate localization Scale bar 5 microm (B) Synthetic effects ofPEX35 and ARF1mutations Pex3ndashGFP was used as a marker to quantify the peroxisome abundance in combinations of Δarf1 and WT Δpex35 or OE-PEX35Scale bar 5 microm (C) Quantification of peroxisomal content reveals that deletion of ARF1 aggravates the Δpex35 phenotype of reduced peroxisome numberNotably Δarf1 deletion compensated for the effect of PEX35 overexpression on peroxisome abundance Error bars indicate sem ngt197 Plt001 comparedwith WT (t-test) (D) Protein secretion analysis of PEX35 and ARF1 mutants Images (left the levels of secretion of Kar2 are indicated) and quantification (right)of a western blot of Kar2 secreted from six colony repeats per strain Induction of the UPR (by deletion of SPF1) or loss of peroxisomes (by deletion of PEX3) aswell as overexpression of GFPndashPex35 or ARF1 significantly reduced secretion levels Deletion of PEX35 compensated for the effect of ARF1 overexpressionError bars indicate sd n=6 Plt01 (not significant) Plt001 (t-test) (E) Deletion ofPEX3 affects cellular Arf1ndashRFP distribution In Δpex3 cells Arf1ndashRFP losesits localization to defined puncta Scale bar 5 microm

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trypanosomes (Maier et al 2000) In addition the COPI inhibitorbrefeldin A does not inhibit peroxisome biogenesis (South et al2000 Voorn-Brouwer et al 2001) However today there is newevidence for the involvement of membrane coats in peroxisomefunction (David et al 2013 Dimitrov et al 2013 Lay et al 2006)and it is becoming clear that peroxisome biogenesis may be distinctfrom peroxisome maintenance by fission Moreover as we saw inour own work fluorescence microscopy alone may not be asensitive enough readout for such changes as sub-diffractionimages can give more detailed information that may be easier toreconcile with genetic dataIn recent years reports on non-vesicular functions of ARFs are

becoming more common (Jackson and Bouvet 2014) We found thateither deletion or overexpression of Pex35 drives Arf1 out of itsphysiological localization mirroring the phenotype of an increasedGAP function or a reduced GEF function because the active GTP-bound form of Arf1 is recruited to the membrane whereas theinactive GDP-bound form is released from themembrane (Donaldsonand Jackson 2011) Of note the Arf1-GEF Sec7 was also found inthe proximity of Pex35 in our DFHR screen (Table S4 rank 29)although the significance of that interaction remains to be establishedWhile deletion of Pex35 elevates Arf1 protein levels (Fig S3A) wedo not understand its mechanistic connection with Arf1 It may verywell be that the upregulation of Arf1 is a consequence of reducedcellular function and cellular compensation mechanisms In supportof this the ability of overexpression of Arf1 to reduce leakage of Kar2is abolished in the absence of Pex35 (Fig 6D)Our findings illuminate both the role of Arf1 as a regulator of

peroxisome abundance and its dependency on healthy peroxisomebiogenesis Pex35 may be the crucial link in these processes loss offocal localization of Arf1 in a peroxisome-defective Δpex3 strain maybe due to the loss of peroxisomal recruitment of Arf1 in this strainIntriguingly Pex35 has domains that show homology with three

human proteins (Fig S4) two of which have membrane-shapingproperties One region of Pex35 is homologous to human Rtn1 anER reticulon and another with Epn4 an epsin-related protein thatbinds the clathrin coat Both proteins shape membrane curvaturewhich is essential for the fission process In addition potentialhomology was also found to Pxmp4 a human peroxisomal proteinThese areas of homology suggest that Pex35 functions have beenconserved in humans and they are suggestive of possiblemechanism of Pex35 functionOur work suggests that regulation of peroxisome size and number

is linked tightly with secretory pathway functions Physiologicalsecretion is dependent on the presence of peroxisomes On theother hand peroxisome formation itself requires a functionalsecretory pathway In this way the metabolic status of the cell couldbe coupled to secretion and growth in a simple and elegantmechanism Such a cross-talk between these two systems couldhave dramatic effects on cellular homeostasis and hence wouldimpact peroxisomes in health and disease

MATERIALS AND METHODSYeast strainsAll yeast strains in this study are based on the BY47412 laboratory strains(Brachmann et al 1998) Strains created in this study are listed in Table S5Allgenetic manipulations were performed using the lithium acetate polyethyleneglycol (PEG) and single-stranded DNA (ssDNA) method for transformingyeast strains (Gietz and Woods 2006) using plasmids previously described(Janke et al 2004 Longtine et al 1998) as listed in Table S6 Primers forgenetic manipulations and their validation were designed using Primers-4-Yeast (Yofe and Schuldiner 2014) as listed in Table S7

A query strain was constructed by introduction of an ER marker Spf1ndashGFP and a peroxisome marker Pex3ndashmCherry (yMS1233) The querystrain was then crossed into the yeast deletion and hypomorphic allelecollections (Breslow et al 2008 Giaever et al 2002) by the syntheticgenetic array method (Cohen and Schuldiner 2011 Tong and Boone2006) Representative strains of the resulting screening library werevalidated by inspection for the presence of peroxisomal mCherry and ER-localized GFP expression This procedure resulted in a collection of haploidstrains used for screening mutants that display peroxisomal abnormalities

Automated high-throughput fluorescence microscopyThe screening collection containing a total of 5878 strains was visualizedin SD medium during mid-logarithmic growth using an automatedmicroscopy setup as described previously (Breker et al 2013)

Automated image analysisAfter acquisition images were analyzed using the ScanR analysis software(Olympus) by which single cells were recognized based on the GFP signaland peroxisomes were recognized based on the mCherry signal (Fig S1)Measures of cell and peroxisome size shape and fluorescent signals wereextracted and outliers were removed A total of 5036 mutant strains weresuccessfully analyzed with a mean of 126plusmn56 (sem) cells per strain(minimum of 30) that passed filtering (Table S1) For each mutant strain themean number of peroxisomes recognized by the software per cell wasextracted as well as the mean area (in pixels) of peroxisomes Note thatthese are arbitrary units that may only be used to compare between strains asresulting values are dependent on the parameters of image analysis and ofthe fluorescent markers used

MicroscopyFor follow up microscopy analysis yeast growth conditions were asdescribed above for the high-content screening Imaging was performedusing the VisiScope Confocal Cell Explorer system composed of a ZeissYokogawa spinning disk scanning unit (CSU-W1) coupled with an invertedOlympus microscope (IX83 times60 oil objective excitation wavelength of488 nm for GFP and 561 nm for mCherry) Images were taken by aconnected PCO-Edge sCMOS camera controlled by VisView software Inthe case of galactose induction of GALp-GFP-PEX35 (Fig 3F) yeast weretransferred to the microscopy plate and after adhering to the glass bottommedium containing 2 galactose was used to wash the cells and during thetime-lapse imaging

STED microscopyStrains were transformed with EYFPndashSKL (plasmid PST1219) andanalyzed as described previously (Kaplan and Ewers 2015) using GFPnanobodies coupled to Atto647N (gba647n-100 Chromotek Planegg-Martinsried) Slides were analyzed on a custom-made STED setupdescribed previously (Goumlttfert et al 2013) Images show unprocessedraw data STED images shown in Fig 3 were rescaled by a factor of twousing bicubic interpolation (Adobe Photoshop) The size of yeastperoxisomes was calculated as follows Random peroxisomes weremarked by manually drawing a ROI and the center of masses wasdetermined Peroxisome diameter was measured after drawing a line scanthrough the minor axis of the peroxisome The full-width at half-maximum(FWHM) of the Gaussian fits for each peroxisomal structurewas determinedusing an ImageJ macro by John Lim Average size was quantified for morethan 50 peroxisomes and statistical significance was calculated using a two-sample t-test (two-tailed unequal variance)

Oleic acid growth experimentsGrowth on oleate oleate consumption and halo formation was analyzed asdescribed previously (Noumltzel et al 2016 Rucktaumlschel et al 2009 Thomset al 2008)

Isolation of peroxisomes and mitochondriaPreparation of peroxisomes and mitochondria followed published protocols(Flis et al 2015 Thoms et al 2011b) Briefly yeast cells were grown in

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YPD (1 yeast extract 2 peptone 2 glucose) to late exponential phasetransferred to YPO medium and grown for 48 h Cells were harvested bycentrifugation (3000 g for 5 min) washed with water and resuspended inbuffer containing 01 M Tris-HCl pH 94 and 15 mgml dithiothreitol(DTT) Spheroblasts were prepared by using 2 mg Zymolyase 20 T per 1 gwet weight Spheroblasts were recovered by centrifugation (3000 g for 10min) washed twice with 12 M sorbitol homogenized and lysed on iceusing a Dounce homogenizer in breaking buffer [5 mM 2-(N-morpholino)ethanesulfonic acid (MES) 1 mM potassium chloride 06 M sorbitol05 mM EDTA 1 mM phenylmethanesulfonylfluoride (PMSF) pH 60(with KOH)] Cell debris and nuclei were removed by centrifugation at3000 g for 5 min The resulting pellet was collected and subjected to twofurther rounds of resuspension in breaking buffer homogenizing andcentrifugation The combined (post nuclear) supernatants (PNS) werecentrifuged at 20000 g in an SS34 rotor (Sorvall) for 30 min The organellepellets containing peroxisomes and mitochondria were collected and gentlyresuspended in a small Dounce homogenizer in breaking buffer plus 1 mMPMSF and centrifuged at low speed (3000 g) for 5 min to remove residualcellular debris The supernatant containing the microsomal fraction wascentrifuged at 27000 g for 10 min The pellet was resuspended in breakingbuffer and loaded onto a Nycodenz step gradient [steps of 17 24 35(wv) in 5 mMMES 1 mM potassium chloride and 024 M sucrose pH 60(with KOH)] Centrifugationwas carried out in a swing out rotor (Sorvall AH-629) at 26000 rpm for 90 min Using a syringe the mitochondrial and theperoxisomal fraction were collected from the top of the 17 and the 24ndash35 interface respectively Purified organelles were diluted in four volumesof breaking buffer and centrifuged for 15 min at 27000 g in an SS34 rotorPellets were stored at minus20degC before determination of protein concentrationand western blot analysis Anti-Por1 rabbit antiserum (courtesy of GuumlntherDaum Institut fuumlr Biochemie Technische Universitaumlt Graz Austria) was usedat a dilution of 12000 to detect mitochondria

Arf1 protein quantificationYeast strains used for protein extraction (BY4741 as wild-type GPD1prom-PEX35natNT2 and Δpex35natNT2) were grown in YPD (with clonat forPex35 strains) to the stationary phase and the diluted in YPD to an opticaldensity at 600 nm (OD600) of 02 and further grown until they reached anOD600 of 06ndash08 25 OD600 units of yeast cells were harvested bycentrifugation (3000 g for 3 min) Cells were resuspended in 100 microl distilledwater 100 microl 02 M NaOH was added and cells were incubated for 5 min atroom temperature pelleted (3000 g for 3 min) resuspended in 75 microl SDSsample buffer (006 M Tris-HCl pH 68 10 glycerol 2 SDS 50 mMDTT 2 mM PMSF and 00025 Bromophenol Blue) boiled for 5 min at95degC and pelleted again (20000 g for 2 min) 25 microl supernatant was loadedper lane for SDS-PAGE (12 acrylamide) and then transferred to anitrocellulose membrane for western blotting Primary antibody was rabbitanti-Arf1 (12000 dilution a gift from Chris Fromme TheWeill Institute forCell and Molecular Biology Cornell University Ithaca USA) andsecondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit-IgG Membranes were exposed in LAS4000 machine usingchemiluminescence reagents Quantification was performed by ImageJrelative to a histone H3 loading control

Protein secretion assayWe analyzed protein secretion as previously described (Copic et al 2009)Specifically yeast strains grown in a 96-colony array format were pinned bythe RoToR colony arrayer onto YEPD agar plates and a nitrocellulosemembrane was applied onto the plate immediately thereafter Overnightincubation at 30degC allowed cell growth and ensured that secreted proteinswere transferred to the nitrocellulose membrane The membrane wasremoved from the plate and then washed (10 mM Tris-HCl 05 M NaClpH 75) to release remaining cells Secreted Kar2 was used as a reporter forsecretion level and was probed with a primary rabbit antibody (rabbitpolyclonal antiserum 15000 dilution kindly shared by Peter WalterDepartment of Biochemistry and Biophysics UCSF San Francisco USA)Next we used secondary goat anti-rabbit-IgG conjugated to IRDye800(926-32211 LI-COR Biosciences) and scanned the membrane for infraredsignal using the Odyssey Imaging System (LI-COR Biosciences)

LipidomicsQuantitative lipid analysis was performed using a Triversa NanoMate ionsource (Advion Biosciences Inc) coupled to a LTQ Orbitrap XL massspectrometer (Thermo Fisher Scientific) as previously described (Ejsinget al 2009)

Glutathione measurementsThe thiolndashdisulfide pair of roGFP2 equilibrates with the glutathione redoxcouple roGFP2 exhibits two fluorescence excitation maxima at sim400 nmand sim480 nm for fluorescence emission is followed at 510 nm The relativeintensity of fluorescence emission at each excitation maxima changes in theopposing direction dependent upon whether the roGFP2 thiols are reducedor forming a disulfide bond thereby allowing ratiometric fluorescenceimaging of the roGFP2 redox state Dynamic EGSH measurements wereconducted as previously described in detail (Morgan et al 2011)

Amino acid analysisAmino acid analysis was conducted as described previously (Noumltzel et al2016)

Protein complementation assay screeningThe proteinndashprotein interaction screen was performed using the yeast DHFRPCA library as previously described (Tarassov et al 2008) In briefstrains with Pex35 tagged at its C-terminus with DHFR[12] (in MATa) orDHFR[3] (in MATalpha) and regulated by either the natural PEX35 promoter(yMS25856) or the strong TEF promoter (yMS258990) weremated with thecomplementary DHFR libraries in 1536 colony array agar plates The resultingdiploids were subsequently selected for growth in the presence ofmethotrexatefor positive DHFR PCA reconstitution for 5 days at 30degC The colony arrayplates were scanned in two repeats and colony sizes were measured using thefreely available Balony software (Young andLoewen 2013) with each colonysize normalized by row and column means (Table S4)

AcknowledgementsWe thank Lihi Gal and Corinna Dickel for technical assistance Monika Schneiderand Ralph Kratzner for help with the amino acid analysis Ariane Wolf and AnneClancy for yeast library handling and Hans Kristian Hannibal-Bach for assistance onlipidomics analysis We would like to thank Ariane Wagner and Gunther Daum foradvice on organelle purification and for anti-Por1 antibodies Peter Walter for anti-Kar2 antibodies and Chris Fromme for antibodies useful discussions and ideas Wethank Nitai Steinberg for help with graphical design of figures and Jutta Gartner forsupport

Competing interestsThe authors declare no competing or financial interests

Author contributionsMS and ST conceived the study IY TPD CSE MS EZ and STconceived and supervised the experiments IY performed the majority of theexperiments in this manuscript S Chuartzman performed the high content screenKS performed the STED experiments and the oleate growth assays BM andTPD measured the redox status of peroxisomes CJE performed the lipidomicsanalysis ST performed peroxisome purification S Cooper KS and STconducted amino acid analyses All authors analyzed the data IY MS EZ andST wrote the manuscript

FundingThis work was supported by the State of Lower-Saxony Hannover Germany[ZN2921 to MS and ST] and by the European Research Council [CoG 646604 toMS] Maya Schuldiner is an incumbent of the Dr Gilbert Omenn and Martha DarlingProfessorial Chair in Molecular Genetics

Supplementary informationSupplementary information available online athttpjcsbiologistsorglookupdoi101242jcs187914supplemental

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Breker M Gymrek M and Schuldiner M (2013) A novel single-cell screeningplatform reveals proteome plasticity during yeast stress responses J Cell Biol200 839-850

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Colasante C Chen J Ahlemeyer B and Baumgart-Vogt E (2015)Peroxisomes in cardiomyocytes and the peroxisome peroxisome proliferator-activated receptor-loop Thromb Haemost 113 452-463

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David C Koch J Oeljeklaus S Laernsack A Melchior S Wiese SSchummer A Erdmann R Warscheid B and Brocard C (2013) Acombined approach of quantitative interaction proteomics and live-cell imagingreveals a regulatory role for endoplasmic reticulum (ER) reticulon homologyproteins in peroxisome biogenesis Mol Cell Proteomics 12 2408-2425

Dimitrov L Lam S K and Schekman R (2013) The role of the endoplasmicreticulum in peroxisome biogenesis Cold Spring Harb Perspect Biol 5a013243-a013243

Dixit E Boulant S Zhang Y Lee A S Y Odendall C Shum B HacohenN Chen Z J Whelan S P Fransen M et al (2010) Peroxisomes aresignaling platforms for antiviral innate immunity Cell 141 668-681

Donaldson J G and Jackson C L (2011) ARF family G proteins and theirregulators roles in membrane transport development and diseaseNat Rev MolCell Biol 12 362-375

Eden E Navon R Steinfeld I Lipson D and Yakhini Z (2009) GOrilla a toolfor discovery and visualization of enriched GO terms in ranked gene lists BMCBioinformatics 10 48

Effelsberg D Cruz-Zaragoza L D Schliebs W and Erdmann R (2016)Pex9p is a novel yeast peroxisomal import receptor for PTS1-proteins J Cell Sci129 4057-4066

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Elbaz-Alon Y Morgan B Clancy A Amoako T N E Zalckvar E DickT P Schwappach B and Schuldiner M (2014) The yeast oligopeptidetransporter Opt2 is localized to peroxisomes and affects glutathione redoxhomeostasis FEMS Yeast Res 14 1055-1067

Emanuelsson O Brunak S von Heijne G and Nielsen H (2007) Locatingproteins in the cell using TargetP SignalP and related tools Nat Protoc 2953-971

Eubel H Meyer E H Taylor N L Bussell J D OrsquoToole N HeazlewoodJ L Castleden I Small I D Smith S M and Millar A H (2008) Novelproteins putative membrane transporters and an integrated metabolic networkare revealed by quantitative proteomic analysis of Arabidopsis cell cultureperoxisomes Plant Physiol 148 1809-1829

Flis V V Fankl A Ramprecht C Zellnig G Leitner E Hermetter A andDaum G (2015) Phosphatidylcholine Supply to Peroxisomes of the YeastSaccharomyces cerevisiae PLoS One 10 e0135084

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2004) mouse liver and kidney (Mi et al 2007 Ofman et al 2006Wiese et al 2007) and human liver (Gronemeyer et al 2013) Inaddition several genome-wide genetic screens in yeast have shedlight on new proteins and their potential functions For examplefitness and transcriptome responses to oleic and myristic acid havebeen used to define the networks underlying the metabolicresponses to these fatty acids and to identify new proteinsinvolved in peroxisome function (Lockshon et al 2007 Smithet al 2002 2006) A collection of kinase and phosphatase genedeletion strains has also been evaluated for the expression of theperoxisomal matrix protein enzyme 3-ketoacyl-CoA thiolase (Pot1)(Saleem et al 2008) Following a similar approach a genome-widescreen for alterations in the expression of a GFP-labeledperoxisomal matrix protein led to the identification of 169 genesrequired for all the processes that lead to faithful biogenesisand propagation of peroxisomes (Saleem et al 2010) Othersystematic approaches including high-throughput experiments withquantitative analysis and modeling have been used to complete theperoxisomal proteome and to improve the understanding ofperoxisome metabolism and biogenesis (Cohen et al 2014Wolinski et al 2009) More recently systematic high-contentscreens and bioinformatics analyses led to the identification of anew peroxisomal targeting protein Pex9 and two new peroxisomematrix proteins Pxp1 and Pxp2 (Effelsberg et al 2016 Noumltzelet al 2016 Yifrach et al 2016 Yofe et al 2016) With each newstudy revealing additional new peroxisomal proteins it is clear thatwe have not yet saturated our understanding of the peroxisomalproteome and new peroxisomal proteins await discoveryIn this work we report the results of a high-content screen in

the yeast Saccharomyces cerevisiae tailored to uncover mutantsaffecting peroxisome abundance By creating a systematic libraryof gene mutants in yeast cells expressing a fluorescent markerfor peroxisomes followed by automated microscopy andcomputational image analysis we identified a previouslyuncharacterized protein affecting peroxisome abundance Weshow that this protein which we name Pex35 is a peroxisomalmembrane protein that controls peroxisome abundance

RESULTSA high-content screen uncovers regulators of peroxisomesize and numberIn order to identify genes that regulate peroxisome number or sizewe wanted to quantify these parameters in mutants of every yeastgene To this end we created a query strain that enables visualizationof peroxisomes and is compatible with the Synthetic Genetic Array(SGA) automated mating approach in yeast (Cohen and Schuldiner2011 Tong and Boone 2006) The strain expressed the peroxisomalmembrane protein Pex3 tagged with the red fluorescent proteinmCherry (Pex3ndashmCherry) and a fluorescent marker for theendoplasmic reticulum (ER) Spf1ndashGFP to enable automated cellsegmentation The query strain was then crossed into nearly 5000strains of the deletion library (Giaever et al 2002) and more than1000 strains of the DAmP (Decreased Abundance by mRNAPerturbation) hypomorphic allele library for essential genes(Breslow et al 2008) Following sporulation and selection ofhaploids we obtained about 6000 unique haploid strains eachmutated in one yeast gene and marked with Pex3ndashmCherry andSpf1ndashGFP This screening library was subjected to automatedimage acquisition and analysis during growth in glucose Using anautomated high-content microscopy setup specifically configuredfor this purpose (Breker et al 2013) we recorded three imagescovering hundreds of cells per strain (Fig 1A) We then developed a

computational pipeline using the green fluorescent ER marker tofind individual cells and counted the mean size of peroxisomes aswell as their number per cell (Fig S1) When analyzing peroxisomesize (Fig 1B for complete data see Table S1) we found that meancell peroxisome size varied through a range of approximately six-fold Although some peroxisomal proteins and proteins associatedwith peroxisome formation were enriched at the edges of thedistribution there was no strong enrichment or near-completecoverage of mutants in peroxisomal proteins or peroxisomebiogenesis factors in the strains with decreased or increasedperoxisome size Rather genes controlling mitochondrial functionor localization were dramatically enriched Among the top 130mutants with enlarged peroxisomes 80 were mitochondrial proteins(five-fold enrichment P-value=2times10minus31) (Eden et al 2009)confirming a tight connection between peroxisomes andmitochondria (Cohen et al 2014 Mohanty and McBride 2013Schrader et al 2013 Thoms et al 2009) Indeed it is known thatwhen mitochondrial metabolism is compromised cells cancompensate by upregulating peroxisome metabolism and size(Eisenberg-Bord and Schuldiner 2016) Interestingly a mutantcausing some of the smallest peroxisomes was defective incarbamoyl phosphate synthase (Cpa1) which is important for thebiosynthesis of the amino acid citrulline These findings indicatethat peroxisome size is not the best parameter to identify newperoxisomal proteins However the information regarding size maybe used in the future to further understand the factors that create thefunctional link between mitochondria and peroxisomes

When focusing on peroxisome number we noticed that mutantswith increased peroxisome number were not enriched forperoxisomal proteins and seemed to have defects in centralcellular processes like translation chromatin control orcytoskeletal dynamics (Fig 1C Table S1) However the strainswith the strongest reduction in peroxisome number were strikinglyenriched in known peroxisomal membrane proteins (105-foldenrichment P-value=10minus26) (Eden et al 2009) (Fig 1C) Amongthose are strains known to affect peroxisome morphology (Δpex19Δpex27 Δinp1 and Δvps1) At the other end of the spectrum loss ofPex17 a protein that is associated with the docking complex forperoxisome matrix protein import (Huhse et al 1998) caused adrastic increase in peroxisome number Importantly severalunknown proteins affected peroxisome number to a similar extentas mutations in known peroxisomal proteins [Fig 1C (inset) andTable 1] Of those the most highly ranking uncharacterized openreading frame was YGR168C

Deletion or overexpression of YGR168C affects peroxisomenumber and functionWe decided to focus on YGR168C since a deletion of this previouslyuncharacterized gene caused a decrease in peroxisome abundance thatis as dramatic as defects in the known peroxisomal biogenesis andinheritance proteins Pex12 and Vps1 (Fig 2A) To ensure that thisphenotype is independent of the peroxisomal marker (Pex3ndashmCherry)that we chose for the initial screen we deleted YGR168C in fourstrains with GFP-tagged peroxisomal markers including both matrixand membrane proteins We found that peroxisome recognition of ourimage analysis program depended on the intensity of the marker usedand the analysis settings Therefore peroxisome abundance valuesfrommeasurement of fluorescent markers should be stated in arbitraryunits that may only be used to compare between strains with the samemarker However regardless of the marker used loss of YGR168Ccaused a significant reduction in peroxisome number per cell whencompared to a control strain (Fig 2B)

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pex35 is a regulator of peroxisome...

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