©2005 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

[Autophagy 1:1, 37-45; April/May/June 2005]; ©2005 Landes Bioscience

Taras Y. Nazarko1,†

Ju Huang2,†

Jean-Marc Nicaud3

Daniel J. Klionsky2

Andrei A. Sibirny1,4,*1Institute of Cell Biology; National Academy of Sciences of Ukraine; Lviv, Ukraine

2University of Michigan; Life Sciences Institute and Departments of Molecular,Cellular and Developmental Biology and Biological Chemistry; Ann Arbor,Michigan USA

3Laboratoire de Microbiologie et de Génétique Moléculaire; CNRS-INRA-INAPG;Centre de Gringon; Thiverval-Gringon, France

4Institute of Biotechnology; Rzeszow University; Rzeszow, Poland

†These authors contributed equally.

*Correspondence to: Andrei A. Sibirny; Institute of Cell Biology; National Academyof Sciences of Ukraine; Drahomanov Street, 14/16; Lviv 79005 Ukraine; Tel.:+380.322.740363; Fax: +380.322.721648; Email: sibirny@biochem.lviv.ua

Received 07/23/04; Accepted 12/30/04

Previously published online as an Autophagy E-publication:http://www.landesbioscience.com/journals/autophagy/abstract.php?id=1512

KEY WORDS

autophagy, TRAPP, early secretory pathway,protein targeting, vacuole, yeast

ACKNOWLEDGEMENTS

This work was supported by INTAS 99-00788grant “Principles of peroxisome biogenesis anddegradation in yeasts” (to A.A. Sibirny) by theINRA (Département de Microbiologie) and CNRS(Département Science de la vie) (to J.-M. Nicaud)and by Public Health Service grant GM53396 fromthe National Institutes of Health (to D.J.Klionsky).

T.Y. Nazarko was also supported by INTASFellowship grant for Young Scientists YSF 2001/2-0094.

Research Paper

Trs85 is Required for Macroautophagy, Pexophagy and Cytoplasmto Vacuole Targeting in Yarrowia lipolytica and Saccharomyces cerevisiae

ABSTRACTYarrowia lipolytica was recently introduced as a new model organism to study

peroxisome degradation in yeasts. Transfer of Y. lipolytica cells from oleate/ethylamineto glucose/ammonium chloride medium leads to selective macroautophagy of peroxisomes.To decipher the molecular mechanisms of macropexophagy we isolated mutants ofY. lipolytica defective in the inactivation of peroxisomal enzymes under pexophagyconditions. Through this analysis we identified the gene YlTRS85, the ortholog ofSaccharomyces cerevisiae TRS85 that encodes the 85 kDa subunit of transport proteinparticle (TRAPP). A parallel genetic screen in S. cerevisiae also identified the trs85mutant. Here, we report that Trs85 is required for nonspecific autophagy, pexophagy andthe cytoplasm to vacuole targeting pathway in both yeasts.

INTRODUCTIONThere are three autophagy-related pathways that deliver cargo proteins and/or organelles

in cytosolic double-membrane vesicles to the vacuole, the membrane compartmentresponsible for degradation, recycling and storage of cellular constituents in yeasts. Theseare macroautophagy of bulk cytosol also referred to here as nonspecific autophagy, selectivemacroautophagy of peroxisomes that is termed macropexophagy, and selective macroau-tophagy of precursor aminopeptidase I (prApe1) by the cytoplasm to vacuole targeting(Cvt) pathway.1 All three of these pathways utilize topologically the same basic mechanismto enclose cargo material into autophagosomes, pexophagosomes and Cvt vesicles, respec-tively. A large number of molecular components that mediate different steps ofautophagy-related pathways were identified in the last decade. Not surprisingly, all threepathways appear to share most of them.2 This is also consistent with the common local-ization of most of the autophagy-related (Atg) proteins at the preautophagosomal structure(PAS).3-5 Assembly of both Cvt vesicles and autophagosomes appears to be dependent ona functional endoplasmic reticulum (ER) and Golgi complex.6-8 Therefore, the early secre-tory pathway was proposed to be the source of lipids for autophagosome and Cvt vesicleformation.7,8 The question of whether the early secretory pathway contributes tomacropexophagy, however, had not been addressed.

Although pexophagy appears to share a number of genes with macroautophagy, itoccurs at a faster rate and to a much greater extent than nonspecific autophagy of bulkcytosol.9 The molecular background of such exclusive efficacy is not clear, because onlyone strictly pexophagy-specific gene was reported until now in yeasts, ATG26/UGT51.ATG26 encodes sterol glucosyltransferase, essential for pexophagy in Pichia pastoris, butdispensable for macroautophagy and both macroautophagy and the Cvt pathway inP. pastoris and S. cerevisiae, respectively;10,11 however, its function in pexophagy may berestricted to P. pastoris or methylotrophic yeasts only, since ATG26 was not required forpexophagy in Yarrowia lipolytica.11 This last study, in addition to other work that hasrevealed differences in the localization and possibly function of other Atg proteins amongdifferent yeasts12 indicates the importance of analyzing autophagy-related pathways inalternative model systems.

Transfer of Y. lipolytica from acetate/oleate/ethylamine to glucose/ammonium sulfatemedium induces selective autophagy of peroxisomes.13 Macropexophagy was proposed tobe the major route of Y. lipolytica peroxisome degradation as determined by both electron13

and fluorescent14 microscopy. Y. lipolytica can use ethylamine as a sole nitrogen source dueto the induction of peroxisomal amine oxidase (AMO). AMO was used previously as amarker enzyme in the isolation of pexophagy mutants,13 but none of the mutant strains

www.landesbioscience.com Autophagy 37

has been further characterized. We isolated Y. lipolytica mutantsaffected in the degradation of peroxisomes (ref. 15, this study) usinginsertional mutagenesis16 and a modified AMO plate assay screeningprocedure.17 Here, we present the identification of the gene dis-rupted in two such mutants, both of which appeared to be affectedin the YlTRS85 gene. A separate screen for mutants defective in theCvt pathway in S. cerevisiae also identified ScTRS85. A deletion ofthis gene resulted in a block in nonspecific autophagy, pexophagyand import of prApe1. These results suggest a function for Trs85 asan Atg protein.

MATERIALS AND METHODSMedia and growth conditions. S. cerevisiae cultures were grown at 30˚C

in YPD (1% yeast extract, 2% peptone, 2% glucose) or SMD (0.67% yeastnitrogen base, 2% glucose, amino acids and vitamins) media. For nitrogenstarvation, SD-N (0.17% yeast nitrogen base without amino acids andammonium sulfate, 2% glucose) medium was used. Solid media were iden-tical with the addition of agar to 2% final concentration. Y. lipolytica strainswere cultured at 28˚C in YPD (1% yeast extract, 1% peptone, 1% glucose)medium, YND (1.7 g/l YNB without amino acids and ammonium sulfatesupplemented with 1% glucose and 0.5% ammonium sulfate) andYND(-N) (identical to YND except for omission of the nitrogen source),YOE (1.7 g/l YNB without amino acids and ammonium sulfate, 50 mMphosphate buffer, pH 6.8 (PiB), 0.05% yeast extract, 1% oleate, 25 mMethylamine) and GA (1.7 g/l YNB without amino acids and ammoniumsulfate, PiB, 1% glucose, 25 mM ammonium chloride) media. We prepared1% oleic acid in 0.025% Tween-80 as a 20-fold sonicated stock emulsion.For growth on solid media, strains were cultured on 2% agar plates usingYND and YEE (1.7 g/l YNB without amino acids and ammonium sulfatesupplemented with 0.05% yeast extract, 0.5% ethanol, 0.2% ethylamine-HCl).

Strains and plasmids. To delete the chromosomal TRS85 locus in S. cere-visiae strain YTS159 the entire coding region was replaced by transformationwith the URA3 gene, which was amplified from the template plasmidpUG7218 by PCR using primers containing 42 nucleotides identical to theregions flanking the TRS85 open reading frame (ORF); primer sequenceswill be made available upon request. The pCuTRS85(416) plasmid wasconstructed by cloning the entire coding region of the TRS85 gene plus 155nucleotides of the 3’ untranslated region into the pCu(416) vector19

between BamHI and HindIII restriction sites after the CUP1 promoter. Theplasmid encoding GFP-ATG8 under the control of the CUP1 promoter waspCuGFPAUT7(416).20

To generate Y. lipolytica mutants defective in amine oxidase (AMO) inac-tivation, the zeta-URA3 mutagenesis cassette (MTC) was amplified with theprimers MTC1 and MTC2,17 specific for the right and left borders of theMTC, respectively, using the plasmid JMP516 as template. Y. lipolytica wastransformed with MTC by the LiAc/LiCl method.21 For each transformationassay we used 0.5 µg of MTC generating up to 5,000 Ura+ transformantsper µg of DNA that were selected on YND plates. Mutants deficient inAMO inactivation (Ain) were isolated by a plate assay screening procedure:17

Ura+ transformants from YND plates were replica-plated onto YEE platesand incubated for 18 h. The plates were then carefully overlaid with 7-8 mlof AMO inactivation mixture containing 0.3% agar, 3% glucose and 1%ammonium sulfate, and incubated for 10 h at 28˚C. Then the plates wereoverlaid with 7–8 ml of AMO assay mixture containing 0.3% agar in 100 mMphosphate buffer, pH 7.0, 0.05% o-dianisidine as a chromogen, 0.5%cetyltrimethylammonium bromide as a permeabilizing agent, 2.3 U/mlperoxidase and 4 mM ethylamine as the substrate for AMO. Colonies withhigh residual activity of AMO were stained red and identified after 14 h ofincubation at 28˚C. Genomic DNA preparation from Y. lipolytica cells21

and Southern blot analysis16 were done as described previously.For complementation studies in Y. lipolytica we constructed the plasmid

JMP61(hph) by replacement of the Y. lipolytica URA3 gene with theKlebsiella pneumoniae hygromycin B phosphotransferase (hph) gene on theplasmid JMP61.22 The hph gene under the control of the synthetic promoter

hp4d was liberated from the plasmid JMP11523 as a BamHI (blunted)- XhoIfragment and ligated to the larger ClaI (blunted)-SalI fragment of JMP61.Then the plasmid JMP61(hph)BamHI with the zeta-hph empty cassette wasconstructed by removal of the POX2 promoter and LIP2 prepro sequence asa BamHI fragment from the plasmid JMP61(hph). The JMP61(hph)-YlTRS85 plasmid with the zeta-hph-YlTRS85 cassette was constructed byreplacement of the POX2 promoter and LIP2 prepro sequence on the plas-mid JMP61(hph) with the BamHI-KpnI fragment containing the YlTRS85ORF with 492 nucleotides of the promoter and 150 nucleotides of theterminator regions, which was amplified from the H222 wild type genomicDNA by PCR; primer sequences will be made available upon request. Ain16and Ain19 mutants were transformed with 0.3 µg of NotI-digested JMP61-(hph)BamHI and JMP61(hph)YlTRS85 plasmids by the LiAc/LiClmethod.21 The transformants with zeta-hph and zeta-hph-YlTRS85 cassetteswere selected on YPD+hygromycin plates with 50 µg/ml of hygromycin B.

Amplification and sequencing of MTC borders. To identify the genesdisrupted in Ain16 and Ain19 mutants, MTC borders were amplified byconvergent PCR. Genomic DNA of the mutants was triple digested withrestriction enzymes producing blunt ends, DraI, MscI and FspI, and ligatedwith specific adapter oligonucleotides.24 Left and right borders were ampli-fied and PCR fragments were gel purified and sequenced. Primer sequenceswill be made available upon request.

Biochemical studies of autophagy and pexophagy. Nonspecificautophagy was monitored in S. cerevisiae by measuring uptake of Pho8∆60through an alkaline phosphatase assay.25 A second method monitored pro-cessing of GFP-Atg8. Wild type (BY4742) and trs85∆ cells were transformedwith pCuGFPAUT7(416) and grown in SMD lacking uracil to OD600 = 1.0.Cells (5 ml) were harvested, washed once in SD-N medium and resuspendedin 5 ml SD-N for 1, 2 and 4 h. At each time point, 1 ml of sample wasremoved and subjected to TCA precipitation, then analyzed by Westernblotting. GFP-Atg8 fusion protein and free GFP were detected with amonoclonal antibody against GFP (Covance Research Products, Berkeley,CA). Pexophagy was analyzed by following the degradation of Fox3 asdescribed elsewhere.9

The survival of Y. lipolytica cells under nitrogen starvation conditions inliquid cultures was examined as described previously.26 For biochemicalstudies, Y. lipolytica cells from YPD cultures in the mid-log growth phasewere washed twice with PiB and inoculated into YOE medium at OD600 =0.3 for 16 h (until the early-log growth phase). Then cells were washed twicewith PiB and transferred to fresh GA medium at OD600 = 1–2. Growthrates, enzymatic activities and protein levels were followed in culture samples

Trs85 is Required for Specific- and Non-Specific Autophagy

Table 1 Yeast strains used in this study

Strain Genotype Source

S. cerevisiaeBY4742 MATα his3∆1 leu2∆0 ResGenTM

lys2∆0 ura3∆0atg1∆ BY4742 atg1∆::KAN ResGenTM

pep4∆ BY4742 pep4∆::KAN ResGenTM

trs85∆ BY4742 trs85∆::KAN ResGenTM

YJH3 YTS159 trs85∆::URA3 This studyYTS159 BY4742 pho8∆60 pho13∆::KAN This studyYTS161 BY4742 pho8∆60 pho13∆::KAN

atg1∆::URA3 This studyY. lipolytica

H222 MATA ylT1-free wild type (21)H222-S4 H222 ura3-302::SUC2 (16)Ain16 H222-S4 trs85-1::zeta-URA3 (15; this study)Ain19 H222-S4 trs85-2::zeta-URA3 (15; this study)

38 Autophagy 2005; Vol. 1 Issue 1

www.landesbioscience.com Autophagy 39

taken after 0, 3, 6, 9 and 12 h of glucose adaptation. For enzymatic assaysand immunoblotting, cells were washed twice with PiB at 4˚C and resus-pended in 0.2 ml of ice-cold PiB with 1 mM PMSF, 0.3 g of glass beads,and frozen at -20˚C. Cell-free extracts were prepared by vortexing for 15min at 4˚C using a Fisher Vortex Genie 2 at speed 7.5. Protein concentra-tions were determined by the Lowry method,27 using bovine serum albuminas the standard. AMO activity was measured in 50 mM phosphate buffer,pH 7.0, using 10 mM ethylamine as the substrate. This reaction led to theproduction of hydrogen peroxide, which was metabolized by horseradishperoxidase (2.5 U/ml) resulting in the oxidation of 1.1 mM 2,2'-azino-bis-(3-ethylbenz-thiazoline-6-sulphonic acid) that was followed by spectrometryat 420 nm.28 The SDS-PAGE29 and immunoblotting30 were performed aspreviously described. Rabbit anti-thiolase (anti-THI) antibodies were kindlyprovided by Dr. Richard A. Rachubinski (Department of Cell Biology,University of Alberta, Edmonton, Canada). Rabbit anti-GroEL antibodieswith cross-reactivity against the mitochondrial Hsp60 homologue ofY. lipolytica were kindly provided by Dr. Marten Veenhuis (GroningenBiomolecular Sciences and Biotechnology Institute, University ofGroningen, Haren, The Netherlands). Antigen-antibody complexes weredetected by enhanced chemiluminescence.

Studies of pexophagy by fluorescence microscopy. Y. lipolytica cells weretransformed with 0.3 µg of NotI-digested pYEG1hyg-POX3-EYFPplasmid14 by the LiAc/LiCl method.21 The transformants with integratedperoxisomal fluorescent protein-encoding cassette 2 (PFC2; 14) were selectedon YPD+hygromycin with 200 µg/ml of hygromycin B for H222 transfor-mants and 50 µg/ml of hygromycin B for transformants of Ain16 andAin19 strains. The induction of pexophagy for fluorescence microscopy,double-fluorescence labeling of peroxisomes and vacuoles, observation of

peroxisome-vacuole dynamics witha single filter set and acquisition ofphotomicrographs were done asdescribed previously.14

Cell labeling and immunopre-cipitation. S. cerevisiae cells weregrown in SMD medium to OD600 =1.0. Cells (10 ml) were collectedand resuspended in 150 µl SMDand labeled with 100 µCi of Tran-35S-label (ICN, Costa Mesa, CA)for 5 min and then subjected to anonradioactive chase by adding6.5 ml medium (SMD supplementedwith 0.2% yeast extract, 2 mMmethionine, and 1 mM cysteine)and incubating at 30˚C. Sampleswere taken at the indicated timepoints, precipitated with 10%trichloroacetic acid and washed withacetone. Protein extracts weregenerated by glass bead lysis andsubjected to immunoprecipitationas described previously.31 Rabbitantisera against Prc1, Pep4, Ape1and Fox3 have been previouslydescribed.9,31,32

Protease-sensitivity analysis. Theprotease-sensitivity experiment wascarried out essentially as describedpreviously.33

RESULTSPeroxisomes are not degraded

in the Y. lipolytica Ain16 and Ain19mutants. Over twenty proteins thatare specific to autophagy have been

identified in the past seven years34 and these likely represent the majority ofthe components involved in autophagy, pexophagy and the cytoplasm tovacuole targeting pathway. For two reasons, however, we decided to pursuean additional screen for autophagy mutants. First, biases inherent in any onescreen may result in certain mutants not being identified. Second, most ofthe mutants were identified in screens that were carried out to identifymutants in nonspecific autophagy and the Cvt pathway; however, one gene,ATG25, has already been identified from analyses of pexophagy inHansenula polymorpha that lacks a homolog in S. cerevisiae, and another,ATG26, from Pichia pastoris that appears to be specific to pexophagy.Accordingly, we undertook a screen for pexophagy-defective mutants in theyeast Yarrowia lipolytica.

The AMO plate assay screening procedure17 was used as a first step inthe selection of mutants deficient in peroxisome degradation. Taggedmutants were generated by insertional mutagenesis16 by inserting the PCRamplified zeta-URA3 mutagenesis cassette (MTC) into the genome of theY. lipolytica H222-S4 strain. We tested more than 30,000 Ura+ transformantsand identified 31 strains with reproducible defects in AMO inactivation onplates.15 We found that YOE (oleic acid, ethylamine) medium induced thehighest levels of peroxisomal enzymes in Y. lipolytica liquid cultures (datanot shown). Subsequent transfer of cells into GA (glucose, ammoniumchloride) medium led to efficient induction of pexophagy that could befollowed by the rates of inactivation of AMO (Fig. 1A and B) and isocitratelyase (ICL; data not shown) and degradation of thiolase (THI; Fig. 1C). Thestable level of the mitochondrial Hsp60 protein showed the specificity ofpexophagy after the transfer of cells from YOE to GA (Fig. 1C). Two mutants,Ain16 and Ain19, exhibited delayed inactivation of both AMO (Fig. 1A and B),and ICL (data not shown), and degradation of THI (see Fig. 1C, for Ain19)

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 1. The Ain16 and Ain19 mutants are defective in pexophagy and nonspecific autophagy. (A–C) Transfer ofY. lipolytica H222 wild type cells from YOE (oleic acid, ethylamine) to GA (glucose, ammonium chloride) mediumfor 12 h led to specific peroxisome degradation as detected by inactivation of peroxisomal AMO, degradation ofperoxisomal THI and the stable level of mitochondrial Hsp60. (A and B) Inactivation of AMO was affected in theY. lipolytica pexophagy mutants Ain16, Ain19 and their random integrative transformants (tr.1 and tr.2) with zeta-hph empty cassette. Transformation with the zeta-hph-YlTRS85 cassette restored the inactivation of AMO in bothmutant strains. The activities were corrected for growth of the cells in GA medium. (C) Degradation of peroxisomalTHI in the Ain19 mutant. (D) Ain16 and Ain19 mutants are starvation-sensitive. Cells were grown in YND medium,shifted to YND(-N) and incubated for the indicated number of days at 28˚C, then spread on YPD plates. The num-ber of viable colonies was determined as a percentage of those present at day zero essentially as described previ-ously.26

A B

C D

after the transfer of YOE-induced cells into GAmedium. In addition, both strains displayed adecreased viability under conditions of nitrogenstarvation relative to the wild type strain, indicatinga defect in nonspecific autophagy (Fig. 1D).

To study the stage of peroxisome degradationaffected in Ain16 and Ain19 strains, we observedtheir peroxisome-vacuole dynamics by fluorescencemicroscopy under pexophagy conditions. For thispurpose the wild type strain H222 and Y. lipolyticaAin16 and Ain19 mutants were transformed witha peroxisomal fluorescent protein-encoding cas-sette 2 (PFC2) and several random transformantsof each strain were selected on YPD+hygromycinplates. Peroxisomes were labeled with a PFC2-encoded fusion of acyl-CoA oxidase 3 andenhanced yellow fluorescent protein.14 All trans-formants exhibited normal growth and peroxisomalfluorescence patterns in YOE medium (data notshown). After the transfer of cells from YOE toGA medium the vacuolar membranes werestained red with the FM 4-64 dye. Peroxisomes ofthe Y. lipolytica wild type strain were delivered tothe vacuoles as seen by a decrease in punctatestaining in the cytosol and a transient increase influorescence within the vacuole lumen (Fig. 2).This decrease in cytosolic punctate staining pre-sumably reflected a macroautophagic mechanism,because the sites of close proximity of peroxisomesand vacuoles did not coincide with the sites ofvacuolar membrane invagination.14 In most casesvacuoles surrounded the lipid bodies but notperoxisomes. Considering that microautophagyof peroxisomes is an easily recognized series ofevents in double labeling experiments,35 we couldexclude this mode of pexophagy in our studies. Incontrast to the H222 wild type strain, almost allof the peroxisomes remained outside the vacuolesin Ain16 (data not shown) and Ain19 (Fig. 2)cells even after 9 h of adaptation to glucose, andthe vacuole lumens remained dark (nonfluorescent). This result indicatedthat macropexophagy was blocked at the stage of pexophagosome formationor fusion of pexophagosomes with the vacuolar membrane. We noted, how-ever, that the vacuoles appeared to be fragmented into smaller vesicles inboth Y. lipolytica Ain16 and Ain19 mutants (see Fig. 2 for Ain19) indicatinga possible defect in vacuole biogenesis. Thus, we could not rule out a non-specific fusion defect that resulted from the aberrant vacuole morphology,which may have reflected a mutation in proteins known to be involved inhomotypic vacuole fusion.36

The Y. lipolytica Ain16 and Ain19 mutants are both disrupted in theTRS85 gene. To identify the mutated gene that caused the pexophagydefect in the Ain16 and Ain19 mutants, we proceeded with an analysis ofthe MTC integration events. The genomic DNA of Ain16 and Ain19strains was analyzed as described in Materials and Methods. We amplifiedand sequenced 370 nucleotides and 570 nucleotides of the left borders fromAin16 and Ain19, respectively. Both DNA fragments appeared to be partsof the same open reading frame that was obtained during the sequencingproject of the Y. lipolytica genome.37 BLAST analysis of the predicted proteinproduct revealed that the MTC disrupted the homolog of S. cerevisiaeTRS85, the 85 kDa subunit of transport protein particle (TRAPP).38 TheScTRS85 gene was originally identified as GSG1 due to an indirect effect onsporulation.39,40 Recently, ScTrs85 was shown to be the component of twoforms of TRAPP, TRAPP I and TRAPP II, which mediate ER-to-Golgi andGolgi transport events, respectively.41 The complete Y. lipolytica TRS85 geneencodes a predicted protein of 573 amino acids displaying 22% identity and41% similarity to Trs85 of S. cerevisiae. Although the score of homology is

not very high, there are no other homologues of ScTRS85 in the Y. lipolyticagenome. Insertion of the MTC in YlTRS85 occurred 171 nucleotides and1106 nucleotides downstream of the predicted translational start codon inAin16 and Ain19, respectively. At the amino acid level, the MTC disruptedYlTrs85 downstream of A57 in Ain16 and downstream of R368 in Ain19,producing a truncated protein in each case. The same overall size of EcoRVMTC borders in Ain16 and Ain19 obtained by Southern blotting (data notshown) excluded the possibility of genomic DNA deletion during MTCinsertion and suggested that the pexophagy phenotype was related to disrup-tion of YlTRS85 in both strains. The latter conclusion was also confirmedby complementation studies that showed the complete restoration of AMOinactivation in both Y. lipolytica trs85 mutants by transformation with acassette containing the YlTRS85 gene (Fig. 1A and B).

The S. cerevisiae trs85∆ mutant is defective in selective autophagy.The identification of YlTRS85 as the mutated gene in the Ain16 and Ain19mutants suggested that a defect in vacuole biogenesis was not the cause ofthe observed pexophagy defect. Nonetheless, the fragmented vacuole pheno-type made it problematic to determine whether the pexophagy defect was adirect result of the mutation in YlTRS85. Independent of the screendescribed above, we had screened the systematic S. cerevisiae gene deletionlibrary for strains that were defective in the transport of precursoraminopeptidase I (prApe1; 42). One of the mutants that displayed a strongblock in processing of the precursor protein was deleted for the ScTRS85gene. In light of the above results, we examined this phenotype more carefully.Protein extracts were prepared from isogenic wild type and mutant cellsgrown under vegetative conditions and examined by Western blot. Wild

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 2. Peroxisomes are not delivered to the vacuoles in the Y. lipolytica Ain19 mutant strain.Peroxisome-vacuole dynamics were followed by fluorescence microscopy after the transfer of cells fromYOE (oleic acid, ethylamine) to GA (glucose, ammonium chloride) medium. Peroxisomes were labelledyellow-green with the fusion of acyl-CoA oxidase 3 and enhanced yellow fluorescent protein and vac-uolar membranes were stained red with FM 4-64 as described in Materials and Methods.

40 Autophagy 2005; Vol. 1 Issue 1

www.landesbioscience.com Autophagy 41

type cells displayed predominantly themature form of Ape1, indicating efficientdelivery to the vacuole and removal of thepropeptide (Fig. 3A). In contrast, theSctrs85∆ cells accumulated only the precur-sor form of the protein indicating a completeblock in vacuolar delivery or proteolyticprocessing (Fig. 3A). The Ape1 phenotype ofthe Sctrs85∆ mutant was restored to that sim-ilar to the wild type strain by transformationwith a plasmid encoding ScTRS85, indicatingthat the initial defect was due to the deletionof ScTRS85 (Fig. 3A).

Because the Y. lipolytica mutant had afragmented vacuole phenotype, we analyzedthe morphology of the vacuole in theSctrs85∆ strain. Although a higher percentageof the Sctrs85∆ cells had a fragmented vacuolephenotype, the total number of cells withnormal vacuoles was not substantially differ-ent from the wild type strain (Fig. 3B); thewild type and Sctrs85∆ strains had 28% and13% of the cells displaying one vacuole,49% and 48% displaying 2–4 vacuoles and23% and 39% displaying more than fourvacuoles, respectively. As a separate means ofassessing the competency of the vacuole, wedecided to examine the transport and pro-cessing of two resident vacuolar hydrolases,Pep4 and Prc1, which are delivered to thevacuole via the CPY pathway through the useof transient transport vesicles.43 S. cerevisiaewild type and trs85∆ cells were analyzed bypulse/chase labelling and immunoprecipita-tion as described in Materials and Methods.The Sctrs85∆ cells showed essentially identicalkinetics for the maturation of both hydro-lases (Fig. 3C) suggesting that the vacuole inthe Sctrs85∆ mutant cells was competent forfusion with transport vesicles. Furthermore,the normal processing of both precursor pro-teins indicated that the Sctrs85∆ vacuolesretained adequate proteolytic activity toremove the prApe1 propeptide.

The above results suggested that prApe1was not delivered to the vacuole in theSctrs85∆ mutant strain. An alternative possi-bility, however, was that the Sctrs85∆ mutantwas defective in the breakdown of the sub-vacuolar vesicle, the Cvt body, that resultsfrom the fusion of the double-membrane Cvtvesicle with the vacuole.44 To differentiatebetween these possibilities, we carried out aprotease-sensitivity analysis. Control Scpep4∆and Scatg1∆ cells and Sctrs85∆ mutant cellswere converted to spheroplasts and lysedunder conditions that retain the integrity ofsubcellular compartments. Exogenous proteasewas added and the state of Ape1 was deter-mined by Western blot. If prApe1 were pre-sent within a completed cytosolic vesicle or asubvacuolar vesicle it would be protectedfrom exogenous protease, whereas prApe1present in a partial vesicle in the cytosolwould be sensitive to removal of the propep-tide.33 The Scpep4∆ mutant is defective in

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 3. (A) The Cvt pathway is blocked in the Sctrs85∆ strain. The wild type (BY4742), Sctrs85∆ strainand the Sctrs85∆ strain harboring the pRS416 empty vector or pCuTRS85(416) plasmid were cultured inSMD medium to mid-log phase. Samples were collected and the proteins were precipitated with TCA andsubjected to SDS-PAGE followed by Western blotting with antiserum to Ape1 as described in Materials andMethods. The positions of precursor and mature Ape1 are indicated. (B) Vacuoles in the Sctrs85∆ strainhave a morphology similar to wild type. Cells were grown in YPD and vacuoles stained with FM 4-64 asdescribed in Materials and Methods. DIC, differential interference contrast. (C) Pep4 (PrA) and Prc1 (CpY)are processed normally in the Sctrs85∆ strain. Wild type and Sctrs85∆ strains were grown to mid-log phaseand then subjected to a pulse-chase analysis at the indicated time points to monitor the kinetics of Pep4 andPrc1 processing as described in Materials and Methods. The positions of precursor and mature forms of bothproteins are indicated.

A

B

C

subvacuolar vesicle breakdown; in this strain prApe1 was protected fromexogenous protease in the absence of detergent (Fig. 4), reflecting its local-ization within Cvt bodies present in the vacuole lumen. In contrast, prApe1was protease-sensitive in the absence of detergent in the Scatg1∆ strain,which is defective in the formation of Cvt vesicles. We found that prApe1was also protease-sensitive in the Sctrs85∆ strain (Fig. 4) indicating thatthere was a defect in formation of the Cvt vesicle rather than in breakdownof the Cvt body. Because the Sctrs85∆ strain did not have a severely frag-mented vacuole we decided to pursue our analysis of the role of Trs85 inS.cerevisiae.

Trs85 is required for pexophagy in S. cerevisiae. The import of prApe1is one type of specific autophagy. The degradation of peroxisomes alsooccurs through a specific mechanism. We used the peroxisomal matrixprotein Fox3 as a marker for peroxisomes to determine whether pexophagyin S. cerevisiae was also dependent on Trs85. Wild type, Scpep4∆ andSctrs85∆ cells were grown in medium containing oleic acid as the sole carbonsource to induce peroxisome proliferation. Cells were then shifted to SD-Nmedium to initiate pexophagy. Protein extracts were prepared at differenttimes and analyzed by Western blot. Fox3 was almost completely degradedin wild type cells after 6 h (Fig. 5). The Scpep4∆ strain is defective invacuolar hydrolase activity and unable to degrade peroxisomes, thus servingas a negative control; the Fox3 protein level remained unchanged through-out the 24 h time course (Fig. 5). Similar to the results with the Scpep4∆strain, the Sctrs85∆ mutant displayed essentially no change in the level ofFox3 (Fig. 5) indicating a defect in pexophagy.

Trs85 is required for nonspecific autophagy inS. cerevisiae. Pexophagy and the Cvt pathway sharemost of the same molecular machinery as nonspecificautophagy; however, some of the components arespecific to each pathway.1 Accordingly, we used threedifferent assays to test whether ScTrs85 was required fornonspecific autophagy. Atg8 is required for Cvt vesicleformation and autophagosome expansion.45 The Atg8protein lines both sides of the forming autophagosomeand a portion of the protein remains associated with thecompleted autophagosome. This population of Atg8 isdegraded in the vacuole lumen following fusion withthe vacuole. GFP-Atg8 can functionally replace theendogenous Atg8 protein. When GFP-Atg8 is deliveredto the vacuole via autophagy the Atg8 portion of theprotein is degraded, releasing free GFP that is moreresistant to proteolysis.46 Cleavage of GFP-Atg8 providesa sensitive measure for autophagosome fusion with thevacuole. We monitored the release of GFP fromGFP-Atg8 in wild type and Sctrs85∆ cells. In the wildtype strain free GFP appeared by the one h time pointand increased over time, with a concomitant decrease inthe level of the full-length fusion protein (Fig. 6A). In contrast, free GFPwas not produced in the Sctrs85∆ mutant indicating a block in autophagy.We also monitored delivery of the GFP-Atg8 protein through fluorescentmicroscopy. In wild type cells GFP-Atg8 could be seen at the pre-autophago-somal structure, the site of autophagosome formation, and within thevacuole lumen after four h in SD-N medium (Fig. 6B). GFP-Atg8 could beseen at the PAS but did not appear in the vacuole lumen in the Sctrs85∆strain (Fig. 6B) indicating a block in autophagic transport in agreement withthe analysis of GFP-Atg8 processing.

Finally, we examined the uptake of a bulk cytosolic marker, Pho8∆60.Pho8∆60 lacks the N-terminal transmembrane domain that allows entry ofwild type Pho8 into the secretory pathway.47 As a result, Pho8∆60 can onlybe delivered to the vacuole via autophagy. Delivery results in removal of theC-terminal propeptide that can be monitored by following an increase inenzymatic activity.25 We measured Pho8∆60-dependent alkaline phosphataseactivity in wild type, Scatg1∆ and Sctrs85∆ cells following a shift to SD-Nto induce autophagy. All three strains showed a similar basal level of activitywhen grown in rich medium. After four h in starvation conditions, therewas a large increase in activity in the wild type strain (Fig. 7). The Scatg1∆

mutant is completely defective in autophagy and maintained a similar levelof Pho8∆60 activity before and after shifting to SD-N medium (Fig. 7).Similarly, the Sctrs85∆ mutant showed essentially no increase in activityunder autophagy-inducing conditions (Fig. 7). Together, these results indi-cate that ScTrs85 is defective in nonspecific autophagy in agreement withthe decreased viability seen in the Yltrs85 strains (Fig. 1D).

DISCUSSIONIn this study, we describe the identification of the TRS85 gene

that was isolated in independent screens in Y. lipolytica and S. cere-visiae. In Y. lipolytica, trs85 mutants Ain16 and Ain19 were isolatedusing a plate screen for strains with delayed AMO inactivation underpexophagy conditions. For both mutants the pexophagy phenotypewas confirmed in liquid cultures by following the inactivation ofperoxisomal AMO and degradation of THI in cell-free extracts(Fig. 1A–C) and by following peroxisome-vacuole dynamics byfluorescence microscopy (Fig. 2) after the transfer of cells from YOE

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 4. The Sctrs85∆ mutant accumulates prApe1 in a protease-sensitiveform. Spheroplasts isolated from Scpep4∆, Sctrs85∆ and Scatg1∆ cells werelysed in osmotic lysis buffer. An aliquot was removed for a total lysate con-trol (T). Supernatant (S) and pellet (P) fractions after a 5,000xg centrifuga-tion were collected, and the pellet fractions were subjected to protease treat-ment in the absence or presence of 0.2% Triton X-100 as described inMaterials and Methods. The resulting samples were subjected to immunoblotanalysis with antiserum against Ape1.

Figure 5. The Sctrs85∆ strain is defective in pexophagy. Wild type, Scpep4∆ and Sctrs85∆strains were grown in YTO medium to induce peroxisomes and then were shifted to SD-N toinduce pexophagy as described in Materials and Methods. Identical volume samples from eachindicated time point were collected and subjected to TCA precipitation. Protein samples wereprocessed for Western blotting using polyclonal antiserum against Fox3. Fox3 was degraded inthe wild type strain but remained stable in the Sctrs85∆ strain and a vacuolar protease-deficientstrain Scpep4∆.

42 Autophagy 2005; Vol. 1 Issue 1

(oleic acid, ethylamine) to GA (glucose, ammonium chloride) mediumthat leads to efficient induction of pexophagy in the Y. lipolytica wildtype strain. In S. cerevisiae, the mutant was isolated in a screen forCvt pathway-defective strains that accumulated the precursor formof the vacuolar hydrolase Ape1.

The Y. lipolytica Ain16 and Ain19mutants appeared to be disrupted at twodifferent locations of the same gene thatcorresponded to the Y. lipolytica homo-logue of S. cerevisiae TRS85. ScTRS85encodes the 85 kDa subunit of TRAPPI and TRAPP II, protein complexes thatare required for ER-to-Golgi and Golgitransport, respectively.38,41 The Yltrs85mutants had a fragmented vacuole thatmade it problematic to demonstrate thatthe pexophagy defect was a direct resultof the absence of YlTrs85. In contrast,the Sctrs85∆ mutant displayed a rela-tively minor defect in vacuole morphol-ogy (Fig. 3B). In addition, this strainshowed normal processing of two vac-uolar hydrolases that transit through theCPY pathway (Fig. 3C), a vacuolar tar-geting route that is mostly independentfrom the autophagic pathway. Thus, thevacuoles of the Sctrs85∆ mutant retainthe ability to fuse with donor compart-ments such as the autophagosome, andalso possess the proteolytic activity nec-essary to process newly delivered zymo-gens such as prApe1. The Sctrs85∆mutant, however, accumulated only theprecursor form of Ape1 (Fig. 3A) in aprotease- accessible state (Fig. 4), indicat-ing a block in the Cvt pathway.Similarly, this mutant was defective inpexophagy (Fig. 5) and nonspecificautophagy (Figs. 6 and 7) similar to theY. lipolytica mutant strains (Figs. 1 and 2).

S. cerevisiae TRAPP I consists of 7different subunits (Bet5, Trs20, Bet3,Trs23, Trs31, Trs33 and Trs85) andTRAPP II contains an additional 3subunits (Trs65, Trs120 and Trs130).41

We can speculate why only trs85mutants were isolated in our screens. InS. cerevisiae, only 3 of the 10 TRAPPsubunits are dispensable for growth:Trs33, Trs65 and Trs85.38 However, theloss of Trs33 and Trs65 did not alter theassembly of the complex and membranetraffic.41 Consequently, only trs85unconditional mutants could be directlyselected for a pexophagy or prApe1-accumulation phenotype. Nevertheless,to our knowledge this is the first reportthat implicates the TRS85 gene inautophagy-related pathways. It has been

suggested that the early secretory pathway is needed for nonspecificautophagy but not for the Cvt pathway in yeast;6,7 however, a recentreport indicates that the early secretory pathway is also required forthe Cvt pathway, suggesting a role in supplying the membraneduring specific types of autophagy.8 We suspect that the early secre-tory pathway is also involved in pexophagy; however, we cannot

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 6. GFP-Atg8 is not efficiently transported into the vacuole in the Sctrs85∆ strain. (A) GFP-Atg8 is notdegraded in the Sctrs85∆ strain. Wild type and Sctrs85∆ cells transformed with the pCuGFPAUT7(416)plasmid were grown in selective minimal medium. Cells at mid-log phase were shifted to SD-N for the indi-cated times. Equal volume samples from each time point were TCA precipitated and processed for Westernblotting. The full-length GFP-Atg8 fusion protein and free GFP moiety were detected with antibody againstGFP. (B) The Sctrs85∆ strain does not accumulate vacuolar GFP-Atg8. The same two strains used in (A) weregrown as described above, then shifted to SD-N for 4 h. Cells were collected and monitored by fluorescencemicroscopy as described in Materials and Methods. Arrows mark some of the sites corresponding to thePAS. DIC, differential interference contrast.

A

B

www.landesbioscience.com Autophagy 43

conclude that result from the present data. The finding that theSctrs85∆ strain showed no defect in the kinetics of Pep4 and Prc1processing (Fig. 3C) suggests normal delivery through the earlysecretory pathway.

The function of Trs85 in autophagy has not been identified yet,but from the evidence that the PAS is normally formed but Atg8 isnot transported into the vacuole in the Sctrs85∆ mutant (Fig. 6) andperoxisomes are not delivered to the vacuole in the Yltrs85 mutants(Fig. 2), we can speculate that it functions before the vesicle dockingand fusion stage, and that it is probably involved in vesicle formation/completion, similar to most of the other Atg proteins. The findingthat the Sctrs85∆ strain accumulates protease-sensitive prApe1supports this model (Fig. 4). Finally, we note that it was reportedthat in a TRS85 deletion strain, ER-Golgi transport of Prc1 isblocked at 37˚C (Sacher et al, 2001), but this phenotype was notseen in our analysis; the Sctrs85∆ strain used in this study displayednormal processing of Prc1 and Pep4 at both 30 (Fig. 3C) and 37(data not shown) degrees. This discrepancy may reflect differencesdue to the strain background. Further study is required to addressthe function of Trs85 and to examine whether other TRAPP subunitsare also involved in the Cvt, autophagy and pexophagy pathways.

References1. Klionsky DJ. In: Klionsky DJ, ed. Autophagy. Georgetown, TX: Landes Bioscience, 2004.2. Reggiori F, Klionsky DJ. Autophagy in the eukaryotic cell. Eukaryot Cell 2002; 1:11-21.3. Noda T, Suzuki K, Ohsumi Y. Yeast autophagosomes: De novo formation of a membrane

structure. Trends Cell Biol 2002; 12:231-5.4. Suzuki K, Kirisako T, Kamada Y, Mizushima N, Noda T, Ohsumi Y. The pre-autophago-

somal structure organized by concerted functions of APG genes is essential for autophago-some formation. EMBO J 2001; 20:5971-81.

5. Kim J, Huang W-P, Stromhaug PE, Klionsky DJ. Convergence of multiple autophagy andcytoplasm to vacuole targeting components to a perivacuolar membrane compartmentprior to de novo vesicle formation. J Biol Chem 2002; 277:763-73.

6. Ishihara N, Hamasaki M, Yokota S, Suzuki K, Kamada Y, Kihara A, Yoshimori T, Noda T,Ohsumi Y. Autophagosome requires specific early Sec proteins for its formation andNSF/SNARE for vacuolar fusion. Mol Biol Cell 2001; 12:3690-702.

7. Hamasaki M, Noda T, Ohsumi Y. The early secretory pathway contributes to autophagy inyeast. Cell Struct Funct 2003; 28:49-54.

8. Reggiori F, Wang C-W, Nair U, Shintani T, Abeliovich H, Klionsky DJ. Early stages of thesecretory pathway, but not endosomes, are required for Cvt vesicle and autophagosomeassembly in Saccharomyces cerevisiae. Mol Biol Cell 2004; 15:2189-2204.

9. Hutchins MU, Veenhuis M, Klionsky DJ. Peroxisome degradation in Saccharomyces cere-visiae is dependent on machinery of macroautophagy and the Cvt pathway. J Cell Sci 1999;112:4079-87.

10. Oku M, Warnecke D, Noda T, Muller F, Heinz E, Mukaiyama H, Kato N, Sakai Y.Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAMdomain. EMBO J 2003; 22:3231-41.

11. Stasyk OV, Nazarko TY, Stasyk OG, Krasovska OS, Warnecke D, Nicaud JM, Cregg JM,Sibirny AA. Sterol glucosyltransferases have different functional roles in Pichia pastoris andYarrowia lipolytica. Cell Biol Int 2003; 27:947-52.

12. Kim J, Kamada Y, Stromhaug PE, Guan J, Hefner-Gravink A, Baba M, Scott SV, OhsumiY, Dunn Jr WA, Klionsky DJ. Cvt9/Gsa9 functions in sequestering selective cytosolic cargodestined for the vacuole. J Cell Biol 2001; 153:381-96.

13. Gunkel K, van der Klei IJ, Barth G, Veenhuis M. Selective peroxisome degradation inYarrowia lipolytica after a shift of cells from acetate/oleate/ethylamine into glucose/ammo-nium sulfate-containing media. FEBS Lett 1999; 451:1-4.

14. Nazarko TY, Nicaud J-M, Sibirny AA. Observation of the Yarrowia lipolytica peroxi-some-vacuole dynamics by fluorescence microscopy with a single filter set. Cell Biol Int2005; In press.

15. Nazarko T, Mala M, Nicaud J-M, Sibirny A. Mutants of the yeast Yarrowia lipolytica defi-cient in the inactivation of peroxisomal enzymes. Visnyk L’viv Univ (Biology Series) 2004;35:128-36.

16. Mauersberger S, Wang HJ, Gaillardin C, Barth G, Nicaud J-M. Insertional mutagenesis inthe n-alkane-assimilating yeast Yarrowia lipolytica: Generation of tagged mutations in genesinvolved in hydrophobic substrate utilization. J Bacteriol 2001; 183:5102-9.

17. Nazarko T, Mala MJ, Sibirny AA. Development of the plate assay screening procedure forisolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeastYarrowia lipolytica. Biopolymers Cell (Kiev) 2002; 18:135-8.

18. Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH. A second set of loxP mark-er cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res2002; 30:e23.

19. Labbé S, Thiele DJ. Copper ion inducible and repressible promoter systems in yeast.Methods Enzymol 1999; 306:145-53.

20. Kim J, Huang W-P, Klionsky DJ. Membrane recruitment of Aut7p in the autophagy andcytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conju-gation complex. J Cell Biol 2001; 152:51-64.

21. Barth G, Gaillardin C. Nonconventional yeasts in biotechnology. In: Wolf K, ed. Yarrowialipolytica. Berlin: Springer, 1996.

22. Nicaud J-M, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, GaillardinC. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res 2002;2:371-9.

23. Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud J-M. New disruption cassettes forrapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J MicrobiolMethods 2003; 55:727-37.

24. Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA. An improved PCRmethod for walking in uncloned genomic DNA. Nucleic Acids Res 1995; 23:1087-8.

25. Noda T, Matsuura A, Wada Y, Ohsumi Y. Novel system for monitoring autophagy in theyeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 1995; 210:126-32.

26. Noda T, Kim J, Huang W-P, Baba M, Tokunaga C, Ohsumi Y, Klionsky DJ. Apg9p/Cvt7pis an integral membrane protein required for transport vesicle formation in the Cvt andautophagy pathways. J Cell Biol 2000; 148:465-80.

27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folinphenol reagent. J Biol Chem 1951; 193:265-75.

28. Sahm H, Wagner F. Microbial assimilation of methanol. The ethanol- and methanol-oxi-dizing enzymes of the yeast Candida boidinii. Eur J Biochem 1973; 36:250-6.

29. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacterio-phage T4. Nature 1970; 227:680-5.

30. Kyhse-Andersen J. Electroblotting of multiple gels: A simple apparatus without buffer tankfor rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem BiophysMethods 1984; 10:203-9.

31. Klionsky DJ, Cueva R, Yaver DS. Aminopeptidase I of Saccharomyces cerevisiae is localizedto the vacuole independent of the secretory pathway. J Cell Biol 1992; 119:287-99.

32. Klionsky DJ, Banta LM, Emr SD. Intracellular sorting and processing of a yeast vacuolarhydrolase: Proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol1988; 8:2105-16.

33. Kim J, Dalton VM, Eggerton KP, Scott SV, Klionsky DJ. Apg7p/Cvt2p is required for thecytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways.Mol Biol Cell 1999; 10:1337-51.

34. Klionsky DJ, Cregg JM, Dunn Jr WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A,Subramani S, Thumm M, Veenhuis M, Ohsumi Y. A unified nomenclature for yeastautophagy-related genes. Dev Cell 2003; 5:539-45.

35. Sakai Y, Koller A, Rangell LK, Keller GA, Subramani S. Peroxisome degradation bymicroautophagy in Pichia pastoris: Identification of specific steps and morphological inter-mediates. J Cell Biol 1998; 141:625-36.

36. Wickner W, Haas A. Yeast homotypic vacuole fusion: A window on organelle traffickingmechanisms. Annu Rev Biochem 2000; 69:247-75.

37. Dujon B, et al. Genome evolution in yeasts. Nature 2004; 430:35-44.38. Sacher M, Barrowman J, Schieltz D, Yates III JR, Ferro-Novick S. Identification and char-

acterization of five new subunits of TRAPP. Eur J Cell Biol 2000; 79:71-80.

Trs85 is Required for Specific- and Non-Specific Autophagy

Figure 7. Nonspecific autophagy is blocked in the Sctrs85∆ strain. Wildtype (YTS159), Scatg1∆ (YTS161) and Sctrs85∆ (YJH3) strains were grownin SMD and shifted to SD-N for 4 h. Autophagy was monitored by measur-ing the Pho8∆60-dependent alkaline phosphatase (AlP) activity as describedin Materials and Methods. The activity of the wild type strain in SD-N wasset as 100% activity and the other strains were normalized relative to thislevel. Error bars represent the standard deviation from three separate exper-iments.

44 Autophagy 2005; Vol. 1 Issue 1

www.landesbioscience.com Autophagy 45

39. Engebrecht J, Masse S, Davis L, Rose K, Kessel T. Yeast meiotic mutants proficient for theinduction of ectopic recombination. Genetics 1998; 148:581-98.

40. Kaytor M.D, Livingston DM. GSG1, a yeast gene required for sporulation. Yeast 1995;11:1147-55.

41. Sacher M, Barrowman J, Wang W, Horecka J, Zhang Y, Pypaert M, Ferro-Novick S.TRAPP I implicated in the specificity of tethering in ER-to-Golgi transport. Mol Cell2001; 7:433-42.

42. Nice DC, Sato TK, Stromhaug PE, Emr SD, Klionsky DJ. Cooperative binding of thecytoplasm to vacuole targeting pathway proteins, Cvt13 and Cvt20, to phosphatidylinosi-tol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy. JBiol Chem 2002; 277:30198-207.

43. Klionsky DJ, Herman PK, Emr SD. The fungal vacuole: Composition, function, and bio-genesis. Microbiol Rev 1990; 54:266-92.

44. Klionsky DJ, Ohsumi Y. Vacuolar import of proteins and organelles from the cytoplasm.Annu Rev Cell Dev Biol 1999; 15:1-32.

45. Abeliovich H, Dunn Jr WA, Kim J, Klionsky DJ. Dissection of autophagosome biogenesisinto distinct nucleation and expansion steps. J Cell Biol 2000; 151:1025-34.

46. Shintani T, Klionsky DJ. Cargo proteins facilitate the formation of transport vesicles in thecytoplasm to vacuole targeting pathway. J Biol Chem 2004; 279:29889-94.

47. Klionsky DJ, Emr SD. Membrane protein sorting: Biosynthesis, transport and processingof yeast vacuolar alkaline phosphatase. EMBO J 1989; 8:2241-50.

Trs85 is Required for Specific- and Non-Specific Autophagy