Endoplasmic Reticulum-Associated Secretory Proteins Sec20p, … · oxisomes in normal human development and physiology (55). Historically, peroxisomes have been classiﬁed with mito-chondria

EUKARYOTIC CELL, June 2009, p. 830–843 Vol. 8, No. 61535-9778/09/$08.00�0 doi:10.1128/EC.00024-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Endoplasmic Reticulum-Associated Secretory Proteins Sec20p, Sec39p,and Dsl1p Are Involved in Peroxisome Biogenesis�

Ryan J. Perry, Fred D. Mast, and Richard A. Rachubinski*Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Received 16 January 2009/Accepted 25 March 2009

Two pathways have been identified for peroxisome formation: (i) growth and division and (ii) de novosynthesis. Recent experiments determined that peroxisomes originate at the endoplasmic reticulum (ER).Although many proteins have been implicated in the peroxisome biogenic program, no proteins in theeukaryotic secretory pathway have been identified as having roles in peroxisome formation. Using the yeastSaccharomyces cerevisiae regulatable Tet promoter Hughes clone collection, we found that repression of theER-associated secretory proteins Sec20p and Sec39p resulted in mislocalization of the peroxisomal matrixprotein chimera Pot1p-green fluorescent protein (GFP) to the cytosol. Likewise, the peroxisomal membraneprotein chimera Pex3p-GFP localized to tubular-vesicular structures in cells suppressed for Sec20p, Sec39p,and Dsl1p, which form a complex at the ER. Loss of Sec39p attenuated formation of Pex3p-derived peroxisomalstructures following galactose induction of Pex3p-GFP expression from the GAL1 promoter. Expression ofSec20p, Sec39p, and Dsl1p was moderately increased in yeast grown under conditions that proliferate peroxi-somes, and Sec20p, Sec39p, and Dsl1p were found to cofractionate with peroxisomes and colocalize withPex3p-monomeric red fluorescent protein under these conditions. Our results show that SEC20, SEC39, andDSL1 are essential secretory genes involved in the early stages of peroxisome assembly, and this work is thefirst to identify and characterize an ER-associated secretory machinery involved in peroxisome biogenesis.

Peroxisomes are ubiquitous multifunctional organelles lim-ited by a single membrane and devoid of genetic material. Theparacrystalline matrix of peroxisomes results from an ex-tremely high concentration of proteins that carry out a numberof metabolic processes, the most conserved among speciesbeing the �-oxidation of fatty acids and the neutralization ofhydrogen peroxide (41). Peroxisomes have a striking capacityto alter their enzyme content according to the metabolic needsof the cell and to increase dramatically in number and sizeunder various environmental conditions. The inability of per-oxisomes to assemble correctly or react appropriately to themetabolic demands of the cell is detrimental. This biologicalrequirement for peroxisomal function is evidenced by a groupof genetic disorders collectively called the peroxisome biogen-esis disorders (PBD). PBD-afflicted individuals fail to assemblefunctional peroxisomes, and the severity and lethality generallyassociated with the PBDs demonstrate the necessity for per-oxisomes in normal human development and physiology (55).

Historically, peroxisomes have been classified with mito-chondria and chloroplasts as semiautonomous organelles thatderived from an endosymbiotic event during evolution (9).Characterization of the peroxisomal fission pathway supportedan autonomous proliferation model of peroxisomes in whichperoxisomes arose by growth and division of preexisting per-oxisomes and were partitioned to and inherited by daughtercells (29, 30). Further evidence in support of the autonomousnature of peroxisomes came from the demonstration that per-oxisomal proteins are synthesized on free polysomes and in the

majority are imported directly to the peroxisome from thecytosol (15–17, 42). Unlike mitochondria and chloroplasts, per-oxisomes were observed to form de novo in mutant cells thatcompletely lacked any identifiable peroxisomal structures uponreintroduction of the wild-type version of the mutated gene(33, 46, 51–53). This observation challenged the concept of anendosymbiotic origin for peroxisomes and the view that per-oxisomes relied solely on the autonomous partitioning of pre-existing peroxisomes for their proliferation and inheritance.

The ability of peroxisomes to form de novo suggests thatanother organelle must provide peroxisomal membranecomponents. Previous morphological evidence showing per-oxisomes in close association with, and as extensions of, theendoplasmic reticulum (ER) suggested a role for the ER asthe donor compartment (18, 36, 57). Biochemical evidencefor the ER origin of peroxisomes (60, 61) was controversialand suggested that a functional role for the ER in the denovo synthesis of peroxisomes was dependent on the exper-imental organism being studied (29). Alteration or loss ofprotein components of the classical secretory machinery gen-erally had little or no effect on peroxisome biogenesis (37, 47,53, 73). Recent findings with the yeast Saccharomyces cerevisiaehave now provided unambiguous morphological and biochem-ical evidence that the ER is the site of de novo peroxisomebiogenesis (21, 58). These studies took advantage of the re-quirement for the integral peroxisomal membrane proteinPex3p for the biogenesis of peroxisomes in peroxisome-defi-cient cells and conclusively showed that Pex3p targets to dis-crete ER-localized puncta, forming a dynamic ER subcompart-ment, en route to the peroxisome. Trafficking of Pex3p throughthe ER is not confined to cells lacking peroxisomes, since thede novo synthesis of peroxisomes from the ER was also foundto occur in normal mammalian cells and to require Pex16p, an

* Corresponding author. Mailing address: Department of Cell Biol-ogy, University of Alberta, Medical Sciences Building 5-14, Edmonton,Alberta T6G 2H7, Canada. Phone: (780) 492-9868. Fax: (780) 492-9278. E-mail: [email protected].

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integral component of the peroxisomal membrane protein im-port machinery (26).

Given the recent incontrovertible evidence showing de novoperoxisome biogenesis from the ER, we sought to identifyproteins involved in the exit of peroxisomes from the ER.Transcriptome profiling, organellar proteomics, comparativegene analysis, and screening of knockout libraries for nones-sential genes of S. cerevisiae have so far failed to implicatecomponents of the secretory machinery in the biogenesis ofperoxisomes from the ER (44, 49, 50, 59, 69, 70, 74; R. J. Perryand R. A. Rachubinski, unpublished results). Therefore, wescreened a library of S. cerevisiae essential genes placed underthe control of a regulatable promoter to identify essential se-cretory (SEC) genes that might be required for the biogenesisof peroxisomes. From this screen we identified SEC20, SEC39,and DSL1 as essential SEC genes encoding ER-associatedproteins involved in the early stages of peroxisome biogenesisand assembly.

MATERIALS AND METHODS

Strains and culture conditions. The S. cerevisiae strains used in this study arelisted in Table 1. Strains were cultured at 30°C unless indicated otherwise. TheTet promoter Hughes clone (THC) collection (Open Biosystems) was used tocontrol the expression of essential genes from a regulatable Tet promoter (34).Repression of the TetO7 promoter was achieved by addition of doxycycline to 10�g/ml from a 10-mg/ml stock in water to the medium and incubation for 18 h orless. Cells incubated in the presence of doxycycline were seeded at an increasedcell number to obtain comparable cell densities at the time of experimentation.Medium components were as follows: YEPD, 1% yeast extract, 2% peptone, 2%glucose; YEP2�D, 1% yeast extract, 2% peptone, 4% glucose; YEPR, 1% yeastextract, 2% peptone, 4% raffinose; YEPG, 1% yeast extract, 2% peptone, 2%galactose; SCIM1, 0.67% yeast nitrogen base without amino acids, 0.5% yeastextract, 0.5% peptone, 0.5% (wt/vol) Tween 40, 1� complete supplement mix-ture, 0.1% glucose, 0.15% (vol/vol) oleic acid; YPBO, 0.3% yeast extract, 0.5%peptone, 0.5% KH2PO4, 0.5% K2HPO4, 0.2% (wt/vol) Tween 40, 1% (wt/vol)oleic acid; and SCIM2, SCIM1 containing 0.3% glucose and 0.3% (vol/vol) oleicacid. Solid media were prepared by addition of 2% agar to liquid media.

Plasmids and integrative transformation of yeast. Plasmids and PCR-basedintegrative transformation used to genomically tag genes and introduce theGAL1 promoter (pGAL1) upstream of the PEX3 gene have been describedpreviously (11, 58). POT1 and PEX3 were genomically tagged with sequencesencoding fluorescent proteins to visualize peroxisomes, and genomic introduc-tion of pGAL1 upstream of PEX3 was used to control the biogenesis of peroxi-somes.

Induction of Pot1p-GFP expression. THC strains expressing a fusion of POT1and the green fluorescent protein (GFP) gene were grown overnight in YEPDand transferred to YEPD with or without 10 �g doxycycline/ml at a cell densityto achieve mid-log growth following a further 18 h of incubation. An aliquot wastaken at time zero, and the remaining culture was used for oleic acid inductionof Pot1p-GFP synthesis by incubation in YPBO with or without 10 �g doxycy-cline/ml for 90 min. Cells were then washed extensively to remove free oleic acid,and images were acquired by epifluorescence microscopy.

Pulse-chase analysis of Pex3p-GFP transport between the ER and peroxi-somes. THC strains expressing PEX3-GFP under the control of pGAL1 weregrown overnight in YEPD, transferred to YEPR at an optical density at 600 nmof 0.005, and incubated for an additional 16 to 18 h in YEPR to allow derepres-sion of pGAL1. The culture was divided and primed in YEPR with or without 10�g doxycycline/ml for the times indicated. Pulse expression of Pex3p-GFP wasachieved by transferring cells to YEPG for 1 h, followed by a chase incubation ofcells in YEP2�D for 4 h and 15 h to monitor the transport of Pex3p-GFP fromthe ER to peroxisomes. An aliquot was taken following each incubation step(prime, pulse, and chase), and cells were fixed in 3.7% formaldehyde and ana-lyzed by epifluorescence microscopy.

Microscopy. Images of live or fixed cells were captured using one of twomethods. In the first, a Plan-apochromat 63�/1.4 NA oil differential interferencecontrast objective on an Axiovert 200 inverted microscope equipped with aLSM510 META confocal scanner (Carl Zeiss) was used. GFP was excited witha 488-nm laser and its emission collected with a 505-nm long-pass filter or, for

colocalization analysis, a 505- to 530-nm band-pass filter, while monomeric redfluorescent protein (mRFP) was excited with a 543-nm laser and its emissioncollected using a 600-nm long-pass filter. In the second method, a Plan-APO100�/1.4 NA oil objective on an IX81 inverted epifluorescence microscope(Olympus) equipped with a CoolSNAP HQ digital camera (Roper Scientific) andan ExFo X-Cite 120 PC fluorescent illumination system was used. Final imageswere prepared using Adobe Photoshop software. To prevent interference ofinternal structures captured in the transmission images, the internal structureswere removed in Adobe Photoshop. For Fig. 2, 6B, 6C, and 6D, images weredeconvolved using Huygens Professional software (Scientific Volume ImagingBV, The Netherlands). For this method, three-dimensional (3D) data sets wereprocessed to remove blur through an iterative classic maximum-likelihood esti-mation algorithm and an experimentally derived point spread function. Imarissoftware (Bitplane) was subsequently used to prepare a maximum-intensity pro-jection of the deconvolved 3D data set (for Fig. 2 and 6D) or single-plane images(for Fig. 6B and C) before final assembly in Adobe Photoshop. For Fig. 2, furtherimage analysis was performed on deconvolved 3D data sets using the “Blend”viewing mode in Imaris (Bitplane). This generates a black-and-white image inwhich the fluorescent signal is standardized to minimize the differences betweenbright and dim fluorescent signals for evaluation of the entire fluorescent signallocated within the cell. For electron microscopy, whole cells were processed asdescribed previously (12).

Subcellular fractionation and isolation of peroxisomes. Subcellular fraction-ation and isolation of peroxisomes were performed essentially as describedpreviously (70). Briefly, strains grown overnight in YEPD were transferred toSCIM2 and incubated for 16 h. Cells were harvested, and spheroplasts wereprepared by digestion with Zymolyase 100T (2 mg/g cells). Spheroplasts werehomogenized, and the homogenate was subjected to centrifugation to yield apostnuclear supernatant fraction. The postnuclear supernatant was further di-vided by differential centrifugation at 20,000 � g to yield supernatant and pellet(20KgP) fractions. Organelles in the 20KgP were separated by isopycnic gradientcentrifugation using a discontinuous gradient consisting of 17, 25, 35, and 50%(wt/vol) Nycodenz. The gradient was subjected to centrifugation at 100,000 � gfor 90 min in a VTi50 rotor (Beckman Coulter). Fractions of 2 ml were collectedfrom the bottom of the gradient, and equal volumes from each gradient wereseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted for the indicated pro-teins.

Antibodies and immunoblotting. Polyclonal antibodies to Sec20p and Dsl1pwere raised in guinea pig. Antibodies to thiolase and Sdh2p have been previouslydescribed (10, 13). Rabbit polyclonal antibodies to Act1p, GFP, Sec39p, Dsl1p,and Sec61p were kind gifts from Gary Eitzen (University of Alberta, Canada),Luc Berthiaume (University of Alberta, Canada), Daniel Unger (University ofYork, United Kingdom), Hans Dieter Schmitt (Max Planck Institute, Germany),and Randy Schekman (University of California, Berkeley), respectively. Yeastwhole-cell lysates were prepared by solubilizing cell pellets in 1.85 M sodiumhydroxide–7.4% 2-mercaptoethanol. Proteins were precipitated from lysates byaddition of trichloroacetic acid, and the resultant protein pellets were solubilizedby successive equal-volume additions of magic A (1 M unbuffered Tris, 13%SDS) and magic B (30% glycerol, 200 mM dithiothreitol, 0.25% bromophenolblue) solutions. Equivalent amounts of protein were resolved by SDS–10%PAGE. Proteins were transferred to nitrocellulose for immunoblotting, andhorseradish peroxidase-conjugated secondary antibodies were used to detect theprimary antibodies by chemiluminescence.

RESULTS

Criteria for selecting essential secretory genes involved inperoxisome biogenesis. Analysis of isogenic yeast gene knock-out libraries has failed to identify a nonessential SEC geneinvolved in the biogenesis of peroxisomes from the ER (44, 49,50, 59, 69, 70, 74; Perry and Rachubinski, unpublished results).To ascertain a role for an essential SEC gene in the ER-to-peroxisome biogenesis pathway, we employed the THC collec-tion library, which allows for the conditional and efficient re-pression of essential S. cerevisiae genes (2, 3, 34). In this library,the endogenous promoter of an essential gene has been re-placed by the regulatable TetO7 promoter, allowing for repres-sion of the essential gene by addition of doxycycline to thegrowth medium. Previous reports have shown that the concen-

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trations of doxycycline required for gene repression have littleeffect on yeast physiology and essentially no effect on globalgene expression (23, 39). Using the Saccharomyces GenomeDatabase (http://www.yeastgenome.org), we selected essentialSEC genes of unknown function or that had previously been

determined to function in ER and/or Golgi apparatus trans-port. Using these criteria, we identified 19 SEC genes to screenfor a role in peroxisome assembly (Table 2).

Repression of SEC20 and SEC39 results in mislocalizationof peroxisomal Pot1p to the cytosol. Growth of yeast in oleic

TABLE 1. Yeast strains used in this study

Strain Genotype Reference

BY4742 MAT� his3�1 leu2�0 lys2�0 ura3�0 19pex3� MAT� his3�1 leu2�0 lys2�0 ura3�0 pex3::KanMX4 19SEC20-GFP-PEX3-mRFP MAT� his3�1 leu2�0 lys2�0 ura3�0 sec20::SEC20-GFP(HIS5) pex3::PEX3-mRFP(URA3) This studySEC39-GFP-PEX3-mRFP MAT� his3�1 leu2�0 lys2�0 ura3�0 sec39::SEC39-GFP(HIS5) pex3::PEX3-mRFP(URA3) This studyDSL1-GFP-PEX3-mRFP MAT� his3�1 leu2�0 lys2�0 ura3�0 dsl1::DSL1-GFP(HIS5) pex3::PEX3-mRFP(URA3) This studyPEX3-GFP-RTN1-mRFP MAT� his3�1 leu2�0 lys2�0 ura3�0 pex3::PEX3-GFP(HIS5) rtn1::RTN1-mRFP(URA3) This studyR1158-POT1-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTa pot1::POT1-GFP(HIS5) This studyTHC-SEC1-POT1-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC1::tet07-TATA(KanMX6)

pot1::POT1-GFP(HIS5)This study

THC-SEC7-POT1-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC7::tet07-TATA(KanMX6)pot1::POT1-GFP(HIS5)

This study


This study


This study


This study


This study


This study


This study


This study


This study


This study


This study


This study

THC-COPI-POT1-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pCOPI::tet07-TATA(KanMX6)pot1::POT1-GFP(HIS5)

This study


This study


This study


This study


This study


This study

R1158-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pex3::PEX3-GFP(HIS5) This studyTHC-SEC14-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC14::tet07-TATA(KanMX6)

pex3::PEX3-GFP(HIS5)This study

THC-SEC61-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC61::tet07-TATA(KanMX6)pex3::PEX3-GFP(HIS5)

This study


This study


This study

THC-DSL1-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pDSL1::tet07-TATA(KanMX6)pex3::PEX3-GFP(HIS5)

This study

THC-SEC39-pGAL1-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC39::tet07-TATA(NAT)pPEX3::pGAL1(KanMX6) pex3::PEX3-GFP(HIS5)

This study

THC-SEC61-pGAL1-PEX3-GFP MATa his3�1 leu2�0 met15�0 URA3::CMV-tTA pSEC61::tet07-TATA(NAT)pPEX3::pGAL1(KanMX6) pex3::PEX3-GFP(HIS5)

This study

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acid as the sole carbon source induces both the de novo for-mation and proliferation of peroxisomes (58, 65, 68). In addi-tion, oleic acid activates the expression of a number of genesthat code for peroxisomal �-oxidation enzymes (20, 48–50, 68).These enzymes are imported directly from the cytosol into theperoxisomal matrix during the normal assembly of functionallymature peroxisomes. Under conditions where peroxisome as-sembly is compromised or when peroxisome biogenesis is in-hibited, oleic acid-induced �-oxidation enzymes, such as 3-ketoacyl coenzyme A thiolase (Pot1p), are mislocalized to thecytosol instead of the peroxisome matrix. Thus, we integratedthe cDNA sequence encoding GFP at the 3� end of the POT1locus to serve as an unambiguous binary reporter (punctateversus nonpunctate) to screen for peroxisome assembly defectsin each of the selected THC-SEC strains (Table 2). We moni-tored the intracellular distribution of Pot1p-GFP using epifluo-rescence microscopy following growth of cells in either glucosemedium (YEPD) or oleic acid medium (YPBO) in the presenceor absence of doxycycline (Fig. 1). Growth of the THC librarycontrol strain, R1158, in YEPD exhibited the expected low ex-pression of Pot1p-GFP. Culturing R1158 cells in YPBO signifi-cantly increased Pot1p-GFP expression, where it localized exclu-sively to discrete punctate peroxisome structures. Addition ofdoxycycline had a modest effect on the induction of Pot1p-GFPexpression in R1158 cells cultured in YEPD but had no effecton Pot1p-GFP expression when these cells were grown inYPBO. This did not affect our screen for peroxisome biogen-esis defects in doxycycline-repressed THC-SEC-POT1-GFPstrains, since the subsequent intracellular localization of Pot1p-GFP to characteristic punctate peroxisome structures in controlR1158 cells was not affected.

Of the THC-SEC-POT1-GFP strains screened, only doxycy-cline-repressed THC-SEC20-POT1-GFP, and THC-SEC39-POT1-GFP cells exhibited significant mislocalization of Pot1p-GFP to the cytosol (Fig. 1A and B and unpublished results).Interestingly, Pot1p-GFP also mislocalized in THC-SEC20-POT1-GFP cells even in the absence of doxycycline, suggesting

that altering the endogenous promoter for SEC20 was suffi-cient to affect normal peroxisome assembly in these cells. In-deed, expression of Sec20p in THC-SEC20 cells in the absenceor presence of doxycycline was significantly reduced comparedto that in the parental strain, R1158 (Fig. 1C). Repression ofSEC7, SEC13, SEC18, SEC61, or COPI did not affect localiza-tion of Pot1p-GFP to peroxisomes; however, a modest effect ofgene repression on peroxisome number and size was observedin the THC-SEC61-POT1-GFP and THC-COPI-POT1-GFPstrains (Fig. 1A and B). Overall, these results show that themislocalization of Pot1p-GFP in repressed THC-SEC20-POT1-GFP and THC-SEC39-POT1-GFP cells was specific to thesegenes and did not result from nonspecific effects caused by theloss of an essential secretory protein associated with the ERand/or Golgi apparatus.

Repression of SEC20, SEC39, and DSL1 mislocalizes Pex3pto tubular-vesicular structures. Since Pot1p-GFP was mislo-calized to the cytosol in cells repressed for SEC20 or SEC39expression, we wanted to characterize further a role for theseSEC genes in peroxisome biogenesis. To do this, a cDNAsequence encoding GFP was integrated at the 3� end of thePEX3 locus in the THC-SEC20 and THC-SEC39 strains. Wechoose PEX3 since the biogenesis and maintenance of peroxi-somes from ER-derived membrane components require Pex3pand are independent of the oleic acid-induced biogenesis andproliferation of peroxisomes (21, 58). Thus, using this system,we could determine the effects of SEC20 and SEC39 generepression on ER-dependent peroxisome biogenesis by moni-toring the intracellular distribution of Pex3p between the ERand peroxisomes. To control for effects of doxycycline additionand for loss of an essential SEC protein at the Golgi apparatusor the ER, the same integration and experimentation wereperformed with the control R1158 strain and with the THC-SEC14 and THC-SEC61 strains, respectively.

Although it was not identified initially using our selectioncriteria (see above), we included the THC-DSL1 strain in thisscreen, because the gene products of SEC20 and SEC39 are

TABLE 2. SEC genes screened for defects in localization of Pot1p-GFP to peroxisomes

Gene name Systematic name Alternate name(s) Description of product or functiona

SEC1 YDR164c Localized to sites of secretion; dependent on SNARE functionSEC7 YDR170c GEF involved in intra-Golgi and ER-to-Golgi transportSEC11 YIR022w 18-kDa subunit of signal peptidase complexSEC12 YNR026c SED2 GEF involved in initiation of transport vesicle budding from ERSEC13 YLR208w ANU3 COPII complexSEC14 YMR079w PIT1 PtdIns/PtdCho transfer protein involved in export of vesicles from the Golgi apparatus;

homolog of mammalian PITPsSEC17 YBL050w Required for transport of vesicles between ER and Golgi apparatusSEC18 YBR080c ANU4 Homolog of mammalian NSF; involved in ER-to-Golgi transportSEC20 YDR498c v-SNARE; Golgi-to-ER retrograde transportSEC21 YNL287w COPI complexSEC26 YDR238c COPI complexSEC27 YGL137w COPI complexSEC32 YLR078c BOS1 v-SNARE; ER-to-Golgi vesicular transportSEC33 YDL145c COPI, RET1, SOO1 COPI complexSEC39 YLR440c Unknown functionSEC59 YMR013c Lipid synthesis; protein N glycosylationSEC61 YLR378c TransloconSEC63 YOR254c PTL1 TransloconSEC65 YML105c Signal recognition particle complex

a GEF, guanine nucleotide exchange factor; PITPs, phosphatidylinositol transfer proteins; NSF, N-ethylmaleimide-sensitive factor.



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part of an essential secretory protein complex that includesDsl1p (27). Furthermore, inclusion of the THC-DSL1 strain aspart of this screen served to provide a more complete analysisof the potential role of these SEC genes and this complex inthe biogenesis of peroxisomes.

As expected, Pex3p-GFP localized to discrete punctate per-oxisomal structures in the R1158 strain regardless of the pres-ence or absence of doxycycline (Fig. 2). Similarly, Pex3p-GFPlocalized normally in the THC-SEC14-PEX3-GFP strain, fur-ther supporting that the loss of an essential Golgi apparatus-localized Sec protein does not have a general inhibitory effecton peroxisome biogenesis. Pex3p-GFP in the THC-SEC61-PEX3-GFP, THC-SEC39-PEX3-GFP, and THC-DSL1-PEX3-GFP strains cultured in the absence of doxycycline also local-ized to discrete peroxisome structures, as was seen in theR1158-PEX3-GFP strain. Pex3p-GFP in the THC-SEC20-PEX3-GFP strain exhibited a heterogeneous distribution be-tween peroxisomes and tubular-vesicular structures under sim-ilar conditions. Doxycycline repression of SEC61 resulted in anincreased number of these Pex3p-GFP-labeled peroxisomestructures, in addition to a weaker localization of Pex3p-GFPto tubular-vesicular structures (Fig. 2). In contrast, repressionof SEC20, SEC39, and DSL1 expression resulted in significantlocalization of Pex3p-GFP to tubular-vesicular structures, inaddition to discrete punctate structures of various sizes (Fig.2). These results corroborate our initial Pot1p-GFP localiza-tion screen showing that peroxisome biogenesis is compro-mised by the loss of SEC20 and SEC39. Moreover, repressionof DSL1 similarly affected Pex3p-GFP localization to peroxi-somes, supporting a relationship between these genes for per-oxisome biogenesis and assembly.

Repression of SEC20, SEC39, and DSL1 alters peroxisomeprofiles and ultrastructure. Due to the mislocalization of per-oxisomal proteins seen in cells repressed for SEC20, SEC39,and DSL1, we next investigated the peroxisome profiles andultrastructure in doxycycline-repressed SEC20, SEC39, andDSL1 cells by electron microscopy (Fig. 3). To clearly visualizeperoxisomes by electron microscopy, cells were cultured inoleic acid to induce the expression and delivery of matrixproteins to the peroxisome, thereby producing the character-istic peroxisome structure of an electron-dense core sur-rounded by a single-membrane bilayer. Characteristic peroxi-some structures and profiles were seen in each of the strainsgrown in oleic acid except for THC-SEC20. In this strain, wewere unable to detect typical peroxisome structures, but ob-

FIG. 1. The peroxisomal matrix protein Pot1p is mislocalized incells repressed for SEC20 and SEC39 expression. (A) Wild-type strainR1158 and strains THC-SEC7, THC-SEC61, THC-SEC20, and THC-SEC39 harboring genomic POT1-GFP were incubated for 18 h inYEPD, at which time cells were transferred to oleic acid-containingmedium (YPBO) for an additional 90 min to induce the expression ofPot1p-GFP. (B) Pot1p localizes to peroxisomes in repressed THC-SEC11, THC-SEC13, THC-SEC17, THC-SEC18, THC-SEC65, and

THC-COPI cells. The indicated THC strains expressing genomicPOT1-GFP were incubated in YEPD as for panel A and then trans-ferred to YPBO medium for 2 h (SEC13) or 4 h (remaining strains).Repression of essential SEC genes under the control of the TetO7promoter in panels A and B was achieved by addition of 10 �g doxy-cycline/ml to the medium. The localization of Pot1p-GFP to peroxi-somes was monitored by epifluorescence microscopy. Bars, 5 �m. (C)Sec20p levels are reduced in doxycycline-treated or untreated THC-SEC20 cells. Equal amounts of protein from whole-cell lysates ofdoxycycline-treated or untreated THC-SEC20 cells and untreatedwild-type R1158 cells were subjected to immunoblot analysis withantibodies to Sec20p. Immunodetection of Act1p was used as a controlfor protein loading.



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served expanded vesicular regions of the ER (Fig. 3D). Thisvesicular expansion of the ER was further enhanced in doxy-cycline-repressed THC-SEC20 cells and was highly reminiscentof the tubular-vesicular structures seen in Fig. 2 (Fig. 3D� andD�). Interestingly, repression of SEC20 also resulted in a sig-nificant expansion and reticulation of the outer nuclear mem-brane, which was continuous with the ER. This proliferation ofthe ER generally resulted in a characteristic honeycomb-likepattern, which has also been observed in yeast cells harboringmutant COPI and COPII coat proteins (1, 45). Repression ofSEC39 also led to a honeycomb-like expansion of the outernuclear membrane and expanded vesicular regions of the ER(Fig. 3E� and E� and unpublished results). Unlike for therepression of SEC20, a few peroxisomes were present in doxy-cycline-treated THC-SEC39 cells; however, these structureswere typically smaller than those seen in R1158 control cells.Peroxisomes were also found in cells repressed for DSL1, and

these structures were usually associated with ER mem-branes (Fig. 3F�). In addition, several atypical peroxisome-like structures were found to extend from ER membranes indoxycycline-repressed DSL1 cells (Fig. 3F� and F�). Thesestructures were smaller and contained a central core ofdecreased electron density that was similar to that seen forthe expanded vesicular structures in cells repressed forSEC20 and SEC39.

The addition of doxycycline had no effect on the peroxisomeprofiles of R1158 and THC-SEC14 cells (Fig. 3A� and B�,respectively), as expected based on the light microscopy anal-ysis (Fig. 2). Also, similar to what was observed by epifluores-cence (Fig. 1 and 2), repression of SEC61 resulted in an in-creased number of peroxisomes as seen by the number ofperoxisomes captured in a single thin-slice electron microscopypreparation (Fig. 3C�). Ultrastructural analysis further re-vealed increased variability in the dimensions of this expanded

FIG. 2. The peroxisomal membrane protein Pex3p is mislocalized to the cytosol and undefined tubular-vesicular structures in cells repressedfor SEC20, SEC39, or DSL1. THC-SEC strains with genomically integrated PEX3-GFP were incubated for 18 h in YEPD (doxycycline).Repression of TetO7 promoter-regulated genes was achieved by addition of doxycycline to a concentration of 10 �g/ml in YEPD (�doxycycline).Cells were fixed with formaldehyde, images were captured by confocal microscopy, and a maximum-intensity projection was created from adeconvolved 3D data set using Huygens software. Black-and-white images were generated using the “Blend” viewing mode in Imaris (Bitplane)to enhance the total fluorescent signal located within the cell compared to the maximum-intensity projection. Repression of SEC20, SEC39, andDSL1 resulted in redistribution of Pex3p-GFP to the cytosol and undefined tubular-vesicular structures (arrows). Bar, 5 �m.



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peroxisome profile observed in gene-repressed THC-SEC61cells.

Loss of Sec39p attenuates the formation of peroxisomesfrom the ER. Since the normal distribution of Pex3p-GFPbetween the ER and peroxisomes was affected in yeast uponloss of the essential secretory genes, SEC20, SEC39, and DSL1(Fig. 2), we next wanted to determine which point along theER-to-peroxisome transport pathway was affected. As Pex3p isinvolved in the early stages of peroxisome biogenesis from theER (21, 58), we again used Pex3p as the reporter for thefollowing pulse-chase experiments. To do this we integratedthe GAL1 promoter (pGAL1) to the 5� end of the PEX3-GFPlocus in THC-SEC39-PEX3-GFP cells, permitting us to regu-late the expression of Pex3p-GFP. This system further permit-ted the study of Pex3p transport out of the ER for de novoperoxisome biogenesis in the absence of preexisting peroxi-somes (21).

To ensure that Pex3p-GFP expression driven by pGAL1 wasnot affected by the transient loss of SEC39 or the presence ofdoxycycline, we first compared galactose-induced Pex3p-GFPexpression in THC-SEC39-pGAL1-PEX3-GFP cells in thepresence and absence of doxycycline (Fig. 4A). THC-SEC39-GAL1-PEX3-GFP cells were primed for Pex3p-GFP expres-sion by incubation in raffinose for 18 to 20 h, and SEC39expression was transiently repressed by inclusion of doxycy-cline during the last 8 h of incubation. As expected, no Pex3p-GFP was detected in THC-SEC39-pGAL1-PEX3-GFP cellsfollowing the priming period, and essentially no Sec39p was

detected when these cells were primed in the presence ofdoxycycline. Importantly, the level of Pex3p-GFP expressionduring the 1-h pulse in galactose was not altered by the loss ofSec39p, and the repression of Sec39p and the expression ofPex3p-GFP were maintained throughout the chase period.

Having determined the validity of our experimental systemwe next monitored the trafficking of Pex3p-GFP from the ERby epifluorescence microscopy (Fig. 4B). Consistent with ourimmunoblot analysis, Pex3p-GFP was not detected initially incells during the priming step. Following the 1-h pulse in galac-tose to induce pGAL1-PEX3-GFP expression, Pex3p-GFP wasdetected and localized mainly to the cytosol along with target-ing to a few localized foci. This localization was independent ofthe repression of SEC39 by doxycycline and was consistent withprevious reports of an ER localization for Pex3p (21, 58).Upon removal of galactose, the transit of newly synthesizedPex3p-GFP between the ER and peroxisomes was monitoredat 4 h and 15 h during the chase period. In the absence ofdoxycycline, Pex3p-GFP was found to traffic efficiently out ofthe ER to punctate peroxisome structures at the 4-h chase timepoint and remained in discrete punctate peroxisome structuresfor the remaining 15-h chase period. Loss of Sec39p resulted inPex3p-GFP localizing initially to tubular-vesicular structures,with apparent perinuclear localization at the 4-h chase timepoint and subsequent localization to discrete punctate perox-isomal structures by 15 h of chase. However, localization ofPex3p-GFP to tubular-vesicular structures was still evident atthe 15-h chase time point in the doxycycline-repressed cells.

FIG. 3. Altered peroxisome profiles and ultrastructure in cells repressed for SEC20, SEC39, or DSL1. (A to F) Strains R1158 (A), THC-SEC14(B), THC-SEC61 (C), THC-SEC20 (D), THC-SEC39 (E), and THC-DSL1 (F) were incubated for 16 h in YEPD prior to incubation in YPBO for7 h. (A� to F�) Repression of TetO7 promoter-regulated genes was achieved by addition of doxycycline to a final concentration of 10 �g/ml to themedium. (D� to F�) Increased magnification of selected areas from panels D� to F�, respectively, to allow better visualization of the expandedvesicular regions associated with the ER (asterisks) resulting from repression of SEC20, SEC39, or DSL1. M, mitochondrion; N, nucleus; P,peroxisome. Bars, 1 �m in panels A to F� and 0.5 �m in panels D� to F�.



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These results show that the loss of Sec39p attenuates the exitof Pex3p-GFP from the ER, thereby delaying the de novoformation of ER-derived peroxisomes.

When the experimental conditions were modified to includedoxycycline during the entire priming step, we found that amore prolonged loss of Sec39p led to a more striking localiza-tion of Pex3p-GFP to tubular structures following the 1-h pulseincubation in galactose (Fig. 4C). Localization of Pex3p-GFPto tubular structures persisted during the 4-h chase period.Interestingly, the apparent fluorescent signal for Pex3p-GFPwas found to be considerably less in THC-SEC39-pGAL1-PEX3-GFP cells when they were treated with doxycycline.

Since the intensity of the fluorescent signal may not be linearwith protein abundance, depending on the intracellular local-ization of the protein, immunoblot analysis was performed onthese samples. The decrease in the apparent fluorescence wastraced to a significant reduction in the expression of Pex3p-GFP from pGAL1 under these conditions (Fig. 4D). The re-duced expression was specific for the prolonged repression ofSEC39, since this was not seen with the shorter repression time

for SEC39 (Fig. 4A) or in THC-SEC61-pGAL1-PEX3-GFPcells treated under similar conditions (Fig. 4D). NormalPex3p-GFP trafficking between the ER and peroxisomes wasnot affected in THC-SEC61-pGAL1-PEX3-GFP cells underthese conditions (Fig. 4D). These results demonstrate thatgene repression of SEC39 affects the early stages of peroxi-some biogenesis by attenuation of Pex3p transit from the ERto peroxisomes and provide additional support for the speci-ficity of SEC39 repression on peroxisome biogenesis, as loss ofthe essential ER secretory gene SEC61 had no effect on Pex3ptrafficking between the ER and peroxisomes.

Oleic acid induces the expression of Sec20p, Sec39p, andDsl1p. A number of genes coding for proteins associated withperoxisome biogenesis and proliferation are induced duringgrowth of yeast in oleic acid as the sole carbon source (25, 49,50, 68, 69). Accordingly, we investigated whether the expres-sion of Sec20p, Sec39p, and Dsl1p was induced by oleic acid(Fig. 5). Indicative of induction of peroxisome biogenesis,Pot1p showed the typical marked increase in expression overtime of incubation of cells in oleic acid. The levels of Sec20p,

FIG. 4. Repression of SEC39 alters Pex3p trafficking. (A) Equal amounts of protein from whole-cell lysates prepared from THC-SEC39 cellscontaining genomically integrated PEX3-GFP under the control of the GAL1 promoter at the PEX3 locus were resolved by SDS-PAGE,transferred to nitrocellulose, and immunoblotted for GFP, Sec39p, and Gsp1p as a loading control. (B) THC-SEC39 cells integrated forpGAL1-PEX3-GFP were fixed with formaldehyde prior to image capture by epifluorescence microscopy. Arrows indicate the accumulation ofPex3p-GFP in tubular-vesicular structures with apparent perinuclear localization. Prime, cells were cultured for 8 h in YEPR to derepress theGAL1 promoter for subsequent galactose induction of PEX3-GFP expression. Pulse, cells were then transferred to YEPG and incubated for 1 hto pulse-label the cells for PEX3-GFP expression. Chase, expression of PEX3-GFP was then stopped by changing the growth medium to YEP2�D,and cells were incubated for an additional 4 h and 15 h to monitor the trafficking of Pex3p-GFP between the ER and peroxisomes. Repressionof SEC39 was regulated by the addition or omission of 10 �g doxycycline/ml to the culture medium. Bar, 5 �m. (C) THC-SEC39-pGAL1-PEX3-GFP, and THC-SEC61-pGAL1-PEX3-GFP cells were subjected to prime, pulse, and chase incubation steps as described for panel B, except thatthe prime step was for 18 h and the chase step was for 4 h. Cells were fixed in formaldehyde prior to image capture by epifluorescence microscopyat the indicated steps. Bars, 5 �m. (D) Equal amounts of protein from whole-cell lysates of the THC-SEC39-pGAL1-PEX3-GFP and THC-SEC61-pGAL1-PEX3-GFP cells used for panel C were resolved by SDS-PAGE, transferred to nitrocellulose and immunoblotted for GFP, Sec39p,Sec61p, Gsp1p, and Act1p. Gsp1p and Act1p served as loading controls.



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Sec39p, and Dsl1p also increased to various degrees with timeof incubation of cells in oleic acid. Sec20p levels increasedthroughout the 6-h time course, whereas Sec39p and Dsl1pexpression peaked at 2 h following oleic acid addition. Theseresults are consistent with a role for these proteins at theearliest stages of peroxisome biogenesis.

Sec20p, Sec39p, and Dsl1p associate with peroxisomes invivo. Having established that Sec20p, Sec39p, and Dsl1p wereinvolved in peroxisome biogenesis and assembly, we nextwanted to determine if these proteins were associated withperoxisomes in vivo. To do this, we isolated peroxisomes fromcells grown in oleic acid for 16 h by isopycnic gradient ultra-centrifugation of a peroxisome-enriched 20,000 � g fraction(20KgP) (Fig. 6A). Similar to results described in previousreports (49, 62, 71), peroxisomes were found in higher-densityfractions of the gradient, localizing mainly to fractions 3, 4, and5 as determined by the presence Pot1p and Pex3p in thesefractions. Sec20p, Sec39p, and Dsl1p were also enriched infraction 4, indicating an association of these proteins withperoxisomes in vivo. The ER-resident proteins Kar2p (lumi-nal) and Sec61p (membrane) were also found in this fraction,raising the possibility that the enrichment of Sec20p, Sec39p,and Dsl1p in the peroxisome fraction was due solely to cofrac-tionation with ER membranes. To determine if the localizationof Sec20p, Sec39p, and Dsl1p in the peroxisome fraction wasdue to a true association with peroxisomes or the indirectresult of cofractionation with ER membranes, we performedthe same fractional analysis on pex3� cells, which lack peroxi-somes. Indeed, Sec20p and Sec39p were no longer enriched infraction 4 and localized to lighter fractions. A similar patternwas also found for Dsl1p but not to the extent of that observedfor Sec20p and Sec39p. In contrast, the ER-resident mem-brane protein Sec61p did not shift to lighter fractions upon lossof peroxisomes, indicating that the enrichment of Sec20p,Sec39p, and Dsl1p in fraction 4 in BY4742 yeast cells was dueto the presence of peroxisomes in this fraction. The absence ofperoxisomes in pex3� cells was verified by the loss of Pot1p inthe 20KgP fractions. Interestingly, the ER luminal proteinKar2p also localized to lighter fractions upon loss of peroxi-somes. In summary, these data provide the first evidence of an

in vivo association of essential secretory proteins with peroxi-somes.

To further investigate the association of Sec20p, Sec39p, andDsl1p with peroxisomes, we coexpressed the peroxisome markerPex3p-mRFP with Sec20p-GFP, Sec39p-GFP, or Dsl1p-GFPin BY4742 yeast cells, cultured the cells in oleic acid for 4 h,and visualized the intracellular localization of the proteins byconfocal microscopy. As expected, the majority of Sec20p-GFP, Sec39p-GFP, and Dsl1p-GFP were found to localize tothe ER, as previously described (24, 43). However, a smallfraction of each protein colocalized with Pex3p-mRFP as seenby single-plane confocal images (Fig. 6B). Colocalization ofSec20p-GFP, Sec39p-GFP, and Dsl1p-GFP with Pex3p-mRFPwas not dependent on growth of cells in oleic acid, as theseproteins were also found to colocalize in cells grown in YEPD(Fig. 6C). In addition to the association of Sec20p-GFP,Sec39p-GFP, and Dsl1p-GFP to peroxisomes, Pex3p-mRFPwas also found frequently juxtaposed to, interspersed between,or at the terminal ends of Sec20p-GFP-, Sec39p-GFP-, andDsl1p-GFP-positive ER membranes (Fig. 6B and C). In con-trast, Pex3p did not colocalize with the structural ER-localizedmembrane protein Rtn1p (8, 72), even in a maximum-intensityprojection that collapses all confocal planes onto a single im-age (Fig. 6D), thus showing that the association of Pex3p withSec20p, Sec39p, and Dsl1p was specific.

DISCUSSION

Recent studies have established that the ER plays an essen-tial role in the biogenesis and maintenance of peroxisomes (21,26, 58). Disruption of intracellular transport between the ERand peroxisomes leads to the complete loss of identifiableperoxisomes. This absolute requirement for ER-derived mem-brane components for the biogenesis of peroxisomes predictsthe existence of secretory machinery acting in this process, buta role for secretory genes involved in membrane traffickingbetween the ER and peroxisomes has yet to be described.Previous studies have ruled out a direct role for COPI- andCOPII-mediated vesicular transport in peroxisome biogenesis(53, 73). To date, a complete loss of peroxisomes in yeast hasbeen shown to occur only upon deletion of either PEX3 orPEX19. However, the products of these genes exhibit no sim-ilarities to proteins that function as secretory machinery. In thecurrent study, we performed a gene repression screen of es-sential secretory genes implicated in ER-localized traffickingand identified a role for SEC20, SEC39, and DSL1 in thebiogenesis of peroxisomes. The results from this study aresignificant because they are the first to describe secretory ma-chinery involved in the de novo formation and maintenance ofperoxisomes. Moreover, these results support the existence ofanother vesicular ER trafficking pathway in addition to the onededicated to ER-to-Golgi transport.

Under wild-type conditions, newly derived Pex3p-containingstructures from the ER fuse with preexisting peroxisomes anddo not serve as templates for the assembly and maturation ofnew peroxisomes (35). The growth and division of preexistingperoxisomes constitute the primary mechanism for the prolif-eration of peroxisomes under these conditions, and preexistingperoxisomes serve as the site of import for peroxisomal matrixproteins (35). In the absence of preexisting peroxisomes, ER-

FIG. 5. Oleic acid induces the expression of Sec20p, Sec39p, andDsl1p. Equal amounts of protein from whole-cell lysates of wild-typeBY4742 cells cultured in oleic acid-containing SCIM1 for the timesindicated were separated by SDS-PAGE, transferred to nitrocellulose,and immunoblotted for Sec20p, Sec39p, Dsl1p, the ER-resident pro-tein Kar2p, and Pot1p.



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derived Pex3p-containing structures serve as a scaffold uponwhich new peroxisomes assemble (35), and the intracellularpool of peroxisomes cannot be maintained without the contin-uous production of ER-derived Pex3p-containing structures(21, 22, 58). Based on this current view of peroxisomes, theresults of the present study strongly support a role for Sec20p,Sec39p, and Dsl1p in the early stages of peroxisome biogenesisand/or assembly.

Repression of Sec20p and Sec39p in cells resulted in signif-icant mislocalization of the peroxisomal matrix protein Pot1pto the cytosol, indicative of a lack of preexisting peroxisomes ora defect in peroxisomal matrix protein import in these cells(Fig. 1). The peroxisomal membrane protein Pex3p relocalizedto tubular-vesicular structures in cells repressed for the expres-sion of SEC20, SEC39, and DSL1 (Fig. 2). This is also indic-

ative of a lack of preexisting peroxisomes in these cells. Anal-ysis of peroxisome ultrastructure by electron microscopyfurther supported the relocalization of Pex3p to tubular-vesic-ular structures in cells repressed for SEC20, SEC39, and DSL1expression and corroborated the lack of mature peroxisomestructures in gene-repressed SEC20 and SEC39 cells (Fig. 3).Even in the absence of gene repression, Sec20p levels in THC-SEC20 cells were below the detection limits of our antibody;however, the viability of these cells indicates that a minimumthreshold of expression for the essential SEC20 gene wasachieved. Of note, under these conditions THC-SEC20 cellsalso exhibited defects in peroxisome biogenesis, consistent witha role for Sec20p in this process (Fig. 1, 2, and 3).

We also found that the normal trafficking of newly synthe-sized Pex3p-GFP from the ER to peroxisomes was disrupted in

FIG. 6. Sec20p, Sec39p, and Dsl1p associate with peroxisomes. (A) A 20KgP fraction was prepared from wild-type BY4742 and mutant pex3�cells grown in oleic acid for 16 h for the isolation of peroxisomes by isopycnic gradient centrifugation on a discontinuous Nycodenz gradient asdescribed in Materials and Methods. Equal volumes of each fraction were separated by SDS-PAGE, transferred to nitrocellulose, and immuno-blotted for the indicated proteins. P, peroxisome fractions; M, mitochondrial fractions. BY4742 cells coexpressing Pex3p-mRFP and Sec20p-GFP,Sec39p-GFP, or Dsl1p-GFP were grown in SCIM2 (B) for 4 h or in YEPD (C) for 16 h and fixed in 3.7% formaldehyde. Fluorescently taggedproteins were visualized for a single plane by confocal microscopy. Narrow arrowheads highlight areas of colocalization, and wide arrowheadshighlight areas where Pex3p-mRFP is closely apposed to Sec20p-GFP, Sec39p-GFP, or Dsl1p-GFP. Bars, 5 �m. (D) BY4742 cells coexpressingPex3p-GFP and Rtn1p-mRFP were grown in YEPD for 16 h. Cells were fixed with 3.7% formaldehyde, images were captured by confocalmicroscopy, and a maximum-intensity projection was created from a deconvolved 3D data set using Huygens software. Bar, 5 �m.



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cells depleted of Sec39p (Fig. 4). This disruption was not ob-served in cells repressed for SEC61 expression and was there-fore specific to the loss of Sec39p (Fig. 4). Pulse-chase exper-imentation provided evidence for a defect in Pex3p traffickingbeing at a point between the ER and peroxisomes, as deliveryof Pex3p to the ER and its insertion into the ER were unaf-fected in Sec39p-repressed cells. Also consistent with a role forSEC20, SEC39, and DSL1 in peroxisome biogenesis, the ex-pression of these genes was induced in cells grown under con-ditions that stimulate peroxisome biogenesis and proliferationin wild-type yeast cells (Fig. 5). Finally, a minor fraction ofSec20p, Sec39p, and Dsl1p associated with peroxisomes in vivo

(Fig. 6B and C), and subcellular fractionation of wild-typeyeast cells cultured in oleic acid confirmed an association ofthese proteins with peroxisomes (Fig. 6A). Taken together,these results strongly support a role for Sec20p, Sec39p, andDsl1p in the early stages of the peroxisome biogenic process.

A role for Sec20p, Sec39p, and Dsl1p in the biogenesis ofperoxisomes from the ER challenges the current view thatthese proteins function exclusively in Golgi-to-ER retrogradetransport (4, 5, 27, 43, 66). New structural evidence showedthat Sec39p and Dsl1p operate together as a vesicle-tetheringcomplex at the ER via interactions with the ER-localizedSNARE (soluble N-ethylmaleimide-sensitive factor attach-

FIG. 6—Continued.



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ment protein receptor) Sec20p (63). A central region in Dsl1pcontains binding sites for two different subunits of the COPIvesicle coat complex, which through its interactions withSec20p serves as a bivalent recognition complex for the recruit-ment of COPI vesicles to the ER (4, 5, 63). This suggests amechanism for Sec20p, Sec39p, and Dsl1p in peroxisome bio-genesis that involves the recruitment of COPI vesicles to theER. However, consistent with previous results (53, 73), generepression of COPI did not significantly affect de novo perox-isome biogenesis as indicated by the localization of Pot1p-GFPto peroxisomes in these cells (Fig. 1), although it did alter thenumber and size of peroxisomes, suggesting that COPI has anunidentified role in the growth and division of preexistingperoxisomes (37). Overall, the interaction of Sec20p, Sec39p,and Dsl1p with the COPI vesicle coat appears to be dispens-able for the de novo biogenesis of peroxisomes and indicatesthat Sec20p, Sec39p, and Dsl1p function via a different mech-anism when acting in peroxisome biogenesis.

The association of Sec20p, Sec39p, and Dsl1p with addi-tional functions is not without precedent. The Sec20p homologin plants has been implicated in the exit of seed storage pro-teins from the ER (31), and the mammalian equivalent ofDsl1p, ZW10, was shown to have a role in cell cycle checkpointcontrol, dynein targeting, and membrane trafficking (64). Apossible explanation for these seemingly diverse functionscould be the recruitment of dynein by this complex to variousintracellular sites. This hypothesis is supported by studies thatshow that the loss of ZW10 function leads to the release ofcytoplasmic dynein from both kinetochores and membranes(54, 67). A role for dynein in peroxisome biogenesis and move-ment in mammalian cells has also been described, and dyneinlocalizes to peroxisomes in yeast (7, 28, 56). Since the move-ment of peroxisomes for inheritance in yeast occurs along actincables and uses a myosin motor (14), the association of micro-tubular dynein with peroxisomes most certainly functions inanother capacity. Our data support a potential role for Sec39p,Dsl1p, and Sec20p in the recruitment of dynein for peroxisomebiogenesis, and this requires further investigation. Yet, therecruitment of other factors by Sec20p, Sec39p, and Dsl1p tothe ER for the biogenesis of peroxisomes cannot be ruled out.

The site of action for Sec20p, Sec39p, and Dsl1p is mostlikely at the ER, as these proteins were found localized mainlyto this compartment (Fig. 6B and C) (24, 43). Sec20p, Sec39p,and Dsl1p, along with the ER-luminal protein Kar2p, were alsoenriched in peroxisome-containing fractions from cells grownin oleic acid (Fig. 6A). The presence of Sec20p, Sec39p, Dsl1p,and Kar2p in these fractions was dependent on the presence ofperoxisomes, because in pex3� cells that lack peroxisomes,these proteins shifted in their localization to fractions of lesserdensity (Fig. 6A). In contrast, the ER-resident membrane pro-tein Sec61p, which was also found in peroxisome-enrichedfractions from wild-type cells, did not exhibit a shift to lighterfractions in pex3� cells. Localization of Sec20p, Sec39p, andDsl1p, together with some amount of soluble Kar2p, to aspecific ER subdomain that is involved in the biogenesis ofperoxisomes and which excludes most membrane componentsof the rough ER could explain this observation (58). The shiftof Sec20p, Sec39p, Dsl1p, and Kar2p to less dense subcellularfractions of pex3� cells further indicates that the integrity ofthis specialized ER subdomain is dependent on the continuous

production of ER-derived Pex3p-containing structures and/orthe presence of peroxisomes. Close apposition of peroxisomeswith ER membranes containing Sec20p, Sec39p, and Dsl1psupports this conclusion.

Minor amounts of Sec20p, Sec39p, and Dsl1p colocalizedwith Pex3p in vivo. To determine the validity of this colocal-ization, we investigated the extent of colocalization betweenPex3p and another ER-resident membrane protein, Rtn1p.Rtn1p is localized throughout the cortical ER and functions inproviding the characteristic reticular structure of that organelle(8, 72). Rtn1p was observed not to colocalize with Pex3p invivo, indicating that the pools of Sec20p, Sec39p, and Dsl1pcolocalizing with Pex3p, although small, were significant. Thefunction of the fraction of Sec20p, Sec39p, and Dsl1p thatassociates with peroxisomes remains to be determined. Theassociation of these proteins with peroxisomes could poten-tially result from their “escape” from the ER subdomain in-volved in peroxisome biogenesis during the peroxisome bio-genic process (38).

The atypical vesicular expansions associated with ER mem-branes in cells repressed for SEC20, SEC39, or DSL1 areconsistent with earlier reports investigating mutant alleles ofDSL1 (4, 66). Similar membrane continuities between the ERand peroxisomes have also been described for mammalianintestinal and dendritic cells (18, 36). The electron density ofthe expanded ER structures in the repressed SEC20, SEC39,and DSL1 cells was considerably less than that of normalperoxisomes, suggesting a lack of matrix protein content andconsistent with the mislocalization of the matrix protein Pot1pto the cytosol in these cells. The absence of normal peroxisomeprofiles in THC-SEC20 cells, even in the absence of doxycy-cline, suggests that the Pex3p-GFP-labeled structures observedin these cells are not peroxisomes. These results are furthersupport for the presence of a specialized subdomain of the ERinvolved in the de novo formation of peroxisomes.

The oleic acid-induced expression of SEC20, SEC39, andDSL1 was consistent with a previous report from a globaltranscriptional analysis to identify genes involved in peroxi-some assembly and function (49). Similar to the observation bySmith and colleagues of the robust activation of SEC39 by oleicacid compared to that of SEC20 or DSL1 (50), we found thatSec39p exhibited the greatest increase in protein levels whencells were incubated in oleic acid (Fig. 5). Interestingly, thereported biphasic activation of SEC39 and DSL1 by oleic acidwas essentially mirrored by the extent of Sec39p and Dsl1pexpression, suggesting that the activation and expression ofthese genes are tightly regulated. The biphasic nature of theoleic acid-induced gene activation of SEC39 and DSL1 alsoimplies that peroxisome biogenesis in response to a fatty acidcarbon source may not be a linear process but occurs in aperiodic fashion based on the length of exposure of yeast cellsto lipid.

An unexpected finding was that gene repression of SEC61resulted in an increase in peroxisome number. South and col-leagues had reported previously that incubation of yeast cellsharboring a temperature-sensitive mutant of SEC61 at thenonpermissive temperature had no effect on peroxisome bio-genesis (51). The apparent discrepancy between these resultsand ours may be explained by the absence of PEX11 in theyeast cells used in their study. Pex11p is a peroxisomal mem-



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brane protein involved in peroxisome proliferation (32), andthe use of pex11� cells most likely masked any effect that theloss of Sec61p activity had on peroxisome division and multi-plication. We are currently investigating how Sec61p regulatesperoxisome number and size.

The moderate accumulation of Pex3p-GFP in tubular-vesic-ular structures in cells repressed for SEC61 may have occurredsecondarily to the primary loss of Sec61p. Sec20p is predictedto be a type II transmembrane protein, and its insertion intomembranes would be dependent on Sec61p (40). Based on thisrelationship, we expect that decreased expression of Sec61pwould affect peroxisome biogenesis by affecting Sec20p’s in-corporation into membranes, thereby indirectly affecting thenewly attributed function of Sec20p in peroxisome biogenesis.

Roles for other essential SEC genes in peroxisome biogen-esis cannot be completely excluded by our study. Although ourexperimental conditions were consistent with those of previousstudies involving repression of essential genes using the THCyeast strains (2, 3, 34), subsequent loss of protein was notdirectly verified for each gene. However, based on experi-mentally measured protein half-lives in the budding yeast(6), we would expect the majority of the essential Sec pro-teins screened in our study to have been knocked downsufficiently under our experimental conditions.

In conclusion, we have identified the ER-resident proteinsSec20p, Sec39p, and Dsl1p as being involved in the initial stepsof peroxisome biogenesis and assembly. Whether these pro-teins function in the ER exit of Pex3p-containing structures orin the delivery of peroxisomal membrane components requiresfurther investigation.

ACKNOWLEDGMENTS

The technical assistance of Elena Savidov, Richard Poirier, andHanna Kroliczak is gratefully acknowledged. We thank members ofthe Rachubinski laboratory for helpful discussion.

R. J. Perry is the recipient of a Full-Time Fellowship from theAlberta Heritage Foundation for Medical Research. F. D. Mast is therecipient of a Frederick Banting and Charles Best Canada GraduateScholarship from the Canadian Institutes for Health Research. R. A.Rachubinski is Canada Research Chair in Cell Biology and an Inter-national Research Scholar of the Howard Hughes Medical Institute.This work was supported by grant 15131 from the Canadian Institutesof Health Research to R. A. Rachubinski.

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