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Mol Biol Cell. Jul 2002; 13(7): 2486–2501.
PMCID: PMC117329
Genomic Screen for Vacuolar Protein Sorting Genes in Saccharomyces cerevisiaeD in Box
Cecilia J. Bonangelino, Edna M. Chavez, and Juan S. Bonifacino*
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
Chris Kaiser, Monitoring Editor
*Corresponding author. E-mail address: juan/at/helix.nih.gov.
Received January 25, 2002; Revised March 19, 2002; Accepted March 25, 2002.
The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains of Saccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.
Lysosomes have long been recognized as the major site for the degradation of both exogenous and endogenous macromolecules within mammalian cells (De Duve and Wattiaux, 1966 blue right-pointing triangle; Kornfeld and Mellman, 1989 blue right-pointing triangle). The biogenesis of lysosomes, however, remains poorly understood. Helpful insights have been gained from the study of the yeast vacuole, which is functionally analogous to the mammalian lysosome. To the extent that they have been compared, the biosynthetic pathways and molecular machineries involved in the formation of mammalian lysosomes and the yeast vacuole appear remarkably conserved (reviewed by Lemmon and Traub, 2000 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). Thus, further studies of the yeast vacuole are likely to provide new insights into the mechanisms of lysosome biogenesis.
The main pathway for the delivery of newly synthesized proteins to the vacuole is referred to as the carboxypeptidase Y (CPY) pathway based on the utilization of this pathway by CPY (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). CPY is a soluble hydrolase that normally resides within the vacuole lumen. Another soluble hydrolase, proteinase A (PrA), as well as the membrane-bound hydrolase carboxypeptidase S are also transported to the vacuole via the CPY pathway. This pathway involves transport from the late-Golgi complex (equivalent to the mammalian trans-Golgi network) to a prevacuolar compartment (PVC). From the PVC, some proteins are subsequently transferred to the vacuole, whereas others return to the late-Golgi complex for further rounds of transport. Membrane-bound enzymes involved in the processing of pro-α-factor, such as the Kex2p dibasic endopeptidase, are also transported along parts of the CPY pathway as they cycle between the late-Golgi complex and the PVC. A second pathway, referred to as the alkaline phosphatase (ALP) pathway, is followed by the membrane-bound hydrolase ALP and the t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Vam3p and Nyv1p (Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Reggiori et al., 2000 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). The ALP pathway differs from the CPY pathway in that it uses a different type of Golgi-derived carrier vesicle (Rehling et al., 1999 blue right-pointing triangle) and bypasses the PVC (Cowles et al., 1997b blue right-pointing triangle; Piper et al., 1997 blue right-pointing triangle) en route to the vacuole.
Genetic screens based on the detection of reduced proteolytic activity of the vacuole (Jones, 1977 blue right-pointing triangle) or missorting of vacuolar hydrolases to the periplasmic space (Robinson et al., 1988 blue right-pointing triangle; Rothman et al., 1989 blue right-pointing triangle) have identified >40 vacuolar protein sorting (VPS) genes. Other screens devised for different purposes uncovered additional genes that, although not termed VPS, also participate in vacuolar protein sorting. The products of most vacuolar protein sorting genes identified to date control transport exclusively along the CPY pathway (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). These include proteins involved in transport from the late-Golgi complex to the PVC (e.g., Vps1p, Vps8p, Vps9p, Vps10p, Vps15p, Vps19p, Vps21p, Vps34p, Vps45p, clathrin, the AP-1 complex, Gga1p/Gga2p, Drs2p, and Mvp1p); PVC maturation, including multivesicular body formation (e.g., Vps2p, Vps4p, Vps20p, Vps22p, Vps23p, Vps24p, Vps27p, Vps28p, Vps32p, and Vps36p); and retrieval from the PVC to the Golgi complex (e.g., Vps5p, Vps17p, Vps26p, Vps29p, Vps30p, Vps35p, Vps51p, Vps52p, Vps53p, Vps54p, and Grd19p). The ALP pathway is controlled by a surprisingly simpler machinery, of which the heterotetrameric adaptor protein complex AP-3 is the only specific component known to date (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). AP-3 participates, together with Vps39p and Vps41p, in the formation of ALP carriers at the late-Golgi complex (Rehling et al., 1999 blue right-pointing triangle). The CPY and ALP pathways converge at the step of fusion with the vacuole, for which reason they share a number of gene products involved in vacuole targeting and fusion (e.g., Vam3p, Vam7p, Vps11p, Vps16p, Vps18p, Vps33p, Vps39p, Vps41p, and Ypt7p).
As numerous as the vacuolar protein sorting genes may seem at present, the total number of genes involved in this process may be even higher. Indeed, studies over the past 2 yr alone have shown that mutations in several novel genes, including Mrl1p (Whyte and Munro, 2001b blue right-pointing triangle), Ric1p (Siniossoglou et al., 2000 blue right-pointing triangle; Bensen et al., 2001 blue right-pointing triangle), Rgp1p (Siniossoglou et al., 2000 blue right-pointing triangle; Bensen et al., 2001 blue right-pointing triangle), Did2p (Amerik et al., 2000 blue right-pointing triangle), Did4p (Amerik et al., 2000 blue right-pointing triangle), Ccz1p (Kucharczyk et al., 2000 blue right-pointing triangle), Mos10p (Kranz et al., 2001 blue right-pointing triangle), and Swa2p (Gall et al., 2000 blue right-pointing triangle) impair CPY sorting to the vacuole. The steady pace of discovery of new vacuolar protein sorting genes suggests that previous screens were not saturating and that additional genes involved in this process might still remain to be identified.
To obtain a global view of all the genes involved in vacuolar protein sorting, we decided to perform a genome-wide screen for VPS genes in Saccharomyces cerevisiae. The approach used in our study consisted of screening a collection of 4653 homozygous diploid gene deletion strains (Winzeler et al., 1999 blue right-pointing triangle) for secretion of CPY into the medium. This approach benefited from the fact that most vacuolar protein sorting genes are nonessential in yeast. We identified 146 mutant strains that secreted strong-to-moderate levels of CPY. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting. The identity of the remaining 93 genes and the phenotypic characterization of the mutant strains reported herein provide surprising new insights into vacuole biogenesis.
Media, Strains, and Molecular Biology Procedures
S. cerevisiae cells were grown in yeast extract-peptone-dextrose (YEPD) medium. DNA manipulations and transformation into Escherichia coli DH5α cells were performed by standard protocols. Homozygous diploid deletion strains and Mat α haploid deletion strains were obtained from Research Genetics (Invitrogen, Huntsville, AL) and compared with the corresponding parental strains, BY4743 (diploid), BY4739 (haploid), and BY4742 (haploid). For genotypes see the Research Genetics Web site at http://www.resgen.com/products/YEASTD.php3.
CPY Colony Blots
The CPY colony blot assay was adapted from Roberts et al. (1991) blue right-pointing triangle. Briefly, yeast strains were transferred from the 96-well plates to 15-cm2 YEPD plates with a pinning tool and placed at 30°C for 2 to 3 d. Once strains had grown in the round patches, they were replica-plated to a fresh YEPD plate and overlaid with a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany), taking care to remove any trapped bubbles. The plates were then incubated at 30°C for 18–22 h. The nitrocellulose membranes were washed several times with double-distilled H2O and then with standard phosphate-buffered saline. The membranes were then subjected to immunoblotting with mouse anti-CPY (Molecular Probes, Eugene, OR) performed as described previously (Mullins and Bonifacino, 2001b blue right-pointing triangle) with slight modifications. Briefly, the mouse anti-CPY was used at 1:1000 dilution. Donkey anti-mouse IgG-horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) at 1:5000 dilution was used as the secondary antibody, followed by visualization with enhanced chemiluminescence reagents (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's protocols.
Metabolic Labeling and Immunoprecipitation
Metabolic labeling of yeast cells with 35S Express label (PerkinElmer Life Sciences), pulse-chase analyses, and immunoprecipitations were performed at 30°C as described by Bonifacino and Dell'Angelica (1998) blue right-pointing triangle. A 10-min pulse, followed by a 10-min chase were used in all the pulse-chase assays. Immunoprecipitations were performed overnight at 4°C and the immunoprecipitates analyzed by SDS-PAGE and autoradiography. Mouse anti-CPY antibodies were purchased from Molecular Probes. Anti-PrA and rabbit anti-ALP antibodies were the generous gifts of Carol Woolford (Carnegie Mellon University, Pittsburgh, PA) and Tom Stevens (University of Oregon, Eugene, OR), respectively. For α-factor experiments, cells were pulse-labeled at 25°C for 7.5 min to visualize α-factor–processing intermediates (Graham and Emr, 1991 blue right-pointing triangle) and immunoprecipitated with anti-α-factor (generously provided by Todd Graham, Vanderbilt University, Nashville, TN).
Labeling Yeast Vacuoles with FM4-64
Yeast vacuoles were visualized in vivo by labeling log-phase cells with 80 μM N-(3-triethylammoniumpropyl)-4-(p-diethyl-aminophenylhexatrienyl) pyridinium dibromide (FM4-64) (Vida and Emr, 1995 blue right-pointing triangle; Bonangelino et al., 1997 blue right-pointing triangle). Cells were viewed with a 100× objective lens on an Olympus IX-70 fluorescence microscope (excitation, 560 nm; dichroic mirror at 595 nm; emission, 630 nm) combined with a low level of transmitted light to reveal cell outlines. Images were captured digitally with an IMAGO charge-coupled device camera controlled by TILLvisION software (TILL Photonics, Eugene, OR). Images were processed using Adobe Photoshop 5.0 (Adobe Systems, Mountain View, CA).
Labeling Yeast Actin with Oregon-Green Phalloidin
Cells were grown in 5 ml of YEPD at 30°C to midlog phase (OD600 ~0.5) and subsequently fixed by the addition of 1/9 volumes of 37.5% formaldehyde (final concentration ~3.7%). This fixation was carried out at room temperature for 1 h with gentle agitation. The fixed cells were harvested and washed twice with phosphate-buffered saline. The cells were then resuspended in 100 μl of phosphate-buffered saline, added Oregon green-phalloidin (Molecular Probes) to 1.1 μM (20 μl of 6.6 μM stock), and incubated at 30°C with agitation in the dark overnight (~16–20 h.). Labeled cells were viewed with a 100× objective lens on an IX-70 fluorescence microscope (excitation, 485 nm; dichroic mirror at 520 nm; emission, 540 nm; Olympus, Tokyo, Japan). Images were captured and processed as described above.
Analysis of α-Factor Secretion
Halo assays for α-factor secretion were performed essentially as described previously (Mullins and Bonifacino, 2001b blue right-pointing triangle). Briefly, the sst1-3 mutant strain RC634 (Mat a) was grown at 30°C to stationary phase in YEPD. Cells were harvested, washed with YEPD and resuspended in fresh medium to a final concentration of ~9.0 OD600/ml. Then, a 120-μl aliquot of the sst1-3 mutant strain was spread evenly on a YEPD agar plate and used immediately in the assay. Strains to be tested for α-factor secretion were grown to 1–1.5 OD600/ml at 30°C, concentrated to 1 OD600/ml, and serially diluted in YEPD to the concentrations indicated in the figure. Three microliters of each dilution was spotted on plates containing RC634 cells and incubated at 30°C for ~72 h to visualize the α-factor–induced growth inhibition (i.e., growth halo). Strains that produced no visible halos when spotted on the sst1-3 lawn were retested in the same way by using Xbhb-2C, a Mat α sst2-1 mutant strain, to ensure that these strains were not secreting a-factor.
Screening of a Collection of Gene Deletion Strains for CPY Secretion
To identify vacuolar protein sorting mutants, we screened a collection of 4653 homozygous diploid deletion strains developed by the Saccharomyces Genome Deletion Project (http://sequence-www.stanford.edu/group/yeast_deletion_project/deletions3.html) (Winzeler et al., 1999 blue right-pointing triangle) and available through Research Genetics (http://www.resgen.com/products/YEASTD.php3). The collection was made by polymerase chain reaction-based disruption of all open reading frames (ORFs) larger than 100 codons in the BY4743 wild-type strain. Because the diploid strains are homozygous for the deletions, only nonessential genes (~82% of the total) are represented in the collection. The primary screening consisted of analyzing each deletion strain for secretion of CPY by using a colony blotting assay (Roberts et al., 1991 blue right-pointing triangle). In wild-type cells, CPY is efficiently sorted to the vacuole and therefore is not secreted. In vps mutants, on the other hand, CPY sorting to the vacuole is impaired and different levels of the protein are secreted. Figure Figure1a,1a, top, shows examples of the CPY colony blotting assay for wild-type (negative control) and vps39, a vps mutant known to secrete high levels of CPY (positive control).
Figure 1
Figure 1
Identification of CPY-secreting strains. The collection of 4653 homozygous diploid deletion strains developed by the Saccharomyces Genome Deletion Project was screened using a CPY colony blot assay (Roberts et al., 1991 blue right-pointing triangle). The membranes were subjected (more ...)
Screening of the complete collection resulted in the identification of 362 mutant strains (7.8% of the total) that secreted various amounts of CPY. The strains were categorized as strong (46 strains), moderate (100 strains), and weak (216 strains) based on a visual evaluation of the signal intensity, as exemplified by the colony blots shown in Figure Figure1,1, a and b. The majority of known VPS genes and other genes previously shown to be involved in vacuolar protein sorting were found distributed among the strong and moderate categories. For this reason, genes whose deletion gave strong and moderate phenotypes were treated as a single class in all subsequent analyses. Tables Tables11 and and22 list the genes with strong-moderate and weak phenotypes, respectively, that were identified in the screen. Supplemental Tables 1 and 2 are available online and contain additional information on these genes, their products and the phenotypes of the corresponding mutant strains. Genes in most tables are grouped according to the known or presumed functions of their products.
Table 1
Table 1
Genes identified in CPY secretion screen (strong or moderate phenotypes)
Table 2
Table 2
Genes identified in CPY secretion screen (weak phenotype)
Genes whose deletions gave strong-moderate phenotypes encoded: most known Vps proteins, other proteins previously implicated in vacuolar protein sorting, vacuolar ATPase subunits, components of the glycosylation machinery, AP-3 subunits, Arf1 and Arf-related proteins, actin-related proteins, monensin and brefeldin A hypersensitivity proteins, ribosomal proteins, miscellaneous proteins, and the products of a number of hypothetical ORFs (Table (Table11 and Supplemental Table 1). Genes whose deletions gave weak phenotypes included members of some of the same groups discussed above, plus genes involved in protein trafficking to compartments other than the vacuole, and a large number of miscellaneous genes and hypothetical ORFs (Table (Table22 and Supplemental Table 2). Because of the greater likelihood that mutations in genes whose deletions have a weak phenotype could affect vacuolar protein sorting indirectly, we focused our subsequent analyses on deletion strains with strong-moderate CPY secretion.
The CPY-secretion phenotypes of 146 of the strong-moderate strains were confirmed at least twice by colony blotting of the homozygous diploid mutants and the corresponding haploid mutants. All diploid, as well as all but two haploid strains (adh1 and bud14), retested positive in these assays. Seventy-four mutant strains were selected for further analyses. Additional properties analyzed included growth at 30° or 37°C (Table (Table3);3); biosynthetic processing of CPY, PrA, and ALP (Figure (Figure2);2); vacuole morphology (Figure (Figure3);3); integrity of the actin cytoskeleton (Figure (Figure4);4); and secretion of processed α-factor (Figure (Figure5).5). Representative results for some of the mutants are presented in Figures Figures225, and a summary of these results is presented in Tables Tables33 and and4.4. The characteristics of selected genes and their products are discussed below.
Table 3
Table 3
Phenotypic characteristics of selected CPY-secreting strains
Figure 2
Figure 2
Analysis of CPY, PrA, and ALP processing. Wild-type (wt) and deletion strains were pulse-labeled with [35S]methionine for 10 min (first lane of each pair) and chased for 10 min (second lane of each pair). Sequential immunoprecipitations (more ...)
Figure 3
Figure 3
Visualization of vacuole morphology in wild-type and mutant strains by light microscopy. Vacuole morphology was examined by labeling with FM4-64 as described in MATERIALS AND METHODS. The photographs were taken with both fluorescence and a low level of (more ...)
Figure 4
Figure 4
Visualization of the actin cytoskeleton in wild-type and mutant strains by light microscopy. The actin cytoskeleton was examined by labeling fixed log-phase cells with 1.1 μM Oregon green-phalloidin at 30°C for ~16–20 h (more ...)
Figure 5
Figure 5
Analysis of α-factor secretion and processing. (a) To test for α-factor secretion, wild-type and mutant strains were grown to log phase and serially diluted to the concentrations indicated, and equal amounts were spotted on YEPD plates (more ...)
Table 4
Table 4
Summary of phenotypes
Known VPS Genes and Other Genes Previously Implicated in Vacuolar Protein Sorting
Forty genes identified in our screen had been previously reported in the literature or listed in the Saccharomyces Genome Database as VPS genes (Table (Table11 and Supplemental Table 1). Of the VPS genes characterized to date (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle), only VPS11 andVPS15 were not identified in our screen, but this was because these genes were not represented in the deletion collection. The recovery of virtually all known VPS genes attests to the accuracy of the collection and the reliability of the CPY secretion assay. From this, we infer that the majority of the other genes have been accurately identified. Thirteen other genes (CCZ1, DID4, GOS1, MRL1, MVP1, PTC1, RGP1, RIC1, SYS1, TLG2, VAM7, YPT6, YPT7) had also been previously implicated directly or indirectly in vacuolar protein sorting (Table (Table11 and Supplemental Table 1). These known genes will not be further discussed herein, except for their relationship to other genes of interest.
Vacuolar ATPase
Twelve genes whose deletions resulted in strong-moderate phenotypes (Table (Table11 and Supplemental Table 1) and six genes whose deletions gave weak phenotypes (Table (Table22 and Supplemental Table 2) encoded either subunits of the vacuolar proton-translocating ATPase or proteins involved in its assembly or regulation (reviewed by Graham et al., 2000 blue right-pointing triangle). The subunits belonged to both the catalytic V1 subcomplex (Vma1p, Vma2p, Vma4p, Vma5p, Vma7p, Vma8p, Vma10p, and Vma13p) and the proton-translocating V0 subcomplex (Vma3p, Vma6p, Vma11p, and Vph1p). The only remaining subunit of the V0 subcomplex, Vma16p, was not represented in the deletion collection used for the screen. The assembly factors included Vma12p, Vma21p, and Vma22p, which facilitate assembly but are not stable components of either subcomplex. Thus, the full integrity of the vacuolar ATPase appears to be required for efficient CPY sorting to the vacuole. The identification of vacuolar ATPase genes in our screen is in line with previous observations that mutations in the VMA2 gene (Yamashiro et al., 1990 blue right-pointing triangle) or treatment with the vacuolar ATPase inhibitor, bafilomycin A1 (Klionsky and Emr, 1989 blue right-pointing triangle), cause partial missorting and secretion of CPY. This points to a possible requirement for acidification of the vacuole and perhaps the PVC for efficient CPY sorting. However, the V0 subcomplex has also been shown to participate directly in vacuole fusion, in a role independent of acidification (Peters et al., 2001 blue right-pointing triangle); thus, it is possible that this subcomplex may also play a more direct role in CPY sorting.
Glycosylation
Nine genes whose deletions resulted in strong-moderate phenotypes (Table (Table11 and Supplemental Table 1) encoded proteins involved in glycosylation or modification of carbohydrate chains. Some of these proteins (e.g., Van1p, Mnn9p, Mnn11p, and Anp1, the latter of which is listed under actin-related proteins) are known to form multiprotein complexes with mannosyl-transferase activity in the Golgi complex (Jungmann and Munro, 1998 blue right-pointing triangle; Kojima et al., 1999 blue right-pointing triangle). At present it is unclear how defects in glycosylation could cause missorting of CPY, although it has been reported that unglycosylated CPY is transported more slowly through the secretory pathway (Winther et al., 1991 blue right-pointing triangle). Thus, it is possible that abnormal glycosylation of CPY or transmembrane components of the vacuolar protein sorting machinery (e.g., Vps10p, Mrl1p) could decrease the efficiency of sorting to the vacuole. Given that glycosylating enzymes are also involved in the biosynthesis of cell wall materials, it is also possible that their deficiency renders the cell wall fragile and causes lytic release of vacuolar CPY.
AP-3
We were surprised to find that strains with deletions of the genes encoding the four subunits of the AP-3 complex (Apl5p, Apl6p, Apm3p, and Aps3p) (Cowles et al., 1997a blue right-pointing triangle; Panek et al., 1997 blue right-pointing triangle; Stepp et al., 1997 blue right-pointing triangle) secreted moderate levels of CPY (Figure (Figure1b1b and Table Table11 and Supplemental Table 1). AP-3 had been shown to be involved in the ALP pathway but not the CPY pathway (Cowles et al., 1997a blue right-pointing triangle; Stepp et al., 1997 blue right-pointing triangle). Pulse-chase analyses revealed delayed processing of the Golgi precursor forms of pro-CPY and pro-PrA in the AP-3 mutant strains, although this was much less severe than that of the strongest vps mutants (Figure (Figure2).2). Processing of ALP was completely blocked in the AP-3 mutants (Figure (Figure2),2), as reported previously (Cowles et al., 1997a blue right-pointing triangle; Stepp et al., 1997 blue right-pointing triangle). The detection of a slight CPY sorting defect in our study could be due to the greater sensitivity of the assays or to the different genetic background of the yeast strains used herein. We suspect that this CPY sorting defect could be an indirect consequence of the drastic inhibition of the ALP pathway. The vacuolar t-SNAREs Vam3p and Nyv1p, for example, normally traffic via the ALP pathway, but function in vesicle fusion in the CPY pathway (Darsow et al., 1997 blue right-pointing triangle; Reggiori et al., 2000 blue right-pointing triangle). Although, Vam3p and Nyv1p are rerouted through the CPY pathway in AP-3 mutants (Darsow et al., 1998 blue right-pointing triangle; Reggiori et al., 2000 blue right-pointing triangle), it is possible that this rerouting does not completely compensate for the loss of the AP-3 pathway.
The apl5, apl6, apm3, and aps3 mutant strains have similar vacuole morphology, with slightly fewer lobes than the wild-type strain and lobes that seem somewhat unfolded (Figure (Figure3).3). This phenotype, although subtle, may indicate a decrease in vacuole fission, or an increase in vacuole fusion. This may also be an indirect consequence of reduced Vam3p at the vacuole, as factors required for normal vacuole morphology may have difficulty reaching the vacuole under these conditions.
ARF1 and ARF-related Proteins
Three strong-moderate genes encoded the small GTP-binding proteins of the Ras superfamily, Arf1p (ADP-ribosylation factor 1 product), Arl1p (Arf-like 1 product), and Arl3p (Arf-like 3 product) (Table (Table11 and Supplemental Table 1). In addition, three weak genes encoded the Arf GTPase-activating proteins Age2p, Gcs1p, and Glo3p (Table (Table22 and Supplemental Table 2). Arf proteins had been previously implicated in the regulation of protein trafficking along the secretory pathway by virtue of their interactions with multiple effectors, including several coat proteins (Donaldson and Jackson, 2000 blue right-pointing triangle). S. cerevisiae expresses two Arf proteins, Arf1p and Arf2p, of which Arf1p is the most abundant (Stearns et al., 1990 blue right-pointing triangle). Disruption of both genes is lethal, whereas disruption of only ARF1 is not (Stearns et al., 1990 blue right-pointing triangle). Although viable, arf1 mutant strains display various phenotypes, including slower kinetics of CPY transport through the secretory pathway (Chen and Graham, 1998 blue right-pointing triangle; Huang et al., 1999 blue right-pointing triangle; Yahara et al., 2001 blue right-pointing triangle).
In agreement with these reports, we found that deletion of the ARF1 gene results in delayed processing of CPY and PrA (Figure (Figure2).2). Some of this delay could be due to a reduced rate of transport from the endoplasmic reticulum (ER) to the Golgi complex. However, our finding that the ARF1-deletion strain secretes CPY (Figure (Figure1)1) indicates that Golgi-to-vacuole sorting is also compromised in this strain. This interpretation agrees with the observation that ARF1 displays genetic interactions with clathrin and auxilin (Swa2p, also identified in our screen; Table Table22 and Supplemental Table 2), two proteins involved in late-Golgi transport events (Chen and Graham, 1998 blue right-pointing triangle). Moreover, Arfs are involved in the recruitment of the GGA adaptor proteins to the late-Golgi complex (Zhdankina et al., 2001 blue right-pointing triangle). We also observed delayed processing of ALP in the ARF1-deletion strain (Figure (Figure2),2), which could be due to inhibition of either ER-to-Golgi or Golgi-to-vacuole transport. In mammalian cells, Arf1 and Arf3 regulate the association of AP-3 with membranes (Ooi et al., 1998 blue right-pointing triangle; Drake et al., 2000 blue right-pointing triangle), and it is possible that Arf1p could play a similar role in S. cerevisiae. In addition to defects in protein trafficking, the arf1 strain exhibited defects in vacuole morphology, actin cytosketelon, and α-factor secretion. The vacuole morphology of arf1 was fragmented, exhibiting a larger number of vacuole lobes than the wild-type (Figure (Figure3).3). However, the degree of fragmentation in arf1 was less severe than that observed for vps39, in which protein traffic is more impaired. This suggests that inefficient trafficking of factors required for vacuole fusion or biogenesis may account for the vacuole morphology observed in arf1. We also observed a decrease in the number and thickness of polarized actin filaments in arf1 mutants (Figure (Figure4).4). Finally, we found that the arf1 mutant strain secreted less mature α-factor, as determined by a halo assay for growth arrest (Figure (Figure5A).5A). This reduced secretion of mature α-factor is likely due to a block in Kex2p recycling to the Golgi complex as glycosylated pro-α-factor accumulates in arf1 (Figure (Figure5B),5B), suggesting a deficiency in α-factor processing.
The three Arf GAPs, Age2p, Gcs1p, and Glo3p, that were identified among the weak secretors might play overlapping roles in the inactivation of yeast Arfs at the late-Golgi complex. In support of this notion, a recent study has shown that yeast mutants lacking both Age2p and Gcs1p function exhibit substantial defects in CPY and ALP sorting to the vacuole (Poon et al., 2001 blue right-pointing triangle).
S. cerevisiae Arl1p and Arl3p are structurally related to Arf1p (55 and 37% amino acid sequence identity, respectively), but their functions are less well understood. An ARL1-deletion strain was previously reported to display normal biosynthetic processing of CPY (Lee et al., 1997 blue right-pointing triangle). Another study showed that deletion of ARL3 also resulted in normal processing of CPY, but processing of ALP was delayed at the nonpermissive temperature of 15°C (Huang et al., 1999 blue right-pointing triangle). We observed that ARL1- or ARL3-deletion strains exhibited secretion of CPY into the medium (Figure (Figure1),1), delayed processing of both CPY and PrA (Figure (Figure2),2), and normal processing of ALP (Figure (Figure2)2) at 30°C. Both arl1Δ and arl3Δ have vacuole morphology defects similar to those of arf1Δ (Figure (Figure3).3). Unlike Arf1p, however, Arl1p and Arl3p do not seem to be required for a normal polarized actin cytoskeleton (Figure (Figure4)4) or in α-factor secretion (Figure (Figure55).
These observations place Arl1p and Arl3p in the CPY pathway of biosynthetic sorting to the vacuole. Like Arf1p, Arl1p and Arl3p would be expected to exert their functions through GTP-dependent binding to effector proteins. Human Arl1 has been shown to bind to various putative effector proteins, including one termed SCOCO (for short coiled-coil) (Van Valkenburgh et al., 2001 blue right-pointing triangle). This protein has two S. cerevisiae homologs, Vps30p and Imh1p, both of which were also identified in our screen (Tables (Tables11 and and22 and Supplemental Tables 1 and 2).
Monensin and Brefeldin A Hypersensitivity Genes
Two genes whose deletions gave strong-moderate phenotypes, MON1 and MON2, and two genes whose deletions gave weak phenotypes, BRE5 and ERG4, identified in our screen had been previously found in a screen for mutant strains hypersensitive to the drugs monensin and/or brefeldin A (Muren et al., 2001 blue right-pointing triangle). We observed that both MON1- and MON2-deletion strains secreted CPY (Figure (Figure1).1). Proteolytic processing of CPY and PrA was completely blocked in mon1, as in the strongest of the vps mutants (Figure (Figure2).2). Processing of ALP was partially blocked in mon1 (Figure (Figure2).2). Processing of all three vacuolar enzymes was also impaired in the mon2 strain, but the pattern differed from that of the mon1 strain in that processing of CPY and PrA was less affected than that of ALP (Figure (Figure2).2). Interestingly, the block in ALP processing in mon2 was similar to that observed in vps39 and the AP-3 mutants (Figure (Figure22).
Consistent with defects in Golgi-to-vacuole traffic, both mon1 and mon2 have fragmented vacuoles (Figure (Figure3).3). However, the vacuoles in mon1 are smaller and tend to remain clustered together (Figure (Figure3),3), whereas those of mon2 are slightly larger but more dispersed (Figure (Figure3).3). The distinct vacuolar phenotypes of these mutants may reflect their preferential function in the CPY and ALP pathways, respectively. Neither Mon1p nor Mon2p is required for secretion of α-factor (Figure (Figure55).
A noteworthy feature of Mon2p is a 115-amino acid segment of homology (26% amino acid sequence identity) to Sec7p, a guanine nucleotide exchange factor (GEF) for Arf (Peyroche et al., 1996 blue right-pointing triangle). The homologous segment, however, does not overlap with the so-called Sec7 domain, which has intrinsic Arf GEF activity (Chardin et al., 1996 blue right-pointing triangle).
Actin-related Proteins
Another intriguing finding was the identification in our screen of 10 genes (ANP1, AOR1, ARP5, ARP6, CAX4, HOF1, MDM20, PSL10, SAC3, and TPM1) encoding proteins that are structurally or functionally related to actin (Table (Table11 and Supplementary Table 1). Arp5p and Arp6p are two of 10 actin-related proteins identified in S. cerevisiae (Schafer and Schroer, 1999 blue right-pointing triangle). In addition to secreting CPY (Figure (Figure1),1), arp5 and arp6 mutant strains displayed delayed processing of CPY and PrA (arp5 more than arp6), but not ALP (Figure (Figure2),2), placing them in the CPY pathway. Both arp5 and arp6 have similar defects in vacuolar morphology, often displaying a large vacuole lobe surrounded by much smaller lobes (Figure (Figure3).3). Furthermore, both mutant strains were defective in secretion of mature α-factor (Figure (Figure5),5), defects that seem to stem from a delay in α-factor processing indicated by the accumulation of pro-α-factor and α-factor–processing intermediates in these strains (Figure (Figure5B;5B; our unpublished data). Neither Arp5 nor Arp6 has severe defects in the actin cytoskeleton structure at 30°C, although polarized actin cables in the mother cells may be slightly more disorganized than those observed in wild type (Figure (Figure4).4). Other ARPs are components of stable complexes with other proteins (Schafer and Schroer, 1999 blue right-pointing triangle). Arp2p and Arp3p, for example, are part of a complex that regulates the assembly of actin networks and also participates in endocytosis in S. cerevisiae (Moreau et al., 1997 blue right-pointing triangle). In mammalian cells, the Arp2/3 complex mediates propulsion of endosomes in the cytoplasm (Taunton et al., 2000 blue right-pointing triangle). It is thus tempting to speculate that Arp5p and Arp6p could play a role in transport to the vacuole analogous to that of Arp2p and Arp3p in endocytosis.
anp1, psl10, cax4, aor1, and hof1 displayed severe defects in the actin cytoskeleton with few or no polarized actin cables visible in the mother cells [Novick et al., 1989 blue right-pointing triangle; Kamei et al., 1998 blue right-pointing triangle; Sekiya-Kawasaki et al., 1998 blue right-pointing triangle; our unpublished data), similar to those observed for tpm1 and mdm20 (Liu and Bretscher, 1989 blue right-pointing triangle; Hermann et al., 1997 blue right-pointing triangle; Figure Figure4).4). All of these mutants also exhibited defects in secretion of mature α-factor (Figure (Figure5).5). cax4, which, like arp5, secretes very little mature α-factor, also accumulates a large amount of α-factor–processing intermediates (Figure (Figure5B).5B). In fact, all the actin mutants analyzed accumulate some of these intermediates, suggesting that the integrity of the actin cytoskeleton may be important for recycling of Kex2 from the prevacuolar compartment back to the Golgi.
The actin cytoskeleton is known to mediate vacuole movements in the process of mitotic vacuolar inheritance (Catlett and Weisman, 2000 blue right-pointing triangle). Thus, it is possible that some of the actin-related proteins identified in our screen could participate in the translocation of vesicular intermediates on their way to the vacuole. This role could be analogous to the well-established function of the actin cytoskeleton in endocytosis in yeast (Munn, 2001 blue right-pointing triangle). It is also possible that some of the genes in this group could be more directly involved in vesicle tethering or fusion events and only indirectly involved in association with the actin cytoskeleton. A case in point is the product of the VPS52 gene, a component of the Vps52p-Vps53p-Vps54p complex involved in tethering endosome-derived vesicles to the Golgi apparatus (Conibear and Stevens, 2000 blue right-pointing triangle; Siniossoglou and Pelham, 2001 blue right-pointing triangle), which was first identified as a suppressor of actin mutations, Sac2p (Novick et al., 1989 blue right-pointing triangle). Interestingly, sac2 also displays similar defects in the actin cytoskeleton as the other mutants, exhibiting a decrease in the number of polarized actin cables visible in the mother cells (Novick et al., 1989 blue right-pointing triangle; our unpublished data).
Ribosomal Proteins
Five strong-moderate genes (Table (Table11 and Supplemental Table 1) and 16 weak genes (Table (Table22 and Supplemental Table 2) encoded components of both the large and small ribosome subunits. One intriguing explanation for the secretion of CPY by these mutants would be the existence of a regulatory network that reduces vacuolar protein sorting when translation is impaired. In this regard, RIC1 was initially identified in a screen for mutations that decrease ribosome synthesis (Mizuta et al., 1997 blue right-pointing triangle), but was later found to encode a protein that, together with the product of RGP1, functions as a GEF for Ypt6p (Siniossoglou et al., 2000 blue right-pointing triangle; Bensen et al., 2001 blue right-pointing triangle) (both RIC1 and RGP1 were identified in our screen; Table Table11 and Supplemental Table 1). We cannot rule out, however, that decreased translation of one or more key components of the vacuolar protein sorting machinery might underlie the CPY sorting defects observed in ribosomal proteins mutants.
Miscellaneous Proteins
This group includes the products of 35 genes whose deletions resulted in strong-moderate phenotypes but that were previously identified in screens other than for vacuolar protein sorting (Table (Table11 and Supplemental Table 1). A subgroup of these genes has been implicated in Golgi function. These include GYP1, PMR1, BRO1, DOR1, COD2, COD3, COD4, and COD5. The products of five of these genes (DOR1, COD2, COD3, COD4, and COD5) have recently been shown to be components of an eight-subunit complex with Sec34p and Sec35p (Whyte and Munro, 2001a blue right-pointing triangle) referred to as the Sec34/35 complex (Kim et al., 1999 blue right-pointing triangle; VanRheenen et al., 1999 blue right-pointing triangle). This complex has been proposed to mediate vesicle tethering to the Golgi complex based on the weak homology of some of its subunits to subunits of the Vps52p-Vps53p-Vps54p (Conibear and Stevens, 2000 blue right-pointing triangle; Siniossoglou and Pelham, 2001 blue right-pointing triangle) and exocyst (TerBush et al., 1996 blue right-pointing triangle) complexes (Whyte and Munro, 2001a blue right-pointing triangle). Deletion of genes encoding some of the subunits of the Sec34/35 complex has been shown to result in abnormal accumulation of intracellular membranes and glycosylation defects (Whyte and Munro, 2001a blue right-pointing triangle). Other observations suggest that the role of this complex may not be restricted to the Golgi complex. For example, the Golgi-plasma-membrane v-SNARE Snc1p became trapped in internal membranes in dor1 mutants (Whyte and Munro, 2001a blue right-pointing triangle). Moreover, a sec34 (also known as grd20) mutant strain exhibited mislocalization of Kex2p and secretion of CPY (Spelbrink and Nothwehr, 1999 blue right-pointing triangle).
We found that the DOR1-, COD2-, COD3-, COD4-, and COD5-deletion strains displayed secretion of CPY (Figure (Figure1)1) and delayed processing of CPY and PrA (Figure (Figure2).2). These observations are consistent with the notion that the Sec34/35 complex could play a role in late-Golgi or post-Golgi sorting events. However, only cod3 had a significant delay in ALP processing (Figure (Figure2).2). In addition, dor1, cod2, cod4, and cod5 had similar vacuolar morphologies, exhibiting fragmented vacuoles (Figure (Figure3),3), whereas the vacuoles of cod3 were smaller and often exhibited unusual tubular structures (Figure (Figure3).3). All of the mutants with the exception of cod2 displayed defects in α-factor secretion with cod3 having the most severe defect (Figure (Figure5).5). The observations that these mutants share only some of the phenotypes and have some distinct phenotypes is consistent with previous reports, and may suggest that some of the proteins have additional roles besides their function in the sec34/35 complex.
Hypothetical ORFs
Fifteen hypothetical ORFs were among the strong-moderate genes (Table (Table11 and Supplemental Table 1). For convenience, we designated these ORFs VPS61-VPS75. Many of these are small open reading frames (under 300 amino acids) and may have been missed in previous screens that depended on random mutagenesis, as the smaller target size of the genes lowered the probability of them being hit. Of these new putative ORFs, the deletion of two, VPS61 (YDR136C) and VPS67 (YKR020W), resulted in secretion of high levels of CPY (Figure (Figure1)1) and caused a significant block in the processing of CPY, PrA, and ALP (Figure (Figure2).2). Interestingly, vps67 has similar vacuolar morphology to cod3, exhibiting small fragmented vacuoles as well as some tubular structures (Figure (Figure3).3). VPS61 is a small ORF opposite of 5′-untranslated region of RGP1, which has previously been implicated in vacuole protein sorting. Thus, it is still unclear whether deletion of VPS61 is responsible for all or some of the phenotypes observed. However rgp1Δ did not display a defect in ALP processing (our unpublished data), suggesting that the lack of Vps61p may be responsible for this defect. Deletion of the other ORFs resulted in secretion of moderate levels of CPY (Figure (Figure1;1; and Table Table3)3) as well as defects in CPY and PrA processing that were less severe (Figure (Figure22 and Table Table3).3). In addition, many of them exhibited defects in α-factor secretion and/or processing (Figure (Figure5).5). vps67 and vps64 (deletion of YDR200C) displayed the most severe phenotype (Figure (Figure5),5), with little to no mature α-factor being secreted. Intriguingly, although vps67 accumulates α-factor–processing intermediates (Figure (Figure5B),5B), vps64, which has a more severe secretion defect, exhibits almost normal levels of mature α-factor and α-factor–processing intermediates compared with wild type. Although in many of the strains analyzed, aberrant Kex2p localization and recycling to the Golgi compartment may result in delayed processing of α-factor and thus indirectly cause an α-factor secretion defect, this is not always the case. Some of the proteins involved in vacuole protein sorting, such as Vps64p, may be directly involved in the secretion of mature α-factor as well.
Of the 15 new ORFs, seven displayed some homology to mammalian gene products. However, only Vps74p (product of YDR372C) exhibited homology to a known protein, having ~40% amino acid sequence identity to a mammalian protein termed GMx33 (Wu et al., 2000 blue right-pointing triangle) or GPP34/Golgi phosphoprotein 3 (Bell et al., 2001 blue right-pointing triangle).
Because several actin-related proteins were found to be important for CPY sorting in our screen, it seemed possible that some of the new ORFs could encode proteins involved in the organization or stability of the actin cytoskeleton. Thus, we examined the actin cytoskeleton through staining F-actin with Oregon green-phalloidin in all of the new open reading frame deletions, as well as some additional control strains (Figure (Figure4;4; our unpublished data). Of the new ORFs examined, only vps61, vps65 (deletion of YLR322W), and vps67 displayed defects in the actin cytoskeleton at 30°C. Interestingly, both vps36 and vps65 had a similar staining pattern, displaying a greatly reduced number of polarized actin cables as well as a large aggregate (indicated by arrowheads) in the mother cells (Figure (Figure4).4). Although it is still unclear whether this aberrant structure is associated with any cellular organelles or whether it is simply a cytosolic actin aggregate, these observations further confirm that the need to establish a normal actin cytoskeleton may be important in intracellular Golgi-to-vacuole trafficking. Further characterization of these proteins and their interaction with the actin cytoskeleton will help to clarify the role of actin in trafficking to the vacuole.
Concluding Remarks
Many more genes had been previously implicated in the CPY pathway than in the ALP pathway (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Lemmon and Traub, 2000 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). In fact, only the genes encoding the four subunits of AP-3 were known to be specifically involved in the ALP pathway (Cowles et al., 1997a blue right-pointing triangle; Stepp et al., 1997 blue right-pointing triangle). Other gene products that controlled the ALP pathway were shared with the CPY pathway. These included Vps39p and Vps41p, which play dual roles in the budding of AP-3–coated intermediates from the late-Golgi complex (Rehling et al., 1999 blue right-pointing triangle; Darsow et al., 2001 blue right-pointing triangle) and tethering/fusion of both CPY and ALP carriers to the vacuole (Price et al., 2000 blue right-pointing triangle; Wurmser et al., 2000 blue right-pointing triangle). They also included other proteins involved in vesicle formation in the late-Golgi complex and tethering to the vacuole (reviewed by Burd et al., 1998 blue right-pointing triangle; Conibear and Stevens, 1998 blue right-pointing triangle; Mullins and Bonifacino, 2001a blue right-pointing triangle). More than two-thirds of all known VPS genes, however, were specific to the CPY pathway.
The work reported herein resulted in the identification of many new genes involved in vacuolar protein sorting. The fact remains, however, that the majority of them controls trafficking along the CPY pathway (summarized in Table Table4).4). This could reflect a bias due to the use of CPY secretion as the assay for identification of the mutants. More likely, however, is that the overabundance of CPY pathway genes reflects the complex roles of the PVC in this pathway. The interposition of the PVC between the Golgi complex and the vacuole enables the recycling of cargo receptors such as the CPY and PrA receptor, Vps10p. In addition, the PVC allows for certain membrane proteins to be transported into the vacuole lumen through invagination of its limiting membrane (Odorizzi et al., 1998 blue right-pointing triangle; Katzmann et al., 2001 blue right-pointing triangle). This process is important for the delivery of some vacuole resident proteins (e.g., carboxypeptidase S) into the lumen and for the regulated turnover of proteins whose primary function is in other compartments (e.g., Kex2p).
Our studies did nonetheless identify several novel proteins involved in the ALP pathway. Mon2p was particularly interesting because its absence affected the ALP pathway more than the CPY pathway. The absence of Mon1p, Cod3p, Vps61p, and Vps67p also impaired both pathways, with the CPY pathway being more affected than the ALP pathway. These proteins could thus mediate either formation of both CPY and ALP carriers at the late-Golgi complex or tethering/fusion of Golgi-derived ALP carriers or PVC-derived CPY carriers to the vacuole. It is likely that screens specifically designed for analysis of ALP, Vam3p, or Nyv1p missorting could result in the identification of novel ALP pathway genes. Given that even AP-3 subunit mutants secrete some CPY, however, it is plausible that some ALP pathway genes may be represented among the “weak” genes identified in our screen (Table (Table22 and Supplemental Table 2).
We do not expect the list of genes identified in our study to represent the complete repertoire of vacuolar protein sorting genes, for the following reasons: 1) Some VPS genes might be essential and therefore would not be represented in the collection. For example, Sec18p is the product of an essential gene that plays a role in vesicle fusion in various pathways, including transport to the vacuole (Graham and Emr, 1991 blue right-pointing triangle). 2) Other VPS genes might be redundant, as is the case for those encoding Gga1p and Gga2p (Dell'Angelica et al., 2000 blue right-pointing triangle; Hirst et al., 2000 blue right-pointing triangle; Costaguta et al., 2001 blue right-pointing triangle; Zhdankina et al., 2001 blue right-pointing triangle). 3) Some knockout strains might cease secretion of CPY due to an adaptive or compensatory response, as is the case for clathrin mutants (Seeger and Payne, 1992 blue right-pointing triangle). 4) Genes <100 codons were not targeted for deletion. 5) Manifestation of the CPY secretion phenotype might be dependent on the genetic background of the parental strains. Most of the deletion mutants used herein were made in the BY4743 background, whereas most previously characterized vps mutants were derived from other strains (e.g., SEY6210, SEY6211, and SF839–1D). 6) Some genes may not have been deleted due to technical problems. For example, vps11 and vps15 mutants are not present in the collection. 7) Some genes could have been erroneously identified, or the mutants mishandled. Given the high accuracy with which VPS genes were identified in our screen, however, we suspect that the cases of mistaken identity are few.
Our studies support the conclusion that the vacuolar protein sorting machinery is highly conserved between yeast and higher eukaryotes. BLAST searches by using the strong and moderate genes as queries revealed that >50% of them have mammalian homologs. This percentage is considerably higher than the 31% of all yeast genes that have definite homologs among mammals (Botstein et al., 1997 blue right-pointing triangle). This confirms the presumption that studies of vacuolar protein sorting in yeast are particularly relevant to the understanding of lysosome biogenesis in mammals. The biochemical and functional characterization of the novel gene products identified in this study should considerably further our understanding of the molecular mechanisms that underlie the biogenesis of the yeast vacuole and mammalian lysosomes.
Supplementary Material
MBC Supplementary Material
ACKNOWLEDGMENTS
We thank Carol Woolford, Tom Stevens, and Todd Graham for generous gifts of reagents; Peter Rubenstein, Kuo-Kuang Wen, and Runa Musib for discussion of actin related genes and protocols; and Cathy Jackson and Chris Mullins for helpful discussions and critical review of the manuscript. C. Bonangelino was supported by Pharmacology Research Associates Training Award from the NIGMS.
Footnotes
D in BoxOnline versions of this article contain complete data sets.
Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.02–01–0005. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.02–01–0005.
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