Genetic Interaction With vps8-200 Allows Partial Suppression of the Vestigial Vacuole Phenotype Caused by a pep5 Mutation in Saccharomyces cerevisiae

Corresponding author: Carol A. Woolford, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, cw2g{at}andrew.cmu.edu (E-mail).

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS Disruption of the VPS8... DISCUSSION LITERATURE CITED

pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. In addition, they show a vestigial vacuole morphology and a sensitivity to growth on media containing excess divalent cations. This pleiotropic phenotype observed for pep5::TRP1 mutants is partially suppressed by the vps8-200 allele. pep5::TRP1 vps8-200 mutants show near wild-type levels of mature-sized soluble vacuolar hydrolases, growth on zinc-containing medium, and a more "wild-type" vacuolar morphology; however, aminopeptidase I and alkaline phosphatase accumulate as precursors. These data suggest that Pep5p is a bifunctional protein and that the TRP1 insertion does not eliminate function, but results in a shorter peptide that can interact with Vps8-200p, allowing for partial function. vps8 deletion/disruption mutants contain a single enlarged vacuole. This genetic interaction was unexpected, since Pep5p was thought to interact more directly with the vacuole, and Vps8p is thought to play a role in transport between the Golgi complex and the prevacuolar compartment. The data are consistent with Pep5p functioning both at the site of Vps8p function and more closely proximal to the vacuole. They also provide evidence that the three transport pathways to the vacuole either converge or share gene products at late step(s) in the pathway(s).

THE yeast vacuole is thought to be analogous to the mammalian lysosome in that it is an acidic compartment and contains a number of major hydrolases of the cell, including the soluble enzymes protease A (PrA), protease B (prB), carboxypeptidase Y (CpY), and the repressible integral membrane alkaline phosphatase (ALP; reviewed in JONES and MURDOCK 1994 ; JONES et al. 1997 ; VAN DEN HAZEL et al. 1996 ). The vacuole also serves as a repository for a number of small molecules, including amino acids, several divalent cations, and phosphate and polyphosphate (COOPER 1982 ; DUNN et al. 1994 ; OHSUMI and ANRAKU 1983 ; SERRANO 1991 ; URECH et al. 1978 ; WIEMKEN and DURR 1974 ; WIEMKEN et al. 1979 ). Most of the hydrolases are delivered to the vacuole as inactive precursors that are then processed to their active forms by vacuolar hydrolases (reviewed in JONES 1991 ; JONES and MURDOCK 1994 ; JONES et al. 1989 , JONES et al. 1997 ; VAN DEN HAZEL et al. 1996 ).

Proteins reach the vacuole in a variety of ways. A number of proteins, including precursors to PrA, PrB, and CpY, travel through the secretory pathway after co- or post-translational translocation into the endoplasmic reticulum (HANN and WALTER 1991 ; NG et al. 1996 ; STEVENS et al. 1982 ). In the lumen of the ER, proteins are modified by core glycosylation and then transported to the Golgi complex, where further modification occurs (STEVENS et al. 1982 ). In the late Golgi (trans-Golgi network), vacuole-bound proteins are sorted away from the secreted proteins and pass through an endosomal compartment en route to the vacuole (for review see JONES et al. 1997 ). The precursor to the vacuolar membrane protein ALP proceeds from the Golgi to the vacuole by a route that bypasses the endosomal compartment (BECHERER et al. 1996 ; BURD et al. 1997 ; HERMAN et al. 1991 ; PIPER et al. 1997 ; WEBB et al. 1997B ). A few enzymes reach the vacuole directly from the cytoplasm, via the cytoplasm-to-vacuole targeting (Cvt) pathway, including -mannosidase (YOSHIHISA and ANRAKU 1990 ) and the precursor to aminopeptidase I (ApI; KLIONSKY et al. 1992 ), which is activated by PrB in the vacuole.

A variety of screens and selections have been used to identify mutants that show deficiencies in vacuolar peptidase activity (pep; JONES 1977 ) or that affect the vacuolar protein sorting pathway (vps; BANKAITIS et al. 1986 ; RAYMOND et al. 1992 ; ROBINSON et al. 1988 ; ROTHMAN et al. 1989 ; ROTHMAN and STEVENS 1986 ). More than 40 complementation groups have been defined, with extensive genetic overlap between the pep and vps mutants. The products of these genes are required for normal transport of vacuolar hydrolases to the vacuole and/or formation of the vacuolar compartment.

In this report, we show a genetic interaction between mutations in two genes that are involved in protein transport to the vacuole. Mutations in these genes, PEP5 and VPS8, result in very different vacuolar morphologies—pep5 mutants have no discernible vacuole and vps8 mutants have a single enlarged vacuole. Yet, when a specific allele of VPS8, vps8-200, is in combination with the pep5::TRP1 insertion allele, one sees, by a variety of criteria, a restoration of vacuolar function. The VPS8 gene product has been reported to act between the Golgi and endosomal compartment, in docking/fusion at the endosome (HORAZDOVSKY et al. 1996 ), and in recycling from the endosome to the Golgi cisternae (CHEN and STEVENS 1996 ), much earlier in the pathway than Pep5p had been placed. Yet the suppression analysis presented here points to a much closer functional relationship than that implied by the morphology. ApI and ALP, however, accumulate as unprocessed precursors in pep5::TRP1 vps8-200 strains. This suggests that these proteins are not being delivered to the site of vacuolar function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS Disruption of the VPS8... DISCUSSION LITERATURE CITED

Materials:
Restriction enzymes were purchased from Boehringer Mannheim (Indianapolis, IN), New England Biolabs (Beverly, MA), or Promega (Madison, WI). Lyticase L-8012, ß-glucuronidase G-7770, and Ponceau S solution were obtained from Sigma (St. Louis). Vent_R DNA polymerase was purchased from New England Biolabs, and Taq DNA polymerase was purchased from Fisher Scientific (Pittsburgh, PA). Goat anti–rabbit IgG horseradish peroxidase conjugate was purchased from Bio-Rad (Richmond, CA). ³⁵S-dATP and the Rediprime random primer labeling kit were purchased from Amersham (Arlington Heights, IL).

Media and strains:
YEPD and synthetic media (JONES et al. 1982 ; ZUBENKO et al. 1982 ) were prepared for yeast cultures. LB medium (MANIATIS et al. 1982 ) was prepared for bacterial cultures. YEPD/0.3 M SrCl₂ medium was prepared by mixing ingredients for 1 liter of YEPD medium in 900 ml, autoclaving, and adding 100 ml of 3 M sterile SrCl₂ after cooling to 60°. YEPD/ZnCl₂ (5 mM) medium was prepared by adding the appropriate volume of a sterile 250 mM ZnCl₂, pH 4, solution to autoclaved and cooled (60°) YEPD medium.

All yeast strains in our laboratory were derived from strain X2180-1B (MAT gal2 SUC2), or from crosses between the strains in our isogenic series and strains congenic to strain X2180-1B, obtained from D. BOTSTEIN or P. HIETER. See Table 1 for strain genotypes. The original unsuppressed pep5::TRP1 disruption (BJ4394, described in WOOLFORD et al. 1990 ) was constructed by transplacing into BJ4334 a DNA fragment from plasmid BJ4325, which contains ~6.5 kb of DNA from the PEP5 locus with the upstream end starting at the SphI site 200 nucleotides 5' to the ATG and with an EcoRI fragment bearing the TRP1 gene inserted at the EcoRI site of PEP5, 1334 nucleotides downstream of the ATG (Figure 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. —Physical maps of the constructed plasmids. (A) Plasmids with PEP5 DNA. (B) Plasmids with VPS8 DNA. Relevant restriction sites are indicated (B, Bgl II; R, EcoRI; S, SphI; F, Afl II; A, AvaI; H, Hind III; P, Pst I) (see Table 1).

Nucleic acid and genetic manipulation:
The procedures used for routine sporulation, dissection, and scoring of nutritional markers have been described previously (HAWTHORNE and MORTIMER 1960 ). The pep5-associated CpY deficiency was scored by the APE overlay test (JONES 1977 ). pep5-associated growth sensitivity to divalent cations was scored on 5 mM Zn²⁺ plates or 300 mM Sr²⁺ plates. Yeast cells were transformed by a modified lithium acetate method (WOOLFORD et al. 1990 ) or by the lithium acetate/dimethyl sulfoxide procedure (HILL et al. 1991 ). Bacteria were transformed by using the CaCl₂ protocol (MANIATIS et al. 1982 ) or by electroporation using a Bio-Rad Gene Pulser apparatus as described by the manufacturer. Plasmid DNA minipreparations were made by the alkaline lysis method (SAMBROOK et al. 1989 ). Yeast genomic DNA was prepared by the method of HOFFMAN and WINSTON 1987 , as was plasmid DNA that was isolated from yeast to be shuttled into Escherichia coli.

The techniques for preparation and analysis of DNA fragments have been described previously (MANIATIS et al. 1982 ). After electrophoresis in 0.8% agarose gels run in TBE buffer, staining in ethidium bromide, and visualization by the use of UV light, gel slices containing DNA fragments were extracted using the Prep-A-Gene DNA purification Kit (Bio-Rad).

Isolation of an unlinked suppressor of a pep5::TRP1 mutation:
Strain BJ4394, bearing a pep5::TRP1 disruption allele, was started as a plate colony and used to inoculate 10 ml of YEPD for a preculture for growth experiments. Upon dilution for regrowth, the culture initially grew very slowly but eventually reached a normal stationary phase (OD₆₀₀ of ~50). When five colonies were tested after streaking, however, all were Trp⁺ Leu^- and MATa as expected, but three of the five were now Cpy⁺ (active carboxypeptidase Y present) rather than Cpy^-, an indication that they were no longer PrA and/or PrB deficient. DNA blot analysis confirmed that the PEP5 locus still carried the TRP1 insertion. To determine whether the suppressor of the Cpy^- phenotype was linked to the PEP5 locus, the suppressed strains BJ4490 and BJ4492 (MATa trp1 pep5::TRP1 leu2 Sup⁺) were crossed to BJ4343 (MAT his3 trp1 PEP5). The results of both crosses indicated that a suppressor mutation unlinked to pep5 was segregating. In seven tetrads from cross BJ4490 x BJ4343, the Trp phenotype segregated 2⁺:2^-. All Trp^- spores were Cpy⁺, and of the 14 Trp⁺ spores, six were Cpy^- and eight were Cpy⁺. Similar results were obtained from the second cross. In segregants from the control cross of BJ4343 to the parent insertion mutant, BJ4394, Trp segregated 2⁺:2^-, and all Trp⁺ spores were Cpy^-. Thus, strains BJ4490 and BJ4492 each contain a mutation unlinked to PEP5 that can suppress the Cpy^- phenotype of the pep5::TRP1 strain. Crosses were made to determine whether the suppressor mutation also suppressed the zinc sensitivity caused by the pep5::TRP1 allele [Pep⁺ strains grow well in the presence of 5 mM zinc, but all of the protease-deficient strains tested, including pep5 mutants, are unable to grow on this medium (WEBB et al. 1997A ; C. WOOLFORD, G. WEBB, A. SRIVASTAVA and M. HILLER, unpublished observations)]. In both crosses, all Cpy⁺ spore clones, whether Trp⁺ (pep5::TRP1) or Trp^- (PEP5), were Zn^R, indicating that the suppressor also suppressed the zinc sensitivity phenotype caused by the pep5::TRP1 allele (Table 2). It was determined that the suppressor mutation was recessive to its wild-type allele by constructing a pep5::TRP1/pep5::TRP1 sup/+ diploid (BJ4490 x BJ5304). The diploid does not show a suppressed phenotype; it is Zn²⁺- and Sr⁺-sensitive and Cpy^-. Complementation tests between suppressed strains from the BJ4490 lineage and the BJ4492 lineage gave Cpy⁺ and Zn^R diploids, indicating that the suppressors are allelic and probably siblings. As the suppressor mutation proved to be an allele of VPS8, we have named it vps8-200.

The DNA sequences at the pep5/TRP1 junctions were determined to rule out the possibility of translational suppression. The upstream junction coded for three novel amino acids (Ser, Thr, and Cys) before encountering a stop codon (TRP1 was inserted in opposite orientation to PEP5). There were no Met codons in the noncoding strand of the TRP1 sequence in frame to PEP5 at the downstream junction.

DNA sequencing:
DNA sequence was determined by the dideoxy chain termination method using Sequenase according to the manufacturer's (United States Biochemical, Cleveland, OH) instructions. Sequencing gels were made and run using the Long Ranger Gel System (FMC BioProducts, Rockland, ME) or Sequagel-6 (National Diagnostics, Atlanta, GA) according to their supplied protocols.

DNA used for double-stranded sequencing was prepared either by Prep-A-Gene purification of plasmid DNA minipreps or using the Wizard Plus Miniprep DNA Purification System (Promega). The double-stranded DNA was then alkali denatured as described by KRAFT et al. 1988 , modified by the addition of 76% volume 2 N NaOH, 2 mM EDTA.

To define the end points of the DNA insert on the plasmid, sequencing primers that match the sequence of the tetracycline resistance gene near the BamHI site found in YCp50 (KUO and CAMPBELL 1983 ) and other derivatives of pBR322 (BOLIVAR et al. 1977 ) were used.

Plasmid construction:
BJ4324 (Figure 1) has the TRP1 ORF oriented in the same direction as the PEP5 ORF. BJ4325, constructed as was BJ4324, contains the TRP1 ORF in the opposing direction to the PEP5 ORF (see Media and strains ; Figure 1). BJ3767 contains a 4-kb HindIII/AvaI fragment bearing sequence from upstream of the PEP5 ORF to the first AvaI site of PEP5, inserted into the HindIII/SalI sites of YCp50. This construct has 2139 of the 3089 nucleotides of the PEP5 ORF, and it extends 805 nucleotides beyond the site (EcoRI) of the TRP1 insertion in BJ4324 and BJ4325. Plasmid constructs BJ8722 and BJ8723 are the BJ4324 and BJ4325 pep5::TRP1 alleles in yeast shuttle vectors, respectively. First, BJ7877, which has a Bgl II/HindIII fragment of the PEP5 region inserted into the BamHI/HindIII sites of pRS316 (SIKORSKI and HIETER 1989 ) was constructed (Figure 1). This plasmid was gapped in the PEP5 gene by digesting with Afl II. The SphI pep5::TRP1 bearing fragments of BJ4324 and BJ4325 were then each cotransformed with gapped BJ7877 into the yeast strain BJ5305, and gap-repaired Ura⁺ transformants were selected (MA et al. 1987 ). Plasmids were shuttled into E. coli and then checked by restriction digest for the expected size fragments. The integrative plasmid BJ8605 (Figure 1) contains the Pst I fragment from nucleotides -119 to +2759 of the VPS8 ORF cloned into the Pst I site of pRS305 (SIKORSKI and HIETER 1989 ). The orientation of the VPS8 ORF is from the polylinker Hind III site toward the polylinker BamHI site. The VPS8 disruption plasmid BJ8717 was constructed with the use of oligonucleotide-directed mutagenesis according to the method of KUNKEL et al. 1987 . A Sal I site was engineered at nucleotide 2607 of the VPS8 ORF in the plasmid BJ8659 (this plasmid has the same insert as BJ8605, but is in the CEN bearing plasmid pRS316; see Figure 1). A Bgl II/Sal I fragment was dropped out of VPS8 and replaced with the LEU 2 gene on a BamHI/Sal I fragment obtained from pJJ250 (JONES and PRAKASH 1990 ). Nucleotides 1540–2613 of the VPS8 ORF were deleted. This plasmid, BJ8717, was digested within the polylinker with HindIII and BamHI to direct transplacement of the vps8::LEU2-bearing fragment into the VPS8 locus in the diploid BJ6280 x BJ6281. The disruption was confirmed by Southern blot analysis on individual transformed diploids.

Yeast extracts and buoyant density preparation of vacuolar pathway components:
Yeast extracts were prepared by a Braun homogenizer as described previously (WOOLFORD et al. 1990 ). Buoyant density "floats" were prepared as described previously for vacuole preparations (WOOLFORD et al. 1990 ) using strains BJ1983 (PEP5 VPS8), BJ4394 (pep5::TRP1 VPS8), and BJ4490 (pep5::TRP1 vps8-200) with modifications. Protease inhibitors (2 mM PMSF, 0.1 mM pepstatin A) were included before and after each homogenization step. Because of the unknown buoyant characteristics of vestigial vacuoles and whatever might be present in the suppressed strain, we decided to collect the floats after longer centrifugation times. The initial float was collected after a 1-hr centrifugation, and the second float was collected after a 2-hr centrifugation.

Immunoblots:
Immunoblots were prepared as described previously (WOOLFORD et al. 1990 ).

Electron microscopy:
Cells subjected to electron microscopy were processed as described in WEBB et al. 1997B . Briefly, cells were grown in YEPD to an OD₆₀₀ of ~0.5. Cells were fixed for 2 hr and then washed. The cells were resuspended in the presence of ß-glucuronidase and lyticase, and were incubated for 2 hr to allow cell wall removal. After washing, cells were embedded, stained, and viewed as described previously described (WEBB et al. 1997B ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS Disruption of the VPS8... DISCUSSION LITERATURE CITED

Cloning of the suppressor gene:
As described in MATERIALS AND METHODS, a mutation that suppressed and segregated independently of a pep5::TRP1 allele was identified. To obtain the wild-type allele of the suppressing gene, a YCp50LEU2 bank was transformed into a pep5::TRP1 sup leu2 strain (BJ4492). Approximately 18,000 Leu⁺ transformants were screened for the loss of the Cpy⁺ phenotype. 127 transformants were rescreened, looking for the restoration of the Cpy⁺ phenotype upon loss of the Leu⁺ plasmid. Only one transformant showed a Cpy phenotypic change that correlated with the presence or absence of the library plasmid.

Partial sequence from both ends of the insert was obtained. Comparison of the sequences obtained with the sequence of the yeast genome using the BLAST program indicated that the DNA insert was from a single continuous chromosomal region. The Saccharomyces Genome Database revealed this to be a stretch of chromosome I that included only three ORFs. The insert contained part of the TEF5 ORF, all of the VPS8 ORF, and part of the TFC3 ORF. Since the VPS8 ORF was the only one present in its entirety, we focused our attention on it. This ORF was originally identified by Y. J. CHEN and T. H. STEVENS (GenBank/EMBL/DDBJ accession number U44026) as the one encoded by VPS8, a vacuolar protein sorting gene important for protein localization of the CpY receptor.

To determine if the VPS8 ORF originated from the sup chromosome region, the integrating plasmid BJ8605, which carries a 3' truncation of the VPS8 ORF (to be noted as VPS8 *; see MATERIALS AND METHODS), was linearized at the SnaBI site internal to the ORF at nucleotide 1659 to direct integration to the homologous region in a pep5::TRP1 suppressed strain (BJ4492). Depending on the site of the mutation in the suppressing allele, either a wild-type (Cpy^-, nonsuppressing) or a mutant (Cpy⁺, suppressing) allele would be reconstituted (Figure 2). If VPS8 corresponds to the sup locus and if the suppressing mutation is located upstream of the SnaBI site, the VPS8 *-bearing plasmid, when integrated into the genome, would result in one allele of VPS8 being truncated and containing the suppressor mutation, and the other allele of VPS8 being a functional wild-type allele. Integrants with this VPS8 genotype would show a nonsuppressing (Trp⁺ Cpy^-) phenotype. If the suppressing mutation is located downstream of the SnaBI site, the VPS8 *-bearing plasmid, when integrated into the genome, would result in one allele of VPS8 being truncated and the other functional allele of VPS8 carrying the suppressing mutation. Integrants with this genotype would show a suppressing (Trp⁺ Cpy⁺) phenotype. If the VPS8 locus is not the site of the suppressing mutation, but is rather a "suppressor of the suppressor," suppression of the suppressor must be a consequence of increased (2x presumably) dosage of the cloned gene. Because the plasmid to be integrated bears a truncated ORF, integration will result in one intact ORF and one truncated ORF in the chromosome. In other words, any possibility of dosage suppression of the suppressor is eliminated. The resultant integrant should have the same phenotype as the parent strain used as the transformation recipient, namely Trp⁺ Cpy⁺. Nine out of 10 integrants were Trp⁺ Cpy^-, indicating that the cloned gene is the wild-type allele corresponding to the suppressor mutation. In confirmation of this inference, in 28 tetrads from a cross between a Trp⁺ Cpy^- integrant and a PEP5 SUP trp1 leu2 strain (BJ5405), all Trp⁺ spores were Cpy^- as expected, since the LEU2 gene, the truncated VPS8 * allele bearing the suppressor mutation, and the wild-type VPS8 gene are all tightly linked in the integrant. Thus, the mutation that suppresses the pep5::TRP1 allele is a VPS8 allele that we have named vps8-200.

View larger version (26K): [in this window] [in a new window]   Figure 2. —Integration of a truncated VPS8. A Pst I fragment lacking the 3' end of VPS8 is cloned into the integrating vector pRS305. Linearization at the SnaBI site (dashed line in the plasmid) to direct homologous recombination in a pep5::TRP1 sup strain to the VPS8 locus may result in the loss or presence of the suppressed phenotype, depending on the location in VPS8 of the suppressing mutation.

	Disruption of the VPS8 gene

TOP ABSTRACT MATERIALS AND METHODS RESULTS Disruption of the VPS8... DISCUSSION LITERATURE CITED

To determine whether a deletion mutation will suppress, we constructed vps8:: LEU2, in which a BamHI/Sal I fragment carrying the LEU2 gene replaced deleted nucleotides 1540–2613 of the ORF (30% of the VPS8 ORF was deleted). This DNA was used to transform the diploid BJ6280/BJ6281 to Leu⁺. Upon meiosis, all 10 four-spored tetrads segregated 2:2 for Leu⁺ Cpy^-:Leu^- Cpy⁺. A PEP5 vps8:: LEU2 spore was crossed to BJ4490 (pep5::TRP1 vps8-200), and the diploid was sporulated and dissected. None of the doubly mutant pep5::TRP1vps8::LEU2 spores showed a suppressed phenotype (11/11 spores were Cpy^- Zn^S Sr^S). Thus, the deletion/disruption allele of VPS8 cannot suppress the pep5 mutant phenotype (Figure 3A, streak 7).

View larger version (41K): [in this window] [in a new window]   Figure 3. —Phenotypes of pep5 vps8 strains. (A) The Cpy, temperature sensitivity, zinc, and strontium growth phenotypes were tested for the various double mutants. Plates were incubated for 3 days. Strains: (1) PEP5 VPS8 (BJ8866); (2) pep5 VPS8 (BJ5304); (3) PEP5 vps8 (BJ8868); (4) pep5::TRP1 VPS8 (BJ6919); (5) PEP5 vps8-200 (BJ8987); (6) pep5::TRP1 vps8-200 (BJ4490); (7) pep5::TRP1 vps8 (BJ8981); (8) pep5 vps8 (BJ9010). (B) Strains harboring the pep5::TRP1 allele on a CEN plasmid were tested for suppression of the zinc-sensitive, Cpy- phenotype. Strains were streaked onto 5 mM zinc plates to test for growth, or onto YEPD plates to determine their Cpy phenotypes. Chromosomal genotypes are: (1) pep5::TRP1 VPS8 (BJ7964); (2) PEP5 VPS8 (BJ4343); (3) pep5::TRP1 vps8-200 (BJ8757); (4) pep5::TRP1 vps8-200 with plasmid BJ8722 (pep5::TRP1 with TRP1 in the same transcriptional orientation as PEP5); (5) pep5::TRP1 vps8-200 with plasmid BJ8723 (pep5::TRP1 with TRP1 in the transcriptional orientation opposite to PEP5); (6) pep5::TRP1 VPS8 with plasmid BJ8723; (7) pep5::TRP1 VPS8 with plasmid BJ8722.

Two different pep5::TRP1 alleles are suppressible:
To facilitate identification of other suppressible alleles, we determined whether pep5::TRP1 was suppressible when plasmid borne rather than chromosomal. Figure 3B shows that plasmid BJ8723 (pep5::TRP1; TRP1 in opposite transcriptional orientation to PEP5), when introduced into a pep5:TRP1 vps8-200 strain (streak 5), but not when introduced into a pep5::TRP1 VPS8 strain (streak 6), resulted in the suppressed Cpy⁺ Zn^R phenotype. Reversal of the orientation of TRP1 within the PEP5 gene (plasmid BJ8722) still resulted in a suppressible pep5 insertion allele (Figure 3B, streak 4).

Restoration of vacuolar hydrolase production by the suppressor is pathway specific:
Mutations in the PEP5 gene are pleiotropic and lead to reduced levels of the vacuolar hydrolases PrA, PrB, and CpY. The appearance of a Cpy⁺ phenotype in the suppressed mutant led us to look at antigen levels for the vacuolar proteases. The pep5::TRP1 disruptant has lower intracellular levels of PrB and CpY antigen than a PEP5 strain, with most of the antigen being of the precursor size. It has been shown that most of the CpY is secreted as the precursor form in pep5 mutants (ROTHMAN et al. 1989 ). The presence of the vps8-200 allele restored nearly wild-type levels of mature-sized PrB antigen in whole-cell extracts. Levels of CpY were also increased, and a larger percentage of it was of mature size (Figure 4).

View larger version (61K): [in this window] [in a new window]   Figure 4. —Mature-sized PrB and CpY antigens are restored in the presence of the suppressor. Cell extracts from strains were prepared and subjected to immunoblot analysis (45 µg for PrB and 5 µg for CpY). The immunoblots were probed with polyclonal, affinity-purified antibodies to PrB and CpY.

To determine whether only the secretory pathway to the vacuole was affected in pep5 mutants, or whether the cytoplasm to vacuole targeting pathway was also affected, we looked at the processing state of ApI. HARDING et al. 1995 have shown that ApI is delivered to the vacuole independently of the secretory pathway and, once in the vacuole, undergoes a maturation process that depends on active PrB. Immunoblot analysis (Figure 5, upper blot) shows that wild-type, vps8, and vps8-200 strains (Figure 5, lanes 1, 2, and 6) all have mature-sized ApI. The pep5 disruption mutation precludes any production of ApI; antigen is present only in precursor form (Figure 5, lane 4). This phenotype is epistatic to the phenotype observed in vps8 or vps8-200 strains (Figure 5, lanes 3 and 5). That is, the vps8-200 allele does not suppress the lack of processing observed in the pep5::TRP1 strain.

View larger version (32K): [in this window] [in a new window]   Figure 5. —Hydrolases that are not processed in pep5 vps8 strains. (upper panel) Aminopeptidase I processing; (lower panel) alkaline phosphatase processing. Cell extracts from strains were prepared and subjected to immunoblot analysis (15 µg of protein, lanes 1–6, upper panel; 125 µg, lanes 1 and 6, lower panel; 50 µg, lanes 2–5, lower panel). Lanes: (1) PEP5 VPS8 (BJ8866 in upper panel, BJ9018 in lower panel); (2) PEP5 vps8 (BJ8868); (3) pep5::TRP1 vps8 (BJ8981); (4) pep5::TRP1 (BJ6919); (5) pep5::TRP1 vps8-200 (BJ4490); (6) PEP5 vps8-200 (BJ8987 in upper panel, BJ8983 in lower panel).

The precursor to ALP, a vacuolar membrane protein, is normally delivered to the vacuole via the Golgi complex (KLIONSKY and EMR 1989 ), where it is then activated by a proteolytic cleavage (JONES et al. 1982 ; KANEKO et al. 1987 ; KLIONSKY and EMR 1989 ). CHEN and STEVENS 1996 have shown that the processing of ALP is not affected in a vps8 strain (see also Figure 5, lower blot, lane 2). We wanted to determine whether the accumulation of precursor-sized ALP found in a pep5::TRP1 strain (Figure 5, lane 4) was altered in the presence of the vps8-200 allele, which restores protease activity. Only precursor ALP accumulated in the suppressed strain (Figure 5, lane 5).

To determine whether any biochemical entity resembling vacuoles or endosomal compartments were present in the pep5::TRP1 and pep5::TRP1 vps8-200 strains, we followed the procedure for isolating vacuoles from wild-type cells. Figure 6 shows that nearly wild-type levels of mature sized PrA, PrB, and CpY antigens are present in buoyant density floats of suppressed strains. In comparison, float fractions from the pep5::TRP1 strain had less total antigen, and more of it was in the precursor form compared to wild-type floats.

View larger version (71K): [in this window] [in a new window]   Figure 6. —Mature-sized proteases are found in the buoyant density floats of pep5::TRP1 vps8-200 strains. Buoyant density floats were prepared as described in MATERIALS AND METHODS. Equal amounts of protein from floats purified from each strain were subjected to immunoblot analysis (2.5 µg for Cpy, 5 µg for PrA, and 10 µg for PrB). The immunoblots were probed with polyclonal, affinity-purified antibodies to CpY, PrA, and PrB.

Because the protease content of the suppressed strain was similar to the wild-type, we determined whether the vacuolar membranes of the suppressed strain differed in any way from those of the parent disruption strain. We probed the float fractions with antibodies to Vph1p, the 95-kD integral membrane subunit of the vacuolar ATPase. The amount of Vph1p antigen in the suppressed strain was intermediate between that in wild-type and pep5::TRP1 mutant strains (Figure 7). [Because the lower stained band is a degradation product of Vph1p (KANE et al. 1992 ), the sums of the two bands should be compared for each lane.]

View larger version (32K): [in this window] [in a new window]   Figure 7. —Vph1p, a vacuolar integral membrane protein, is restored to nearly wild-type level in the buoyant density float of pep5::TRP1 vps8-200 strains. Buoyant density floats were prepared as described in MATERIALS AND METHODS. Samples (5 µg) of the floats from each strain were subjected to immunoblot analysis as described by MANOLSON et al. (1992).

Immunoblot analysis has shown that suppression of the pep5 mutant phenotype is incomplete. The soluble vacuolar hydrolases that travel to the vacuole via the endosome, namely PrA, PrB, and CpY, are present at near wild-type levels and are localized to a light membrane fraction. Similarly, Vph1p, an integral vacuolar membrane protein that also transits via the endosome, is restored to a nearly wild-type level in the suppressed strain and colocalizes to the same light membrane fraction as the soluble hydrolases. However, precursors to ApI, which reaches the vacuole directly from the cytoplasm, and ALP, which bypasses the endosome en route from the Golgi to the vacuole, do not get processed in the suppressed pep5 mutant strain.

Morphology of pep5 vps8 vacuoles:
pep5 mutants show a classic Type C vestigial vacuole morphology (BANTA et al. 1988 ; WOOLFORD et al. 1990 ). They lack any visibly recognizable vacuolar structure by electron microscopy (Figure 8B) although punctate densely staining material can be seen. A PEP5 strain carrying the vps8-200 allele has wild-type–looking vacuoles (Figure 8C), each cell having two to three densely staining bodies. The vacuoles in vps8 cells appear to be greatly enlarged and one to a cell (Figure 8D), similar to the class D phenotype (RAYMOND et al. 1992 ). In the double mutant, pep5::TRP1 vps8, the pep5 vestigial vacuole morphology is epistatic to the single enlarged vacuole phenotype of the vps8 mutant (Figure 8E); the double mutant lacks vacuoles. However, cells of genotype pep5::TRP1 vps8-200 (Figure 8F) show a restoration of some vacuolar-like, densely staining vesicles. Thus the vps8-200 mutation is able to suppress the vestigial vacuole morphology caused by the pep5::TRP1 mutation.

View larger version (154K): [in this window] [in a new window]   Figure 8. —Electron micrographs of yeast cells stained for vacuoles. Strains were prepared and stained for electron microscopy as described in MATERIALS AND METHODS. (A) PEP5 VPS8 (BJ8866); (B) pep5::TRP1 VPS8 (BJ6919); (C) PEP5 vps8-200 (BJ8987); (D) PEP5 vps8::LEU2 (BJ8868); (E) pep5::TRP1 vps8::LEU2 (BJ8981); (F) pep5::TRP1 vps8-200 (BJ4490).

Other aspects of the phenotypes:
ade2 mutants form red colonies resulting from the accumulation of a purine intermediate that is transported into the vacuole, probably as a glutathione-S conjugate (CHAUDHURI et al. 1997 ), where it forms a red, fluorescent pigment; pep5 ade2 mutants, however, are pale pink. pep5::TRP1 vps8-200 ade2 spore clones were intermediate in color between the red of PEP5 ade2 and the pale pink of pep5::TRP1 ade2 spores. The vps8-200 allele did not suppress the inability of pep5 mutants to grow using glycerol as a carbon source ( JONES 1983 ). As would be expected from this latter observation, homozygosity for vps8-200 did not allow pep5::TRP1 homozygotes to sporulate.

The vps8-200 mutation in a wild-type PEP5 background has no discernible phenotypic consequences: properties tested included sensitivity to divalent cations, thermo- or cold sensitivity, growth on glycerol as a carbon source, and CpY activity. The suppressor did not suppress any of the mutant phenotypes caused by a deletion disruption allele of PEP5. A plasmid (BJ3767) carrying part of the PEP5 gene (4 kb of upstream sequence and 69% of the ORF, extending 805 nucleotides beyond the point of the TRP1 insertion into the EcoRI site) had no effect on suppression. When this plasmid was in a strain deleted for PEP5 but carrying the vps8-200 allele, no suppression was observed. When this plasmid was in the pep5::TRP1 vps8-200 strain, the strain was still suppressed. So the presence of the Pep5p truncation did not interfere with suppression. We suspect that the peptide encoded by the pep5::TRP1 disruption is fortuitously stable and therefore has function, or can be stabilized to have function, in the presence of the suppressor mutation, but that we have not been able to molecularly create any other stable peptide.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS Disruption of the VPS8... DISCUSSION LITERATURE CITED

The pep5 mutant was originally isolated as being unable to catalyze cleavage of acetylphenylalanine ß-napthyl ester, an indication of a decrease in CpY activity, and it was also shown to have decreased levels of the other soluble vacuolar hydrolases, PrA and PrB (JONES 1977 ; JONES 1983 ). Since then, the biological effects of a mutation in PEP5 have been shown to be far reaching. They include perturbation of copper and iron homeostasis (AMILLET et al. 1996 ; SZCZYPKA et al. 1997), sensitivity to amino acid analogs (JONES 1983 ), low amino acid pools (JONES 1983 ), sensitivity to divalent cations, including Ca²⁺, Zn²⁺, and Sr²⁺ (HILLER 1997 ), an inability to use glycerol as a carbon source at high temperatures (JONES 1983 ), and a sporulation deficiency (JONES 1983 ).

We had previously reported Pep5p to be enriched in vacuole preparations (WOOLFORD et al. 1990 ), which we now know are almost certainly enriched in endosomal compartments also (WEBB et al. 1997B ). Specifically, Pep5p was found to be a peripheral membrane protein found in buoyant density "floats." This finding, in conjunction with the observation that electron microscopic examination of a pep5 mutant did not reveal the presence of any normal vacuolar morphology, and that abnormally small, indeed tiny vesicles accumulate the vacuole-specific dyes lucifer yellow and dichlorocarboxyfluorescein (PRESTON et al. 1992 ; WOOLFORD et al. 1990 ), led us to hypothesize that PEP5 encodes a protein whose function is directly required for vacuole biogenesis and the maintenance of the vacuole structure, and probably acts in association with the vacuole membrane. The genetic evidence we present here forces us to reconsider this hypothesis.

Although predicted to be a hydrophilic protein, cell fractionation studies indicated that Vps8p associated with both P13 and P200 membrane fractions, with the majority of it in the P200 (CHEN and STEVENS 1996 ; HORAZDOVSKY et al. 1996 ). It appears to be peripherally associated with membranes. CHEN and STEVENS 1996 showed that vps8 mutants missort and secrete precursors to CpY as well as PrA. They also showed that the CpY sorting receptor, Vps10p, was mislocalized and proteolyzed in the vacuole in the vps8 mutant. They classified the vps8 mutant as having a class A, normal-looking vacuole, and concluded that Vps8p functions in the retrieval of Golgi membrane proteins from the prevacuolar compartment (endosome).

HORAZDOVKSY et al. (1996) found that Vps8p functionally interacts with Vps21p, a member of the Rab5/Ypt1/Sec4 family of small GTPases. They found that although the precursors to the soluble hydrolases CpY and PrA were missorted, the precursor to alkaline phosphatase, an integral vacuolar membrane protein, was sorted and processed normally. They classified vps8 as being a class D mutant, having a single, enlarged vacuole, and concluded that Vps8p plays a role in the transport of soluble vacuole proteins from the Golgi to the prevacuolar endosome.

The work presented in this study provides genetic evidence that these two proteins, Pep5p and Vps8p, may interact. We demonstrated that the vps8-200 allele alleviated several features of the phenotype caused by the pep5::TRP1 allele. Nearly wild-type levels of CpY are present in whole-cell extracts. Soluble hydrolases were recovered in buoyant density "floats" (vacuole-like light membrane compartments) at nearly wild-type levels, although some PrB precursor was present. In addition, the presence of vps8-200 restored levels of Vph1p, a vacuolar integral membrane protein, from barely detectable levels in "floats" prepared from the pep5::TRP1 mutant to at least 50% of wild-type in "floats" from the pep5::TRP1 vps8-200 suppressed strain. Enhanced ability to grow on Zn²⁺ or Sr²⁺-containing media and increased accumulation of red pigment in the ade2 pep5::TRP1 vps8-200 strains also indicated restored vacuole function.

The vps8-200 allele, although itself without effect on vacuolar morphology, resulted in the restoration of moderately large, darkly staining, vacuole-like structures through suppression of the pep5::TRP1 allele. Large, unstained entities reminiscent of objects present in the cytoplasm of cells undergoing autophagy (TAKESHIGE et al. 1992 ) are also present in the suppressed mutant; we do not know what these entities are.

Taken together, these findings suggest that Pep5p may function together with Vps8p in the Golgi-to-endosome step in the vacuolar pathway, and that this function is operative in the pep5::TRP1 vps8-200 strain. If the class D vacuolar morphology of the vps8 mutant (HORAZDOVSKY et al. 1996 ; this work) proves a reliable indicator, then this function of Pep5p is more likely to be related to consumption of transport vesicles at the endosome than to generation of vesicles at the Golgi (BECHERER et al. 1996 ; COWLES et al. 1997 ; PIPER et al. 1994 ; WEBB et al. 1997B ).

The pep5 mutant proved to be defective in maturation of the ALP and ApI precursors, indicating either that the Golgi-to-vacuole pathway that skirts the endosome (ALP) and the cytoplasm-to-vacuole pathway (ApI) are both defective in this mutant, or that the levels of processing proteases are too low to catalyze much conversion. Precursors to both hydrolases are matured properly in the vps8 mutant, indicating that, as expected, Vps8p is not required for these pathways. Interestingly, the suppressed pep5::TRP1 vps8-200 strain remains unable to properly proteolytically process the ApI and ALP precursors, despite the fact that the suppressed strain has nearly normal levels of the processing proteases PrA and PrB. Since we know from other studies that the ALP precursor is a good substrate for the maturation proteases (BECHERER 1991 ; GARLOW 1989 ), we infer that neither the ALP nor the ApI precursor is present in the same compartment as PrA and PrB and, by extension, CpY. One possibility is that the ApI and ALP precursors are not localized to and/or translocated into the "suppressed" vacuoles of pep5:: TRP1 vps8-200 strains. A second possibility is that the "suppressed" vacuoles are not vacuoles, but rather, enlarged endosomes akin to those found in class E mutants such as vps27 and vps28 (PIPER et al. 1995 ; RIEDER et al. 1996 ). In these mutants, the enlarged endosome becomes acidified, and precursors to soluble hydrolases mature within the endosome (PIPER et al. 1995 ). ALP is not present in enlarged endosomes in these class E mutants (RAYMOND et al. 1992 ). It seems unlikely that ApI would be present in endosomes either, since the endosome is not on the cytoplasm-to-vacuole pathway.

There are several implications of the failure of the suppressed strain to process ApI and ALP to their mature forms. The most obvious is that the pathway for delivery of soluble hydrolase precursors to the vacuole via the endosome converges not only with the endocytic pathway, but also with the ALP delivery pathway and with the cytoplasm-to-vacuole pathway (presumably at a step near the vacuole). A second implication, however, is that Pep5p function is required for a step in this common pathway at the point of or after convergence in addition to its role in Golgi-to-endosome trafficking. (The alternative explanation, that Pep5p acts late in three parallel pathways, cannot be excluded, but seems unlikely.) The final implication is that the C-terminal half of Pep5p is required for this common step in the convergent pathway.

The fact that the pep5::TRP1 alleles are suppressible but the deletion/disruption allele is not indicates that vps8-200 is not a bypass suppressor. It also strongly suggests that the pep5::TRP1 alleles are not null mutations. Pep5::Trp1p must supply partial wild-type function in combination with Vps8-200p. The simplest explanation is that Pep5p is a bifunctional protein, and that the insertion of the TRP1-containing fragment, in either orientation in the EcoRI site, somehow results in a truncated peptide that is stable in the presence of Vps8-200p. We have attempted to generate a similar, possibly stable, peptide with other constructs (for instance, by EcoRI digestion of PEP5 and then filling in the ends and religation, which creates an out-of-frame downstream sequence), and we have been unsuccessful; the "fill-in" allele is not suppressible. We infer that the altered Vps8-200p must be able to interact with and stabilize the particular truncated peptides produced from the two insertion alleles.

The pep and vps mutants have been placed into six classes, based mainly on vacuolar morphological analysis (RAYMOND et al. 1992 ). Studies to date suggest that the severity of the protein-sorting defect correlates with the morphological phenotype. In addition, it has been found that mutants within a class that share phenotypes may also be defective in the same part of the secretory pathway; interactions among mutations within a class have been observed (BURD et al. 1997 ; HORAZDOVSKY et al. 1996 ; NAKAMURA et al. 1997 ; STACK et al. 1993 ; WEBB et al. 1997A , WEBB et al. 1997B ). Thus, often the function of proteins defined by mutations within a particular class (based on morphology) are hypothesized to function in the same steps and/or protein complexes. In this paper, we present evidence for the interaction of mutations in two genes that when completely nonfunctional, result in very disparate phenotypes—PEP5, whose loss results in a class C vestigial vacuole null phenotype; and VPS8, whose loss results in a class D enlarged vacuole null phenotype.

Our favored hypothesis to account for the observations is that Pep5p is bifunctional and contributes one function at the Golgi-to-endosome step, where it interacts with Vps8p, and a second function at the endosome-to-vacuole step (Figure 9). In the suppressed strain, function is restored only for the Golgi-to-endosome step, and what is seen as a "vacuole" is an enlarged endosome in which Vph1p accumulates and in which hydrolase maturation occurs. The ApI and ALP precursors are not delivered into this endosomal compartment since it is not on their delivery pathways and, hence, they remain unprocessed. The suppressed strain would then share features with the class E mutants that contain an enlarged endosome that contains Vph1p (RAYMOND et al. 1992 ), but would differ from class E mutants in not having a vacuole as well.

View larger version (30K): [in this window] [in a new window]   Figure 9. —Proposed sites of function of Pep5p. The observations of pep5::TRP1 and vps8-200 genetic interaction, as well as the lack of processing of ApI and ALP in pep5::TRP1 vps8-200 strains can be explained if Pep5p functions in two places. Pep5p and Vps8p interact at the Golgi-to-endosome step along the Golgi–endosome–vacuole path that the soluble vacuolar hydrolases and Vph1p travel. ProALP and proApI transit separate vesicle-dependent pathways to the vacuole. The three paths converge at or near the vacuole. Pep5p is required at or after this point of convergence.

These findings make clear that the intracellular location of Pep5p merits reexamination. Our initial inference that Pep5p acted at the vacuolar membrane because it was enriched in vacuole preparations may reflect association with the vacuolar membrane. Alternatively, it may reflect association with another component, such as endosomes or transport vesicles, that cofractionates with vacuolar membranes in light membrane fractions or, association with more than one membrane compartment if indeed Pep5p proves to act in more than one reaction. Of obvious interest will be the distribution of Vps8p in relation to the Pep5p distribution. Determination of these distributions, together with biochemical analysis of the Pep5p and Vps8p interaction, and the analysis of temperature-sensitive mutants, should allow us to discern more definitively the role(s) and relationship of these two proteins.

	ACKNOWLEDGMENTS

We thank JOE SUHAN for his assistance with electron microscopy, members of the Jones lab for their helpful discussions, and ROBERT PRESTON for never throwing out contaminants until checking whether they are actually contaminants. C.A.W. would like to thank RAJESH NAIK and MARLOES HOEDT-MILLER for their assistance in making figures and AMIT SRIVASTAVA for carrying out the ApI immunoblot. ApI antisera was a gift from DR. D. KLIONSKY, and ALP antisera was a gift from DR. G. PAYNE. This research was supported by a grant from the National Institutes of Health (GM29713 to E.W.J.).

Manuscript received July 17, 1997; Accepted for publication September 26, 1997.

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