Yeast Mn²⁺ Transporter, Smf1p, Is Regulated by Ubiquitin-Dependent Vacuolar Protein Sorting

Corresponding author: Stephen Garrett, UMDNJ-New Jersey Medical School, ICPH Bldg., 225 Warren St., P.O. Box 1709, Newark, NJ 07101-1709., garretst{at}umdnj.edu (E-mail)

Communicating editor: B. J. ANDREWS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Conditional cdc1(Ts) mutants of S. cerevisiae arrest with a phenotype similar to that exhibited by Mn²⁺-depleted cells. Sequence similarity between Cdc1p and a class of Mn²⁺-dependent phosphoesterases, as well as the observation that conditional cdc1(Ts) growth can be ameliorated by Mn²⁺ supplement, suggests that Cdc1p activity is sensitive to intracellular Mn²⁺ levels. This article identifies several previously uncharacterized cdc1(Ts) suppressors as class E vps (vacuolar protein sorting) mutants and shows that these, as well as other vps mutants, accumulate high levels of intracellular Mn²⁺. Yeast VPS genes play a role in delivery of membrane transporters to the vacuole for degradation, and we show that the vps mutants accumulate elevated levels of the high-affinity Mn²⁺ transporter Smf1p. cdc1(Ts) conditional growth is also alleviated by mutations, including doa4 and ubc4, that compromise protein ubiquitination, and these ubiquitination defects are associated with Smf1p accumulation. Epistasis studies show that these suppressors require functional Smf1p to alleviate the cdc1(Ts) growth defect, whereas Smf1p is dispensable for cdc1(Ts) suppression by a mutation (cos16/per1) that does not influence intracellular Mn²⁺ levels. Because Smf1p is ubiquitinated in vivo, we propose that Smf1p is targeted to the vacuole for degradation by ubiquitination-dependent protein sorting.

THE trace element Mn²⁺ is essential for growth of the yeast Saccharomyces cerevisiae (LOUKIN and KUNG 1995 ). Although the essential Mn²⁺-dependent process is not known, Mn²⁺-requiring enzymes include a Golgi-resident glycosyltransferase that is required for bud growth at extreme temperatures (NAKAYAMA et al. 1992 ) and an essential metalloprotease that is involved in mitochondrial protein import (SUPEK et al. 1996 ). Because Mn²⁺ is also toxic at high concentrations, microbes have complex mechanisms to regulate intracellular Mn²⁺ levels in the face of wide fluctuations in environmental Mn²⁺.

Yeast high-affinity Mn²⁺ uptake is mediated by Smf1p, a protein that bears structural homology with the Nramp (natural resistance associated macrophage protein) family of mammalian metal ion transporters (SUPEK et al. 1996 ). Strains containing SMF1 on a high-copy plasmid exhibit elevated levels of Mn²⁺, whereas deletion of SMF1 lowers intracellular Mn²⁺ concentration and imparts sensitivity to the chelator EGTA (SUPEK et al. 1996 ). Functional Smf1p is a plasma membrane protein that is tightly regulated by external Mn²⁺ (WEST et al. 1992 ; SUPEK et al. 1996 ; LIU and CULOTTA 1999A , LIU and CULOTTA 1999B ). In Mn²⁺-limiting medium, Smf1p accumulates on the plasma membrane, whereas in Mn²⁺-rich medium Smf1p undergoes rapid proteolysis in the vacuole (LIU and CULOTTA 1999B ).

The mechanism by which Mn²⁺ regulates Smf1p localization is not fully understood, although recent studies have implicated the endoplasmic reticulum (ER) resident protein, Bsd2p (LIU and CULOTTA 1994 ; LIU et al. 1997 ). bsd2 mutants contain elevated Mn²⁺ levels and accumulate Smf1p, even when extracellular Mn²⁺ levels are high (LIU et al. 1997 ; LIU and CULOTTA 1999B ). These results support a model in which Bsd2p is required for proper sorting of Smf1p between the plasma membrane and the vacuole (LIU and CULOTTA 1999B ). Interestingly, a mutant form of the plasma membrane protein Pma1p, which is sent to the vacuole along the biosynthetic route of the vacuolar protein sorting (VPS) pathway, is rerouted back to the plasma membrane in a bsd2 mutant (LUO and CHANG 1997 ). This last observation suggests that Bsd2p plays a general role in membrane protein targeting, controlling the trafficking of Pma1p and Smf1p between the plasma membrane and the vacuole. Although it is also consistent with the notion that Smf1p is delivered to the vacuole by the same targeting pathway as aberrant Pma1p, current evidence suggests only that endocytosis does not contribute significantly to Smf1p localization or turnover (LIU and CULOTTA 1999B ).

Mn²⁺ homeostasis is also regulated by intracellular storage. For example, the Golgi protein Pmr1p, a Ca²⁺-ATPase homolog, regulates cytosolic Mn²⁺ levels by pumping Mn²⁺ from the cytosol into the Golgi (LAPINSKAS et al. 1995 ). This has the dual property of reducing cytosolic Mn²⁺ levels and increasing Mn²⁺ levels in the Golgi, where it is required and can be exported out of the cell (LAPINSKAS et al. 1995 ; DURR et al. 1998 ; WEI et al. 1999 ). A second Golgi protein, Atx2p, plays a positive role in Mn²⁺ homeostasis (LIN and CULOTTA 1996 ). ATX2 was first identified in a high-copy screen for genes whose overexpression increased intracellular Mn²⁺ (LIN and CULOTTA 1996 ) and was subsequently shown to be essential for the maintenance of normal intracellular Mn²⁺ levels (LIN and CULOTTA 1996 ). The mechanism by which Atx2p regulates intracellular Mn²⁺ levels is not known. Finally, Mn²⁺ is transported from the cytoplasm to the vacuole by the vacuolar membrane protein Ccc1p (LAPINSKAS et al. 1996 ; LI et al. 2001 ).

Another yeast protein implicated in Mn²⁺ metabolism/function is Cdc1p. That attribution was based on the observation that cdc1(Ts) mutants exhibit a small-bud growth arrest that is identical to the phenotype displayed by Mn²⁺-depleted cells (LOUKIN and KUNG 1995 ; PAIDHUNGAT and GARRETT 1998A ). Consistent with that hypothesis, cdc1(Ts) growth is exacerbated or alleviated by manipulations that respectively deplete or supplement intracellular Mn²⁺ (LOUKIN and KUNG 1995 ; SUPEK et al. 1996 ; PAIDHUNGAT and GARRETT 1998A ). Although we initially proposed that Cdc1p played a central role in Mn²⁺ homeostasis (PAIDHUNGAT and GARRETT 1998A ), these observations can also be accommodated by a model in which Cdc1p is a Mn²⁺-dependent protein (SUPEK et al. 1996 ). The latter model is supported by the weak, but significant, sequence similarity between Cdc1p and Mn²⁺-dependent phosphoesterases (RUSNAK 2000 ), as well as by the observation that Cdc1p activity does not correlate with cellular Mn²⁺ levels (PAIDHUNGAT and GARRETT 1998A ).

Several suppressors of the cdc1(Ts) growth defect identified genes implicated in vacuole protein sorting (PAIDHUNGAT and GARRETT 1998B ). Strains lacking VPS gene function exhibit a myriad of defects in vacuolar protein trafficking and function (BRYANT and STEVENS 1998 ). In this article, we show that genes corresponding to three previously uncharacterized cdc1(Ts) suppressors (PAIDHUNGAT and GARRETT 1998B ) are identical to members of a group of VPS genes (the so-called class E VPS genes) that are required for proper processing of membrane proteins through the late endosome, or prevacuolar, compartment (BANTA et al. 1988 ; RAYMOND et al. 1992 ; BABST et al. 1998 ) into the vacuole. These and other vps mutants accumulate high levels of intracellular Mn²⁺, as well as elevated amounts of the high-affinity Mn²⁺ transporter, Smf1p. Because cdc1(Ts) suppression is dependent upon Smf1p, these results suggest that the vps mutations suppress the cdc1(Ts) growth defect by increasing Smf1p-dependent Mn²⁺ levels. A second group of cdc1(Ts) suppressors defines a class of proteins that function in ubiquitination of proteins targeted for vacuolar turnover. These ubiquitin processing mutants also accumulate Smf1p, which is consistent with the observation that Smf1p is ubiquitinated in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media and standard genetic techniques:
Standard yeast media were prepared as described (KAISER et al. 1994 ). LiCl was added to rich yeast medium (YEPD) after autoclaving. Medium containing CaCl₂ or MnCl₂ was made by autoclaving YEPD, buffering to pH 5.5 with 50 mM succinate/NaOH, and adding CaCl₂ or MnCl₂ to the indicated concentration. Genetic and molecular manipulations of yeast strains were carried out by standard techniques (KAISER et al. 1994 ).

Yeast strains:
Yeast strains are listed in Table 1. Disruption derivatives of FY11, FY70, FY388, and YR98 were made by transformation with digested plasmid DNA or gel-purified PCR products from the Yeast Deletion Consortium KanMx disruption strains using the recommended A and D primers (Open Biosystems). Strain 27038a (generously provided by C. Michaels) contains the rsp5/npi1 mutation and was crossed with FY11 to determine if the cdc1-1(Ts) growth defect could be suppressed by the rps5 mutation. Strain Y152 was isolated from a cross between FY11 and 12992 (BY4742 end3::KanMx) from Open Biosystems.

Plasmids:
The high-copy (YEp13-CDC1) and low-copy (YCp50-CDC1) CDC1 plasmids have been described (PAIDHUNGAT and GARRETT 1998B ). The high-copy SMF1 (YEp24-SMF1) and smf1 disruption (smf1::URA3) plasmids (SUPEK et al. 1996 ), as well as the low-copy SMF1-HA₂ (pSF10) plasmid (LIU and CULOTTA 1999A ), were generous gifts of V. Culotta (The Johns Hopkins University). Plasmid YEp105 contains a myc-tagged allele of ubiquitin gene UBI4 expressed from the copper-inducible CUP1 promoter (MEDINTZ et al. 1998 ). The high-copy SMF1-HA plasmid pLE59 was constructed by cloning the SacI-XhoI fragment containing SMF1-HA from pSF10 into the same sites of the high-copy URA3 vector pRS202. A high-copy SMF1-GFP plasmid was constructed in several steps. SMF1 was amplified by PCR using primers 5'-CTCGAGCGAAATCTCTCCAAAGGGTG-3' and 5'-GGATTCCACTGATATCACCATGAGACATGCC-3', which left XhoI and BamHI sites 700 bp upstream of the promoter and at the last codon, respectively. The PCR fragment was digested with XhoI and BamHI and cloned into the corresponding sites of pRS306 to generate pYZ18. A SMF1-GFP fusion was created by inserting an in-frame 800-bp BglII-NotI fragment containing green fluorescent protein (GFP) into the BamHI and NotI sites to generate pYZ20. Finally, the 2.8-kb XhoI-NotI fragment containing SMF1-GFP was removed from pYZ20 and inserted into the same sites of pRS202 to generate pLE61. The vps18::HIS3, vps4::RA3, and cos16::LEU2 disruption plasmids (pFB107, pFB275, and pFB371) have been described (PAIDHUNGAT and GARRETT 1998B ).

Cloning VPS36 (COS3), VPS32/SNF7 (COS14), and VPS20 (COS1):
The wild-type VPS36 (COS3), VPS32 (COS14), and VPS20 (COS1) genes were isolated from a low-copy, yeast genomic library (ROSE et al. 1987 ) by their ability to confer Ca²⁺ and Li⁺ tolerance to cdc1-1(Ts) cos3-3 [YEp13-CDC1], cdc1-1(Ts) cos14-3 [YEp13-CDC1], and cdc1-1(Ts) cos1-1 [YEp13-CDC1] strains, respectively. One VPS36 plasmid (pFB171) was recovered from 6000 Ura⁺ transformants. The vps36::LEU2 plasmid, pFB281, was generated by cloning the 2.9-kb SalI fragment from plasmid pFB171 into pUC8 and replacing the 1.3-kb EcoRV fragment of VPS36 with the 2.2-kb HpaI LEU2 fragment. Plasmid pFB281 was digested with HindIII prior to transformation.

The VPS32/COS14 gene was identified from four complementing plasmids with overlapping genomic inserts (pFB175-pFB178) that were recovered from a total of 4000 cdc1-1(Ts) cos14-103 [YEp13-CDC1] Ura⁺ transformants. The snf7::URA3 plasmid pUU4 (TU et al. 1993 ) was obtained from M. Carlson, Columbia University. A vps32/snf7::LEU2 plasmid was constructed by inserting the 2.2-kb HpaI LEU2 fragment at the unique StuI site in the URA3 region of pUU4. The resulting plasmid, pFB316, was digested with PvuII and SphI prior to transformation.

The VPS20/COS1-containing plasmid (pFB173) was recovered from 5000 Ura⁺ transformants of the cdc1-1(Ts) cos1-1 [YEp13-CDC1] strain. Plasmid pFB209 was constructed by inserting a 2.3-kb XhoI-BamHI fragment containing VPS20 into the low-copy URA3 vector pRS316 (SIKORSKI and HIETER 1989 ). The vps20::LEU2 plasmid was constructed in two steps. The 2.3-kb XhoI-BamHI VPS20 fragment from pFB209 was subcloned into pBSKII⁺ (Stratagene, La Jolla, CA) to obtain plasmid pFB244. The 0.3-kb EcoRI fragment in pFB244 was replaced with a 1.2-kb EcoRI URA3 fragment to produce plasmid pFB245. Finally, a 2.2-kb HpaI LEU2 fragment was inserted into the StuI site within the URA3 marker in plasmid pFB245 to generate the vps20::LEU2 plasmid pFB337. pFB337 was digested with XbaI prior to transformation.

Cellular Ca²⁺ and Mn²⁺ content:
Cellular Ca²⁺ content was measured using radionuclide ⁴⁵Ca²⁺ tracer as described (PAIDHUNGAT and GARRETT 1997 ). Cellular Mn²⁺ content was measured by atomic absorption spectroscopy as described (PAIDHUNGAT and GARRETT 1998B ).

Western blot analysis and immunoprecipitation:
The hemagglutinin (HA)-tagged Smf1 protein was detected by Western blot analysis according to published protocols (LUI and CULOTTA 1999b). Strains containing the low-copy SMF1-HA₂ plasmid, pSF10, or the high-copy SMF1-HA₂ plasmid, pLE59, were grown in selective medium until midlog phase. Extracts were prepared by alkalai lysis and then separated by SDS-polyacrylamide electrophoresis. The Smf1p-HA₂ protein was visualized using 1/1000 dilution of ascites fluid anti-HA monoclonal antibody 12CA5 (Covance) diluted 1/1000 and 1/10,000 secondary HRP-congugated goat-anti-mouse antibody (Covance).

The Smf1-HA₂ protein was immunoprecipitated from extracts using ascites fluid of the anti-HA monoclonal antibody 12CA5 diluted 1/200. Extracts were prepared by bead beating and centrifugation (GARRETT et al. 1991 ) and then incubated with anti-HA antibody at 1/200 dilution for 1 hr at 4°. Antigen-antibody complexes were isolated by adding protein G Sepharose (Invitrogen, San Diego) to the extract for 1 hr at 4° and then washing six times in immunoprecipitation buffer (GARRETT et al. 1991 ) before separating proteins by SDS-polyacrylamide electrophoresis and then transferring to nitrocellulose by Western blot. The nitrocellulose filters were incubated with 1/1000 dilution of the monoclonal anti-Myc antibody 9E-10 to visualize Myc-tagged ubiquitin, stripped, and then incubated with 1/1000 dilution of the monoclonal anti-HA antibody 12CA5 to visualize HA-tagged Smf1p.

Immunofluorescence:
Cells containing the Smf1-GFP protein were grown to midlog phase (OD₆₀₀ = 0.5), incubated with Hoechst 33258 (for DNA) or FM4-64 (for vacuole membrane or class E compartment) for 10 min, washed, and then incubated for 30 min before visualizing by epifluorescence or light microscopy.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

cdc1(Ts) suppression by class E vps mutants:
Genetic analysis showed that the cdc1-1(Ts) growth defect can be alleviated by mutations in class C and class D VPS genes (PAIDHUNGAT and GARRETT 1998B ). Three unidentified cdc1 suppressors (cos1, cos3, and cos14) conferred sensitivity to ions such as Ca²⁺ and Li⁺ (data not shown) and missorted the vacuolar protein CPY (PAIDHUNGAT and GARRETT 1998B ). Wild-type genes for these suppressors were cloned by isolating plasmids that could restore Li⁺ and Ca²⁺ resistance to the appropriate CDC1 cos strain, as well as temperature-sensitive growth to the corresponding cdc1-1(Ts) cos mutant. Physical and genetic analysis (MATERIALS AND METHODS) revealed that all three contain mutations in class E VPS genes: COS3 was allelic with VPS36, COS14 was identical to VPS32, and COS1 was allelic with VPS20 (data not shown). Disruption of each gene recapitulated cdc1-1(Ts) suppression of the original recessive suppressors (Fig 1A). To determine if cdc1(Ts) suppression and ion sensitivity are general characteristics of class E vps mutations, we tested several phenotypes of the class E mutants listed in Table 2. Interestingly, although cdc1(Ts) suppression and temperature-sensitive growth are general attributes of class E vps mutations (Table 2), a different pattern emerged when class E mutants were characterized according to ion sensitivity. Mutants lacking individual components of ESCRT-I and ESCRT-II were extremely sensitive to Ca²⁺, Li⁺, and Mn²⁺, as were strains lacking ESCRT-III components Vps20p and Vps32p (see Fig 1B and Table 2). By contrast, mutants lacking ESCRT-III components Vps24p and Vps2p, components of the Vps27p/Hsc1p complex, and the AAA-ATPase Vps4p exhibited wild-type resistance to Ca²⁺ and Li⁺ and only moderate sensitivity to Mn²⁺ (Fig 1B; Table 2).

View larger version (90K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Characterization of class E vps mutants. (A) Suppression of the conditional cdc1-1(Ts) growth defect. The cdc1-1(Ts) strain FY11 (VPS) and its vps36::LEU2 (vps36), vps32::LEU2 (vps32), and vps20::LEU2 (vps20) derivatives were plated onto rich medium and incubated at either 23° or 30° for 3 days. (B) Ca2+, Li+, and Mn2+ tolerance of class E vps mutants. Strain FY70 (VPS) and its indicated derivatives were plated onto YEPD medium, YEPD medium containing 100 mM LiCl (Li+), or YEPD pH 5.5 medium containing either 200 mM CaCl2 (Ca2+) or 3 mM MnCl2 (Mn2+) and incubated at 30° for 3 days.

vps mutations and ion homeostasis:
Conditional cdc1(Ts) growth can be partially alleviated by mutations that restrict Ca²⁺ accumulation (PAIDHUNGAT and GARRETT 1997 ); however, Ca²⁺ levels of vps36::LEU2, vps32::URA3, and vps20::URA3 mutants were similar to the Ca²⁺ level of the wild-type parent (Table 3), indicating that vps mutations do not alleviate the cdc1-1(Ts) growth defect by altering intracellular Ca²⁺ levels. Elimination of the vacuolar proton gradient by deletion of genes VPH6 or VMA2 (YAMASHIRO et al. 1990 ; HEMENWAY et al. 1995 ; BRYANT and STEVENS 1998 ) was synthetically lethal with the cdc1-1(Ts) conditional mutation (data not shown), indicating that vps mutations do not alleviate the cdc1-1(Ts) growth defect by inactivating V-ATPase function.

We previously proposed that class C and D vps mutants suppressed the cdc1(Ts) growth defect by mislocalizing a putative vacuolar Mn²⁺ transporter, Cos16p, thereby elevating cytoplasmic Mn²⁺ levels (PAIDHUNGAT and GARRETT 1998b). However, cos16 deletion strains exhibit wild-type Mn²⁺ levels (PAIDHUNGAT and GARRETT 1998B ) and disruption of a known vacuolar Mn²⁺ transporter, CCC1 (LI et al. 2001 ), failed to suppress conditional growth of either the cdc1-1(Ts) or the cdc1-2(Ts) mutant (Fig 3B; Table 4). Thus, vacuole Mn²⁺ sequestration does not appear to play an important role in cdc1(Ts) growth.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Smf1p protein levels in vps mutants. The wild-type strain BY4742 (wt) and its indicated derivatives were grown to midlog phase, harvested, and subjected to denaturing electrophoresis and Western analysis with anti-HA primary antibody as described in MATERIALS AND METHODS. Strains contained either the Smf1-HA2 fusion plasmid pSF10 (+) or the control plasmid pRS316 (–).

View larger version (86K): [in this window] [in a new window] [Download PPT slide]   Figure 3. cdc1(Ts) suppression. (A) High-copy SMF1 expression and cdc1(Ts) growth. cdc1-1(Ts) strain FY11 containing the indicated plasmids was incubated at 23° and 30° for 3 days. Plasmids were pRS316-SMF1-HA2 (LC-SMF1); YEp24-SMF1 (HC-SMF1); YEp24 (HC); and YCp50-CDC1 (LC-CDC1). (B) Smf1p requirement and cdc1(Ts) suppression. Indicated derivatives of cdc1-2(Ts) strain FY388 were streaked onto rich medium agar and incubated for 3 days at 23° and 34°.

To determine if vps mutations alter intracellular Mn²⁺ levels, we examined Mn²⁺ contents of several vps mutants by atomic absorption spectroscopy. The vps32 and vps20 mutants exhibited threefold higher intracellular Mn²⁺ levels than the isogenic wild-type strain, and accumulation was independent of the CDC1 allele (Table 3). At least two other cdc1-1(Ts) suppressors that affect vacuole protein sorting, vps18 and vps4, also resulted in high levels of intracellular Mn²⁺ (Table 3), suggesting that Mn²⁺ accumulation is not restricted to class E vps mutants.

vps mutants accumulate the high-affinity Mn²⁺ transporter, Smf1p:
Except under Mn²⁺-limiting conditions, the high-affinity Mn²⁺ transporter, Smf1p, is directed to the vacuole where it is rapidly degraded (LIU and CULOTTA 1999A , LIU and CULOTTA 1999B ). The route and mechanism of Smf1p vacuolar delivery is unknown; however, endocytosis does not play a significant role (LIU and CULOTTA 1999A ). To determine if Smf1p accumulation could account for elevated Mn²⁺ levels and cdc1(Ts) suppression of the vps mutants, we measured levels of a functional HA-tagged Smf1p protein (LIU and CULOTTA 1999A ). Western analysis showed that the vps18 mutant accumulated Smf1p to levels similar to those found in a pep4 control, whereas Smf1p was undetectable in the wild-type strain (Fig 3). Even class E vps mutants vps20, vps32, and vps24, which accumulate less Mn²⁺ (Table 3 and data not shown) and display a less dramatic trafficking defect than class C vps mutants (BRYANT and STEVENS 1998 ), contained higher levels of the HA-tagged protein than the wild-type strain (Fig 2). Thus, the increase in intracellular Mn²⁺ is correlated with an increase in Smf1p.

Smf1p accumulation is necessary and sufficient for cdc1-1(Ts) suppression:
If vps mutations alleviate the cdc1-1(Ts) growth defect by increasing Smf1p levels, genetic alterations that result in Smf1p accumulation in the plasma membrane should restore growth. A high-copy SMF1 plasmid is capable of elevating intracellular Mn²⁺ levels and alleviating the EGTA sensitivity of several cdc1 mutants (SUPEK et al. 1996 ; PAIDHUNGAT and GARRETT 1998A ). Temperature-sensitive growth defects of cdc1-1(Ts) (Fig 3A; Table 4) and cdc1-2(Ts) (Table 4) strains were alleviated by a high-copy YEp24-SMF1 plasmid, but were unaffected by the high-copy vector YEp24 and the low-copy SMF1 plasmid pSF10. Moreover, a bsd2 mutant exhibited Smf1p-dependent Mn²⁺ accumulation (LIU et al. 1997 ), and both cdc1-1(Ts) bsd2 (Table 4) and cdc1-2(Ts) bsd2 (Fig 3B; Table 4) strains grew at the nonpermissive temperature.

To determine if Smf1p is necessary for suppression, we asked if the cdc1(Ts) growth defect could be alleviated in strains lacking Smf1p. A cdc1-1(Ts) smf1 double mutant is inviable at all temperatures (PAIDHUNGAT and GARRETT 1998A ), and this synthetic lethality was insensitive to each of three vps mutations [none of >40 predicted triple-mutant spores were viable in crosses between a MATa smf1 strain and MAT cdc1-1(Ts) strains containing vps32, vps20, or vps18]. By contrast, the cos16 mutation, which alleviates the cdc1-1(Ts) growth defect without affecting intracellular Mn²⁺ levels (PAIDHUNGAT and GARRETT 1998B ), suppressed synthetic lethality and the temperature-sensitive growth of the cdc1-1(Ts) smf1 double mutant [six of six predicted cdc1-1(Ts) smf1 cos16 triple mutants grew at both 23° and 30°]. Tests of epistasis were also carried out in the cdc1-2(Ts) mutant, which exhibits wild-type growth at 23° and is inviable at 34° (PAIDHUNGAT and GARRETT 1998A ). Introduction of each of several vps mutations (vps20 and vps32), as well as the bsd2 mutation, into the cdc1-2(Ts) strain alleviated the 34° growth defect (Fig 3B). By contrast, the same suppressors were unable to alleviate the 34° growth defect of the cdc1-2(Ts) smf1 double mutant (Fig 3B; Table 4).

cdc1-1(Ts) suppression and ubiquitin-dependent protein turnover:
To identify new cdc1-1(Ts) suppressors, we transformed a high-copy yeast genomic library into the cdc1-1(Ts) strain and isolated six plasmids that conferred temperature-resistant growth. Four plasmids identified full-length VPS genes: VPS4, a class E gene (BABST et al. 1997 ), was isolated three times, and VPS13, a class A gene (BRYANT and STEVENS 1998 ), was isolated once (Table 4). The isolation of VPS4 as a high-copy suppressor is consistent with our previous observation that VPS4 overexpression results in a vps-like defect (PAIDHUNGAT and GARRETT 1998B ). A fifth plasmid contained ATX2, which was previously identified in a high-copy screen for genes that result in Mn²⁺ accumulation (LIN and CULOTTA 1996 ).

The sixth high-copy suppressor contained the ubiquitin hydrolase gene, UBP1 (Table 4). The Ubp1p hydrolase removes ubiquitin from protein substrates (TOBIAS and VARSHAVSKY 1991 ), and a UBP1 homolog from Kluyveromyces lactis was recently isolated as a high-copy suppressor of a temperature-sensitive centromere-binding protein mutant of S. cerevisiae (WINKLER et al. 2000 ). Identification of UBP1 as a high-copy suppressor suggested a role for ubiquitination in Smf1p regulation. Although proteosomal degradation does not contribute to Smf1p turnover (LIU and CULOTTA 1999B ), ubiquitination has recently been implicated in other cellular functions, including ubiquitin-mediated endocytosis (HICKE and RIEZMAN 1996 ; HICKE 1999 ) and protein delivery from the Golgi to the vacuole (AMERIK et al. 2000 ; HELLIWELL et al. 2001 ; KATZMANN et al. 2001 ). To determine if other ubiquitin-related functions affected Smf1p accumulation, we asked if the cdc1(Ts) growth defect could be alleviated by mutations in genes previously implicated in ubiquitination of vacuole-targeted proteins [DOA4, ubiquitin recycling (AMERIK et al. 2000 ); RSP5, E3 ubiquitin ligase (GALAN et al. 1996 ); UBC4, E2 conjugating enzyme (HOCHSTRASSER 1996 ); and BRO1, unknown activity (SPRINGAEL et al. 2002 )]. All four mutations alleviated the cdc1-1(Ts) and/or cdc1-2(Ts) growth defects, and suppression was dependent on the presence of a functional SMF1 gene (Fig 3; Table 4). Ubiquitination does not appear to influence cdc1(Ts) growth by affecting endocytosis because the cdc1-2(Ts) growth defect was not alleviated by an end3 mutation (Table 4).

Smf1p is targeted to the vacuole by ubiquitination:
To determine the role of ubiquitination in cdc1(Ts) suppression, we examined Smf1p levels in ubiquitin-processing mutant ubc4 and a doa4 pep4 double mutant. The DOA4 gene encodes a ubiquitin recycling protease, loss of which results in diminished ubiquitin pools. Because Doa4p rapidly removes ubiquitin from modified proteins, its loss paradoxically stabilizes the ubiquitin-modified form of a protein if the protein itself is not subject to turnover (SWAMINATHAN et al. 1999 ). Because Smf1p is degraded in the vacuole, the pep4 mutation was used to stabilize all forms of Smf1p. As predicted, both ubc4 and the doa4 pep4 double-mutant strains accumulated more Smf1p protein than did the wild-type parent (Fig 4A). Moreover, a significant proportion of the cross-reacting activity from the doa4 pep4 double mutant migrated as a broad, high-molecular-weight smear (Fig 4A). Smf1p also migrated as a higher-molecular-weight species in the isogenic bsd2 mutant. These species are apparent in several figures of two earlier publications and were thought to be due to unknown protein-processing events (LIU and CULOTTA 1999A , LIU and CULOTTA 1999B ).

View larger version (60K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Smf1p accumulation and ubiquitination. (A) Smf1p accumulation in ubiquitination mutants. Extracts prepared from the indicated strains were run on a 10% polyacrylamide gel, transferred to nitrocellulose, and then probed with anti-HA antibody. Isogenic strains were derived from YR98 (WT) by transformation with ubc4::kanMx (ubc4), doa4::kanMx and pep4::HIS3 (doa4 pep4), or bsd2::kanMx. Strains contained the high-copy plasmid Smf1-HA2, plasmid pLE59 (+), or the control vector pRS202 (–). (B) Smf1p ubiquitination. Protein extracts from strain YR98 doa4::kanMx pep4::HIS3 containing the high-copy plasmid Smf1-HA2, plasmid pLE59 (+), or the control vector pRS202 (–) were prepared after incubating cells in the presence or absence of Cu2+ to induce production of the MYC-tagged ubiquitin from plasmid p105. Protein extracts were incubated with anti-HA antibody and protein G Sepharose, collected and washed by centrifugation, separated by polyacrylamide electrophoresis, transferred to nitrocellulose, and then sequentially probed with anti-MYC (-MYC) and anti-HA (-HA) antibody for ubiquitin and Smf1p, respectively.

To determine the nature of the broad Smf1p-specific smear, we examined the ubiquitination status of Smf1p protein immunoprecipitated from the doa4 pep4 double mutant. Because the doa4 mutation limits available ubiquitin, the strain contained a plasmid (YEp105) that encodes a Cu²⁺-inducible, myc-tagged ubiquitin moiety (MEDINTZ et al. 1998 ). As can be seen in Fig 4B, the Smf1-HA tagged protein immunoprecipitated from the doa4 pep4 double mutant migrated as a broad, high-molecular-weight smear that cross-reacted with the myc-specific antibody. Thus, Smf1p appears to be ubiquitinated.

To determine the disposition of the Smf1p that accumulates in the ubc4 mutant, we localized a functional Smf1-GFP fusion protein in wild-type and ubc4 strains. Whereas wild-type cells displayed little to no fluorescence, the ubc4 mutant exhibited significant staining of a contiguous internal structure that surrounded the nucleus (as delineated by Hoechst staining), as well as patches at or just beneath the cell surface (Fig 5A). This localization is consistent with the majority of Smf1p accumulating in the ER. Modest perimeter staining in the ubc4 mutant (Fig 5A) colocalized with the lipophilic dye FM4-64 after a brief pulse (data not shown), suggesting that some Smf1p was also accumulating at the plasma membrane; however, intense staining of ER structures at the perimeter made it impossible to state definitively that Smf1 protein was accumulating on the plasma membrane. Previous studies have shown that bsd2 mutants accumulate the bulk of Smf1p within the Golgi (LIU and CULOTTA 1999B ). Because bsd2 mutants divert misfolded Pma1p from the vacuole to the plasma membrane (LUO and CHANG 1997 ), accumulate high levels of Smf1p-dependent intracellular Mn²⁺ (LIU et al. 1997 ; LIU and CULOTTA 1999B ), and suppress the cdc1(Ts) growth defect (Fig 3B), it seems likely that Smf1p also accumulates on the plasma membrane of bsd2 cells. Thus, at least some Smf1p may accumulate on the plasma membrane of ubc4 cells.

View larger version (112K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Smf1p localization in cdc1(Ts) suppressor mutants. (A) Smf1p localization in ubiquitination mutant ubc4. The wild-type strain YR98 (WT) or its ubc4::KanMx derivative (ubc4) containing the high-copy Smf1-GFP plasmid pLE61 were incubated with Hoechst to visualize DNA and then analyzed by epifluorescence or differential interference microscopy. (B) Smf1p localization in class E mutant vps32. The wild-type strain BY4742 (WT) or its vps32::KanMx derivative (vps32) containing the high-copy Smf1-GFP plasmid pLE61 were pulsed with FM4-64 and then visualized by epifluorescence or phase microscopy.

To examine the effect of a different class of cdc1(Ts) suppressors on Smf1p accumulation, we localized Smf1p in several vps mutants. In the class E mutants vps32 (Fig 5B) and vps20 (data not shown), Smf1-GFP fluorescence appeared as one or two spots that colocalized with FM4-64 staining of the class E compartment (BRYANT and STEVENS 1998 ). In the wild-type strain (Fig 5B), by contrast, Smf1-GFP was not apparent and FM4-64 was found in the limiting membrane of the vacuole. Several other membrane proteins (Ste2p and Ste6p) accumulated in both the class E compartment and the plasma membrane of class E vps mutants (LI et al. 1999 ; KRANZ et al. 2001 ), suggesting that at least some Smf1p is diverted to the plasma membrane of the vps mutants.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cdc1p and Mn²⁺ homeostasis:
The yeast Cdc1p protein is required for growth and has been implicated in such processes as recombination, actin polarization, bud growth, shmoo formation, and spindle pole body duplication (HALBROOK and HOEKSTRA 1994 ; PAIDHUNGAT and GARRETT 1998A ; ROSSANESE et al. 2001 ). Although the biochemical activity of Cdc1p is not known, the essential function involves the divalent cation Mn²⁺, as judged by the observations that Mn²⁺ supplement alleviates the cdc1(Ts) temperature-sensitive growth defect and that the chelator sensitivities of cdc1 mutants are ameliorated by overproduction of the high-affinity Mn²⁺ transporter Smf1p (LOUKIN and KUNG 1995 ; SUPEK et al. 1996 ; PAIDHUNGAT and GARRETT 1998B ). Consistent with this conclusion, we show here that the temperature-sensitive cdc1(Ts) growth defect can be alleviated by genetic manipulations that increase intracellular Mn²⁺, including additional copies of SMF1 (Fig 3A), as well as mutations that stabilize Smf1p in a functionally active form (Fig 2A and Fig 3; Table 3 and Table 4). The genetic relationship between Cdc1p-dependent growth and intracellular Mn²⁺ levels is significant because previously characterized suppressors alleviated the cdc1(Ts) growth defect by mechanisms that are not obviously related to intracellular Mn²⁺ (PAIDHUNGAT and GARRETT 1997 , PAIDHUNGAT and GARRETT 1998A ). The relationship between Mn²⁺ and Cdc1p function is also intriguing in light of the weak, but significant, sequence similarity between Cdc1p and a family of Mn²⁺-dependent phosphotransfersase proteins (RUSNAK 2000 ).

Although the biochemical activity and biological function of Cdc1p have yet to be defined, the observation that a cos16/per1 deletion can alleviate the cdc1 growth defect without altering Mn²⁺ levels and do so independently of Smf1p suggests that Cos16p/Per1p function might lie downstream of Cdc1p. Previous fractionation studies localized a Cos16-ßGal fusion to the vacuole (PAIDHUNGAT and GARRETT 1998B ), whereas a large-scale localization study identified the ER as the resident site of GFP-tagged Cos16p/Per1p (HUH et al. 2003 ). Further work will be needed to reconcile the differences between these two studies; however, residence in the ER would be consistent with the observation that a per1(cos16) disruption induces the unfolded protein response pathway of the ER (NG et al. 2000 ). Precisely how this would alleviate the physiological defects of a cdc1 mutant is not known. Nevertheless, it is intriguing to note that organisms such as Arabidopsis thaliana, Schizosaccharomyces pombe, and Neurospora crassa contain proteins that bear limited, but significant, sequence identity with Cos16p/Per1p and that Mn²⁺ has been implicated in the function of a Cdc1p homolog (frost) from N. crassa (SONE and GRIFFITHS 1999 ).

Ubiquitination and Smf1p targeting:
In yeast, Mn²⁺ homeostasis is maintained through regulated targeting of the high-affinity Mn²⁺ transporter Smf1p to the vacuole (LIU and CULOTTA 1999A , LIU and CULOTTA 1999B ). Strains lacking the major vacuolar protease Pep4p accumulate high levels of Smf1p in the vacuole in an inactive and Mn²⁺-insensitive form (PAIDHUNGAT and GARRETT 1998B ; LIU and CULOTTA 1999B ). Thus, localization, rather than abundance per se, is critical to Smf1p function. Because vacuole delivery did not require a functional endocytic pathway, LIU and CULOTTA 1999B proposed a model in which Smf1p was either secreted to the plasma membrane or, under Mn²⁺ excess, diverted to the vacuole for degradation. In support of this model, we show here that mutations that disrupt the biosynthetic route of the vacuolar protein sorting pathway stabilize Smf1p in a functionally active form, as judged by an increase in intracellular Mn²⁺ (Table 3), suppression of the cdc1(Ts) growth defect (Fig 1A and Fig 3; Table 2 and Table 4), and Smf1p accumulation (Fig 2 and Fig 5). Because defects in vacuolar protein sorting also affect endocytosis, we cannot rule out the possibility that a defect in endocytosis contributes to Smf1p accumulation in the vps strains; however, our work (Table 4) and the work of others (LIU and CULOTTA 1999B ) suggests that endocytosis does not play a major role in Smf1p localization. Of course, these results do not address the possibility that endocytosis plays a role in rapid elimination of Smf1p from the plasma membrane during transition from Mn²⁺-limiting to Mn²⁺-rich conditions.

Many proteins that are directed to the vacuole for degradation are ubiquitinated. Proteins such as the pheromone receptor Ste2p are ubiquitinated on the plasma membrane as a signal for entry into the early stage of the endocytic pathway (HICKE and RIEZMAN 1996 ; HICKE 1999 ). Later in the endocytic pathway, ubiquitination serves as a molecular pass for recruitment into multivesicular bodies and subsequent deposition into the vacuole lumen (KATZMANN et al. 2001 ). Ubiquitination also plays an essential role in the vacuolar delivery of the general amino acid permease Gap1p (HELLIWELL et al. 2001 ). When nitrogen is limiting, Gap1p is targeted to the plasma membrane where it serves as a nonspecific amino acid permease. During growth on nitrogen-rich medium, Gap1p is diverted to the vacuole along the vacuolar protein sorting pathway (HELLIWELL et al. 2001 ). As it does in endocytosis, ubiquitination directs biosynthetic membrane proteins such as Gap1p into multivesicular bodies and the vacuole lumen (KATZMANN et al. 2001 ); however, recent observations are also consistent with ubiquitination serving as a molecular marker in the Golgi complex to divert Gap1p from the late secretory pathway to the biosynthetic route of the vacuolar protein sorting pathway (HELLIWELL et al. 2001 ). Although this latter role is not as well understood as the analogous role in endocytosis, it is clear that ubiquitination plays an important function in membrane protein localization and degradation. Thus, we are intrigued by the observation (Fig 3B; Table 4) that the cdc1-1(Ts) growth defect can be alleviated in a SMF1-dependent manner by alterations (doa4, ubc4, bro1, rsp5, and high-copy UBP1) that result in reduced ubiquitination of membrane proteins (TOBIAS and VARSHAVSKY 1991 ; GALAN et al. 1996 ; AMERIK et al. 2000 ; WINKLER et al. 2000 ). Suppression by these mutations does not reflect an indirect effect on Cdc1p function because Smf1p is ubiquitinated in vivo (Fig 4) and a ubiquitination defect results in accumulation of Smf1p in the ER and the cell surface (Fig 4 and Fig 5).

Although ubiquitination is commonly described as a signal for degradation by the proteosome, previous results suggest that proteosomal degradation does not contribute significantly to Smf1p turnover or function (LIU and CULOTTA 1999B ). Moreover, Smf1p trafficking and function are unaffected by several endocytosis defects (LIU and CULOTTA 1999A ; Table 4). Thus, we propose that ubiquitination serves as an early determinant in Smf1p targeting, directing it to the VPS pathway rather than to the plasma membrane, and/or as a necessary signal for delivery to multivesicular bodies and the vacuole lumen.

If ubiquitination serves as an early determinant in Smf1p trafficking, factors involved in this Mn²⁺-regulated step should reside in or before the Golgi complex. Although the ER-resident protein Bsd2p seems an unlikely candidate for this role, it might mark Smf1p for vacuolar targeting in the Golgi. At odds with this notion, bsd2 mutations affect the vacuolar targeting of proteins that are unresponsive to Mn²⁺ (LUO and CHANG 1997 ), suggesting that Bsd2p plays a more general role in protein trafficking. By contrast, the Atx2p protein resides in the Golgi apparatus and has been associated only with Mn²⁺ homeostasis (LIN and CULOTTA 1996 ; Table 4). Because ATX2 effects require Smf1p (LIN and CULOTTA 1996 ), Atx2p may play a direct role in Mn²⁺-regulated Smf1p trafficking and degradation. In this scenario, Atx2p would direct Smf1p to the plasma membrane in medium depleted of Mn²⁺, either by sequestering Smf1p before it was ubiquitinated or by overriding a ubiquitin signal that was present under all conditions.

Class E vps mutants define two phenotypic classes:
Individual class E mutants display qualitative differences in processing of carboxypeptidase S (CPS), a selective cargo of the MVB (BABST et al. 2002 ). Mutants such as vps4, vps2, and vps24, which accumulate ESCRT-III subcomplex Vps20p-Vps32p on the class E compartment membrane, exhibit a dramatic defect in CPS processing, whereas strains lacking individual components of the Vps20p-Vps32p subcomplex mature CPS with wild-type kinetics (BABST et al. 2002 ). Interestingly, class E mutants vps4, vps2, and vps24, along with strains lacking components of the Vps27p/Hse1p complex, exhibit wild-type (Ca²⁺ or Li⁺) or moderate (Mn²⁺) resistance to monovalent and divalent cations, whereas mutants lacking components of ESCRT-I, ESCRT-II, and ESCRT-III subcomplex Vps20p-Vps32p are extremely sensitive to those treatments (Fig 1B; Table 2). We do not fully understand the molecular natures of these ion sensitivities, although they may represent documented defects in removing high-affinity ion transporters from the plasma membrane as well as defects in ion sequestration. Nevertheless, we are intrigued by the correlation between ion sensitivity and CPS processing.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present Address: Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021.

	ACKNOWLEDGMENTS

We thank C. Michaels, M. Carlson, V. Culotta, and M. Rose for yeast strains and plasmids. M.P. thanks T. Graf and J. Johnson for help and instruction with atomic absorption spectroscopy, and Y.-S. Chung thanks V. Culotta for suggestions concerning Western analysis with the HA-tagged Smf1p protein.

Manuscript received November 10, 2003; Accepted for publication January 30, 2004.

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