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© The Rockefeller University Press,
0021-9525/2000//915 $5.00
The Journal of Cell Biology, Volume 148, Number 5,
, 2000 915-924
Original Article |
Regulation of Hmg-Coa Reductase Degradation Requires the P-Type Atpase Cod1p/Spf1p
rhampton{at}biomail.ucsd.edu
The integral ER membrane protein HMG-CoA reductase (HMGR) is a key enzyme of the mevalonate pathway from which sterols and other essential molecules are produced. HMGR degradation occurs in the ER and is regulated by mevalonate-derived signals. Little is known about the mechanisms responsible for regulating HMGR degradation. The yeast Hmg2p isozyme of HMGR undergoes regulated degradation in a manner very similar to mammalian HMGR, allowing us to isolate mutants deficient in regulating Hmg2p stability. We call these mutants cod mutants for the control of HMG-CoA reductase degradation. With this screen, we have identified the first gene of this class, COD1, which encodes a P-type ATPase and is identical to SPF1. Our data suggested that Cod1p is a calcium transporter required for regulating Hmg2p degradation. This role for Cod1p is distinctly different from that of the well-characterized Ca2+ P-type ATPase Pmr1p which is neither required for Hmg2p degradation nor its control. The identification of Cod1p is especially intriguing in light of the role Ca2+ plays in the regulated degradation of mammalian HMGR.
Key Words: hydroxymethylglutaryl CoA reductase • Ca2+-transporting ATPase • ubiquitin • endoplasmic reticulum • Saccharomyces cerevisiae
© 2000 The Rockefeller University Press
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Introduction |
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The yeast HMGR isozyme Hmg2p undergoes regulated degradation with many similarities to the mammalian enzyme, including control by a signal derived from the mevalonate pathway product farnesyl pyrophosphate FPP (Hampton and Rine 1994; Hampton and Bhakta 1997; Gardner and Hampton 1999a). We have identified genes required for the degradation of Hmg2p, referred to as HRD genes (pronounced "herd", for HMG-CoA reductase degradation). The HRD genes are also required for the degradation of numerous other proteins, none of which are regulated by the mevalonate pathway (Wilhovsky et al. 2000; Hiller et al. 1996; Bordallo et al. 1998; Galan et al. 1998; Plemper et al. 1998).
Our studies suggest that regulation of Hmg2p stability does not occur by modulation of the HRD-encoded degradation machinery (Hampton et al. 1996a; Gardner and Hampton 1999b). Rather, we posit that separate genes are required for specifically regulating degradation (see Fig. 1). We refer to these regulatory genes as COD genes, for the control of HMG-CoA reductase degradation. cod mutants deficient in regulation of Hmg2p degradation could fall into two phenotypic classes. Those in which Hmg2p is constitutively stable even when the degradation machinery is intact, and those in which Hmg2p is constitutively degraded even when degradation signals are low. This second class of mutants is the subject of this work. These mutants would allow degradation of Hmg2p in the presence of drugs such as lovastatin, an inhibitor of HMGR, that normally slow degradation by decreasing production of the signal. By our model (see Fig. 1), Hmg2p degradation in this type of cod mutant should still be blocked by hrd mutations (see Fig. 1).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmid Construction and DNA Manipulation
Plasmid pRH468 (integrating) expressed 1myc-Hmg2p from a GAPDH promoter. pRH468 was constructed by removing the NcoI-AatII fragment containing part of the URA3 open reading frame from pRH423 (integrating, URA3; Hampton and Bhakta 1997). The Hmg2p-GFP reporter protein was expressed from plasmid pRH680 (integrating, LEU2) or pRH469 (integrating, URA3; Gardner et al. 1998). pRH680 was constructed from pRH469 by replacing the AatII-ApaI segment of the URA3 gene in pRH469 with the AatII-ApaI fragment from pRS312 containing the LEU2 gene (Sikorski and Hieter 1989). Hmg1p-GFP was expressed from pRH475 (integrating, URA3; Gardner et al. 1998).
Plasmid pRH397 (2µ, URA3) expressed HA-tagged ubiquitin from the GAPDH promoter and was constructed as follows. The HA-ubiquitin coding region was excised from YEp112 (Ellison and Hochstrasser 1991) with EcoRI-ClaI and cloned into the same sites in Bluescript KS II to make pRH381. The HA-ubiquitin coding region was then cloned as a BamHI-ClaI fragment into the same sites of pRH98-3 (2µ, URA3; Donald et al. 1997) to yield pRH397.
Plasmids pRH810 (ARS/CEN, LEU2) and pRH811 (2µ, LEU2) expressed COD1. COD1 was amplified from yeast genomic DNA by PCR along with 300 bp of flanking sequence. The PCR product was cloned into the vector pCR2.1 (Invitrogen). The BamHI fragment containing COD1 was subcloned into YEp13 (2µ, LEU2; Broach et al. 1979) to generate pRH811 or into pRS315 (ARS/CEN, LEU2; Sikorski and Hieter 1989) to generate pRH810.
pCS186, used for disruption of the COD1/SPF1 open reading frame, was provided by Chise Suzuki (National Food Research Institute, Tsukuba, Japan; Suzuki and Shimma 1999).
Yeast Culture and Strains
Yeast strains were grown in minimal media (Difco Yeast Nitrogen Base without Amino Acids) with glucose and the appropriate supplements as described previously, except that leucine supplementation was increased to 60 mg/liter (Hampton and Rine 1994). Experiments were performed in minimal media at 30°C unless otherwise noted. For experiments with EGTA, strains were grown in YNB without CaCl2 from Bio 101, Inc. buffered with potassium MES to pH 6.5. Yeast was transformed with plasmid DNA using the LiOAC method (Ito et al. 1983). The parental genotype of all of the yeast strains used in this study was MATa ade2-101, his3-
200, lys2-801, met, hmg1::LYS, hmg2::HIS3, ura3-52, and leu2. Only distinguishing mutations are listed in Table and discussed below.
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RHY1127 (cod1-1/hrd1) was constructed by crossing RHY911 (cod1-1) with an isogenic strain with hrd1::URA3, followed by sporulation and recovery of RHY1127 as a meiotic segregant. RHY1076 (cod1-1, ubc7) was made by crossing RHY911 to RHY1056 (ubc7). The ubc7::URA3 allele carried by RHY1056 was made by PCR disruption with HIS3 and subsequent replacement of HIS3 with URA3 with pHU10 (Cross 1997).
RHY1473 (cod1-1) and RHY1475 (COD1) expressed Hmg1p-GFP from the GAPDH promoter. They were constructed in a cross of RHY911 to RHY550 (ura3-52::URA3::HMG1::GFP). RHY550 was constructed by integrative transformation of RHY532 with pRH475 (integrating, URA3, HMG1::GFP).
RHY1203 (cod1-1) expressing K6R-Hmg2p-GFP was constructed by plating RHY811 on 5-fluoro-orotic acid to remove pRH469, and subsequent transformation with pRH671 (integrating, K6RHMG2::GFP, URA3; Gardner and Hampton 1999a). The isogenic Cod+ strain RHY1205 was recovered from a cross of RHY1203 to RHY872.
SPF1/COD1 was disrupted by transformation of RHY791 with the BamHI-NheI fragment from pCS186. A corresponding Cod+ strain (RHY1232), was constructed by integrative transformation of RHY791 with linearized pCS186 to produce a strain with the cod1::LEU2 disruption allele in tandem with functional COD1. RHY2201, RHY2202, RHY2203, and RHY2204 were constructed by transforming pRH469 into CS601A, CS601B, CS601C, and CS601D, respectively (Suzuki and Shimma 1999).
The COD1 paralogue YOR291w was deleted by transformation of the haploid strain RHY791 with a KanMX disruption cassette with 40-bp flanks homologous to the YOR291w locus (Wach et al. 1994). Disruption was confirmed by PCR.
Optical Assays
The optical techniques used in this study are described in full detail elsewhere (Cronin and Hampton 1999). For assaying colony fluorescence, a Kodak Carousel® 4400 slide projector with a 488-nm narrow bandpass filter (Omega Optical) placed in the slot for slides provided illumination. Fluorescence of colonies was assessed visually with a long bandpass filter (Kodak Wratten No. 12) to remove blue light.
Analysis of Hmg2p-GFP fluorescence by flow microfluorimetry was performed on a Becton Dickinson FACScalibur® flow microfluorimeter and Cell Quest software. Strains were typically grown into early log phase in minimal media. After addition of drugs, cultures were incubated 4 h before analysis. Data from 10,000 cells were used for each histogram. In flow microfluorimetry experiments testing the effects of ions, cultures were incubated in exogenous CaCl2 or other salt 
12 h before the addition of mevalonate pathway inhibitors. For ion chelation experiments, EGTA was added to a final concentration of 780 µM in cultures diluted to an optical density of 0.001 at 600 nm 12 h before the addition of mevalonate pathway inhibitors. When indicated, MgCl2 or CaCl2 was added to EGTA-treated cultures to a concentration of 1 mM simultaneously with mevalonate pathway inhibitors, 4 h before analysis. For analysis by fluorescence microscopy, cultures grown as described for flow microfluorimetry were viewed using a Nikon Optiphot II microscope with a B2-A filter.
Cycloheximide Chase
To analyze regulated degradation directly, a cycloheximide chase followed by lysis and immunoblotting was used as described previously (Gardner et al. 1998).
Ubiquitination Assay
To aid in detection of ubiquitin, the strains tested were transformed with plasmid pRH379 (2µ, URA3) that expressed HA epitope-tagged ubiquitin from the GAPDH promoter. Ubiquitination of Hmg2p was assayed by immunoprecipitation of Hmg2p followed by immunoblotting with 12CA5 anti-HA antibody to detect covalently linked ubiquitin and with 9E10 anti-MYC antibody to detect immunoprecipitated 1myc-Hmg2p, as described previously (Gardner et al. 1998; Gardner and Hampton 1999b).
Mutagenesis and COD Screen
EMS (methane sulfonic acid ethyl ester) mutagenesis was based on the protocol of Lawrence 1991. Mutagenized RHY541 cells (75% killing by EMS) were sequentially evaluated for four regulatory phenotypes. The mutagenized cells were plated onto minimal solid medium supplemented with 30 mg/liter adenine sulfate, 30 mg/liter methionine, and 12.5 µg/ml lovastatin. The wild-type strain is normally bright when plated on this medium due to lovastatin-induced stabilization of Hmg2p-GFP. After 3 d, colonies were screened visually for low fluorescence compared with surrounding colonies. Candidate cod mutants with low fluorescence were isolated, rescreened for the optical phenotype, and then assayed for growth sensitivity to 200 µg/ml lovastatin by replica plating. Patches of candidate mutants were grown on YPD plates, then replica plated onto a YPD master plate. The master plate was immediately replica plated onto minimal plates with 200 µg/ml lovastatin and then onto minimal plates without lovastatin. This second dilution plating step insured low, uniform density of test patches on the lovastatin plates. Candidates that were sensitive to 200 µg/ml lovastatin were isolated from the YPD master plate. Dark, lovastatin-sensitive candidates recovered from the no-drug master were next tested directly for ability to regulate Hmg2p-GFP levels using flow microfluorimetry as described above. Finally, candidates exhibiting poor regulation of Hmg2p-GFP levels were assayed for regulation of 1myc-Hmg2p using the cycloheximide chase assay.
Genetic Analysis
Cod– candidates were crossed to the wild-type strain RHY542, to analyze segregation of the mutant phenotype. All mutants were recessive and belonged to a single complementation group.
The wild-type COD1 was cloned by plasmid complementation of a cod1-1 mutant with a URA3, ARS-CEN library (Rose et al. 1987). Ura+ transformants were tested for restoration of Cod+ phenotype using the screening assays described above.
Plasmids recovered from the revertants were retested by transformation into the mutant strain. Insert flanks were sequenced and the sequences were compared with the Saccharomyces Genome Database. Transformation of RHY811 (cod1-1) with plasmids pRH810 and pRH811 that contained only the COD1 coding region rescued the Cod– phenotype. Linkage of SPF1 to cod-1 was tested genetically by crossing a Leu–cod1-1 strain to a strain with LEU2 (from pCS186) integrated in tandem with functional COD1 allele at the COD1 locus. The resulting diploid was sporulated to confirm anti-segregation of the Cod– and Leu+ phenotypes in the haploid progeny.
Growth Curves
Susceptibility of cod1-1 to the mevalonate pathway inhibitors lovastatin, L659,699, zaragozic acid and Ro48-8071 was tested in minimal media. Dilute liquid cultures of RHY791 or RHY811 used to serially dilute the tested agent. The resulting cultures were incubated at 30°C and measured at various times for optical density at 600 nm.
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Results |
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The optical reporter protein Hmg2p-GFP undergoes normal, regulated degradation that can be observed by examining cellular fluorescence by microscopy or flow microfluorimetry (Hampton et al. 1996b; Cronin and Hampton 1999). Hmg2p-GFP was expressed from the constitutive glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter so that changes in the steady-state level of the protein and the resultant cellular fluorescence depended solely on changes in its degradation rate. Hmg2p-GFP expressed in wild-type cells is stabilized when the cells are treated with lovastatin, which lowers degradation signals from the mevalonate pathway by inhibiting HMGR, leading to increased cellular fluorescence (Cronin and Hampton 1999; Gardner and Hampton 1999a). A cod mutant unable to stabilize Hmg2p-GFP in response to lowered regulatory signals would fail to increase fluorescence in the presence of lovastatin. To score such mutants, we used our previously described GFP colony fluorescence assay (Cronin and Hampton 1999). Wild-type colonies expressing Hmg2p-GFP fluoresced brightly when plated on media containing a small dose of lovastatin (12.5 µg/ml), whereas cod mutants remained dark even in the presence of lovastatin.
A second, independent phenotype of poor Hmg2p regulation was also scored. We have shown that strains expressing only a poorly stabilized cis mutant of Hmg2p are much more sensitive to lovastatin than otherwise isogenic strains expressing normally regulated Hmg2p (Hampton et al. 1996a; Gardner et al. 1998). Thus, constitutive degradation of normal Hmg2p caused by a trans cod mutation would similarly be expected to render the mutant hypersensitive to lovastatin, compared with an isogenic wild-type strain. The parent strain for the COD screen expressed the normally regulated 1myc-Hmg2p from a constitutive promoter as its only source of HMGR activity (Gardner et al. 1998). Misregulation of 1myc-Hmg2p by a cod mutant was scored as hypersensitivity to lovastatin.
We combined the optical and pharmacological assays described above using a strain coexpressing Hmg2p-GFP and 1myc-Hmg2p. A successful cod candidate would be dark when plated on a low dose of lovastatin and be hypersensitive to the toxic effects of higher doses of lovastatin. By screening for both phenotypes (see Materials and Methods), we were able to rule out cis mutants of either reporter, as well as trans mutants that affected processes other than regulation of Hmg2p degradation. For example, a mutant unable to stabilize Hmg2p-GFP upon lovastatin treatment due to impermeability to lovastatin would be dark, but would be resistant to lovastatin as opposed to hypersensitive, and so would fail as a cod candidate.
COD1 Was Required for the Regulated Degradation of Hmg2p
A total of >300,000 colonies were screened, from which we isolated cod1-1 and 38 other members of the same complementation group. In all assays the cod1-1 mutant was defective in regulating Hmg2p-GFP degradation. In wild-type cells, inhibition of the mevalonate pathway with lovastatin stabilized Hmg2p-GFP resulting in increased fluorescence which was seen by microscopy and flow microfluorimetry (Fig. 2A and Fig. B). In contrast, addition of lovastatin hardly increased the fluorescence of cod1-1 mutant cells even though the concentration (25 µg/ml) used in these experiments was >10 times that normally needed to cause a maximal stabilization of Hmg2p-GFP (Gardner et al. 1998; Cronin, S., unpublished observations). Unlike lovastatin, addition of the squalene synthase inhibitor zaragozic acid increases degradation of Hmg2p-GFP by increasing a signal derived from the mevalonate pathway product farnesyl pyrophosphate (Hampton and Bhakta 1997; Gardner et al. 1998; Gardner and Hampton 1999a). Hmg2p regulation in cod1-1 mutants was also unresponsive to zaragozic acid when compared with wild-type cells (Fig. 2 B).
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We directly tested the ability of the cod1-1 mutant to regulate Hmg2p degradation with cycloheximide chase assays. In these experiments, protein synthesis was blocked at time zero by the addition of cycloheximide and degradation was allowed to proceed. 1myc-Hmg2p level was determined by immunoblotting at various times to assess degradation. Addition of lovastatin drastically slows the degradation of 1myc-Hmg2p in wild-type cells (Hampton and Rine 1994; Fig. 3), whereas in cod1-1 lovastatin had little or no effect.
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and ubc7 null mutations completely blocked the constitutive degradation caused by the cod1-1 mutation, such that fluorescence histograms of the double mutants were superimposable with the histograms of the hrd1 and ubc7 single mutants (Fig. 5 B). The stability of Hmg2p-GFP in each strain was directly tested by addition of cycloheximide followed by flow microfluorimetry to evaluate loss of cellular fluorescence due to Hmg2p-GFP degradation. This loss of fluorescence was completely inhibited by the presence of ubc7 (middle panels) or hrd1 (right panels). ubc7 (Fig. 5 C) or hrd1 (data not shown) also blocked degradation of 1myc-Hmg2p.
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cells, 6myc-Hmg2p-GFP was degraded rapidly with an estimated half-life of less than an hour, though it appeared to be slightly more abundant in the Cod– cells. In similar experiments, we found that the degradation rate of the regulated 1myc-Hmg2p did not differ substantially between wild-type and Cod– cells in the absence of mevalonate pathway inhibitors (data not shown). Clearly, cod1 mutation did not generally affect the stability of ER proteins. Rather, it specifically affected the feedback regulation of Hmg2p degradation by signals from the mevalonate pathway.
Recognition of cis Determinants for Hmg2p-regulated Degradation in cod1-1
Recently, we have shown that the regulated degradation of Hmg2p is critically dependent on two lysines in the transmembrane region of the protein. Replacement of either lysine 6 or lysine 357 of Hmg2p with arginine (or any other amino acid) strongly stabilizes Hmg2p or Hmg2p-GFP (Gardner and Hampton 1999b). Furthermore, the function of these lysines is extremely sensitive to alterations in Hmg2p structure. Since these lysines play a critical role in regulation of stability, we wondered if they would still function in a cod1-1 background. In both cod1-1 and wild-type genetic backgrounds, the K6R mutant of Hmg2p-GFP was more stable than wild-type Hmg2p, though some degradation is apparent in the cod1-1 mutant (Fig. 6 C). The same result was seen with the K357R replacement, and with the double mutant (data not shown). These data indicated that the degradation of Hmg2p in a cod1-1 mutant was still largely dependent on the same cis determinants that are necessary for regulating Hmg2p degradation in a wild-type cell. However, the incomplete stability of the K6R mutant in cod1-1 suggested that Cod1p has a subtle effect on the structure of Hmg2p or the recognition of its distributed degron (Gardner and Hampton 1999b).
COD1 Encoded a P-Type ATPase
The wild-type COD1 gene was isolated by plasmid library complementation of the cod1-1 mutation and was shown by linkage analysis to be YEL031w, previously isolated as SPF1 (sensitivity to Pichia farinosa). We deleted COD1 in a haploid strain and the null mutant was viable. In all assays for regulation of Hmg2p degradation, the cod1 mutant behaved exactly as the cod1-1 mutant (Fig. 7, data not shown). Additionally, overexpression of COD1 from a 2µ plasmid failed to produce any observable change in regulation of Hmg2p levels.
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The more distantly related P-type ATPase Pmr1p (22% identity, 39% similarity across 733 amino acids of homology) has recently been implicated in the degradation of the misfolded ER lumenal protein CPY*. Deletion of PMR1 prevents the degradation of CPY* by the ubiquitin proteasome pathway (Duerr et al. 1998). We examined the effect of pmr1 on Hmg2p-GFP degradation and found that Hmg2p-GFP stability was identical in an isogenic series including wild-type, pmr1, cod1, and pmr1/ cod1 strains (Fig. 7 A).
We tested the effects of pmr1 on Hmg2p-GFP regulation. In the wild-type H2071 genetic background used in these studies, Hmg2p-GFP degradation was relatively slow, but could be hastened by the addition of zaragozic acid (Fig. 7 B). While the response of Hmg2p-GFP to zaragozic acid was severely blunted in cod1, Hmg2p-GFP degradation was regulated normally in pmr1. Interestingly, the defective regulation of Hmg2p seen in cod1 was partially suppressed by simultaneous deletion of PMR1 suggesting that Pmr1p and Cod1p may both play a role in Ca2+ homeostasis, though in distinctly different ways.
Manipulating Ca2+ Affected Regulation of Hmg2p Stability
Phenotypic defects in some yeast P-type ATPase mutants can be overcome or exacerbated by manipulating the concentration of ions in the growth media (Duerr et al. 1998; Suzuki and Shimma 1999). We examined the effect of such manipulations on the cod1-1 mutant and wild-type strains. Incubation of a cod1 deletion mutant in 200 mM CaCl2 partially restored regulation of Hmg2p-GFP (Fig. 8). No other ions similarly tested (MnCl2, CaCl2, KCl, or NaCl) restored regulation of Hmg2p-GFP degradation. The high concentration of CaCl2 also caused a drop in the pH of the growth media, but lowering the pH of the media with HCl instead of CaCl2 failed to produce any effect (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The resulting mutant, cod1-1, had the desired phenotype: Hmg2p and Hmg2p-GFP each undergo constitutive degradation that is largely refractory to regulatory signals. Despite the lack of regulation, Hmg2p in the cod1 mutant was degraded at roughly the same rate as in the wild-type under normal growth conditions. Importantly, the constitutively degraded Hmg2p in a cod1 mutant was strongly stabilized by the simultaneous presence of a hrd1 or ubc7 mutant, showing that indeed regulation can be uncoupled from degradation. The cod1 mutant did not globally alter the stability of ER proteins, since cod1 mutation neither destabilized the normally stable Hmg1p-GFP nor altered the degradation rate of the misfolded, constitutively degraded 6myc-Hmg2p-GFP.
The degradation of Hmg2p can be slowed with drugs that block early in the mevalonate pathway or hastened by inhibition of squalene synthase with zaragozic acid (Hampton and Rine 1994; Hampton and Bhakta 1997; Gardner and Hampton 1999a). Both actions arise from alteration of the mevalonate-derived molecule farnesyl pyrophosphate (FPP; Gardner and Hampton 1999a). The cod1 mutants showed strongly blunted responses to both lovastatin and zaragozic acid. Thus, COD1 is required for coupling the rate of Hmg2p degradation to levels of the FPP-derived signal.
Cod1p is a P-type ATPase. Members of this widely conserved family function in ATP-dependent pumping of ions across biological membranes. The ion specificity of a given P-type ATPase can not yet be determined from sequence information alone. However, our studies indicate that Cod1p may be a Ca2+ transporter. The cod1 phenotype is reversed by addition of Ca2+ to the growth medium of mutant cells, and no other divalent ions tested could do this. Furthermore, treatment of wild-type cells with the Ca2+-preferring chelator EGTA caused aberrant regulation that was similar to the Cod1– phenotype, and specifically reversed by calcium.
This connection between calcium and HMGR regulation is especially intriguing given that regulated degradation of HMGR in mammals is similarly sensitive to perturbations of cellular calcium (Roitelman et al. 1991; Roitelman and Simoni 1992). In mammalian cells Ca2+ deprivation specifically inhibits the action of the degradation signal derived from FPP (Roitelman and Simoni 1992; Meigs et al. 1996). Similarly, cod1-1 mutants cannot respond to the FPP-derived signal for Hmg2p degradation.
Cod1p appeared to have a fairly specific function. The COD1 gene is not essential and the viable cod1 null mutant has a phenotype identical to that of the cod1-1 allele. A null mutation in COD1's closest paralogue, YOR291w, had no observable effect on yeast growth or Hmg2p regulation, alone or in combination with the cod1 null. Our ongoing studies have localized the Cod1p protein to the ER (Cronin, S.R., and R.Y. Hampton, manuscript in preparation), and our current model is that Cod1p is a Ca2+ transporter that is important for establishing a lumenal environment appropriate for control of Hmg2p stability. Changes to the ER environment in a cod1 mutant might alter Hmg2p stability by affecting the presentation of the highly specific structural determinants needed for regulated degradation (Gardner and Hampton 1999b). Alternatively, Cod1p activity may be critical for the function of trans factors regulating Hmg2p degradation.
A model in which Cod1p functions in the ER might explain some of the other phenotypes reported for cod1 mutants. COD1 was previously identified as SPF1 in an apparently unrelated screen for mutants resistant to a killer toxin. Other phenotypes reported for spf1 null mutants include defective glycosylation of invertase, resistance to vanadate, and sensitivity to hygromycin and calcofluor white (Suzuki and Shimma 1999). The sensitivity to hygromycin and calcofluor white, indicating defective cell wall synthesis, and the defective glycosylation of invertase all support a role for Cod1p in maintaining the lumenal environment of the ER, an environment thought to be controlled principally by PMR1.
The Golgi-localized P-type ATPase Pmr1p is thought to play a major role in maintaining Ca2+ levels in the secretory pathway (Strayle et al. 1999). PMR1 is required for numerous ER functions and has recently been identified as DER5, a gene necessary for the degradation of the misfolded lumenal protein CPY* (Duerr et al. 1998). In contrast to the effect of pmr1 on CPY* degradation, pmr1 had no effect on Hmg2p degradation or its feedback regulation. Thus, Pmr1p and Cod1p appear to have distinct roles, at least in this ER function. There are metazoan P-type ATPase family members of unknown function that have higher similarity to COD1 than to PMR1. It is tempting to speculate that they may have similar, specialized functions in a variety of organisms.
We currently do not know the mechanism of regulated stability, and one possibility is that there are proteins that specifically protect Hmg2p from degradation when degradation signals are lowered. If such protection factors exist, they would be particularly important both in the basic understanding of regulated ER degradation, and as possible targets for cholesterol lowering drugs. Loss of a protection factor by mutation would cause signal-independent, constitutive degradation of Hmg2p, and so would score as a cod candidate, like cod1-1. However, >300,000 mutagenized colonies of the parent strain were screened yet only alleles (39) of COD1 were recovered. Thus, it may be that the mechanism of Hmg2p regulation does not involve protection factors. Alternatively, it is possible that this version of the COD screen was biased towards recovery of COD1 alleles.
In summary, the above work demonstrates that the genetic approach to understanding regulated degradation of HMGR is a viable one. Integration of the COD1 gene's function into the scenario of Hmg2p regulation and ER function will be an important aspect of completing the picture of HMGR regulated degradation, in yeast and most likely in other eukaryotes as well.
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Acknowledgments |
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This work was supported by National Institutes of Health grant no. DK5199601 (R.Y. Hampton), an Affymax fellowship (S.R. Cronin), and a Searle Scholarship (R.Y. Hampton).
Submitted: 26 August 1999
Revised: 31 January 2000
Accepted: 31 January 2000
Abbreviations used in this paper: FPP, farnesyl pyrophosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; HMGR, HMG-CoA reductase.
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