Correspondence to: Randolph Y. Hampton, UCSD Department of Biology, 9500 Gilman Dr. #0347, La Jolla, CA 92093-0347. Tel:(858) 822-0511 Fax:(858) 534-0555 E-mail:rhampton{at}biomail.ucsd.edu.
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Abstract |
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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
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Introduction |
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The ER resident, integral membrane protein, hydroxymethylglutaryl-coenzyme A reductase (HMGR)1, catalyzes the first committed step of the mevalonate pathway from which sterols and other essential isoprenoids are produced. HMGR is subject to numerous modes of regulation, including feedback control of HMGR stability (
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 (
Our studies suggest that regulation of Hmg2p stability does not occur by modulation of the HRD-encoded degradation machinery (
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We have isolated the first cod mutant and cloned the relevant gene, COD1. The cod1-1 mutant failed to properly regulate Hmg2p degradation but did not generally affect the degradation of ER proteins. COD1 was identical to SPF1, a gene previously identified in an unrelated screen (
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Materials and Methods |
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Materials
Restriction enzymes, Vent DNA polymerase and T4 DNA ligase were obtained from New England Biolabs. Lovastatin, L659,699 and zaragozic acid were generously provided by Dr. James Bergstrom (Merck, Rahway, NJ). Ro48-8071 was a gift from Dr. Olivier Morand (F. Hoffman-LaRoche). The 9E10 cell culture supernatant was produced in our lab from cells (CRL 1729; American Type Culture Collection) grown in RPMI1640 culture medium (GIBCO BRL) with 10% fetal calf serum and supplements. 12CA5 anti-HA antibody was obtained from Dr. Don Rio (UC Berkeley, Berkeley, CA). Affinity-purified HRP-conjugated goat antimouse antibodies were purchased from Sigma. ECL chemiluminescence immunodetection reagents were from Amersham. All other chemical reagents were obtained from Sigma or Fisher.
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;
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 (
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;
pCS186, used for disruption of the COD1/SPF1 open reading frame, was provided by Chise Suzuki (National Food Research Institute, Tsukuba, Japan;
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 (200, lys2-801, met, hmg1::LYS, hmg2::HIS3, ura3-52, and leu2
. Only distinguishing mutations are listed in Table 1 and discussed below.
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RHY541, the parent strain for the COD screen, expressed the reporter proteins 1myc-Hmg2p and Hmg2p-GFP. RHY541 was constructed from the mevalonate auxotrophic strain RHY468 by integration at the hmg2::HIS3 locus of plasmid pRH468 expressing 1myc-Hmg2p and selection for mevalonate prototrophy. The resulting strain was then transformed with pRH469 cut at the BsgI site and selected for uracil prototrophy to give the RHY541 parent. Various genotypes of cod1-1 were constructed by crossing with appropriate isogenic strains and isolation of haploid progeny.
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 (
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;
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 (
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 (
Optical Assays
The optical techniques used in this study are described in full detail elsewhere (
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 (
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 (
Mutagenesis and COD Screen
EMS (methane sulfonic acid ethyl ester) mutagenesis was based on the protocol of
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 (
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 COD Screen
We designed a screen to identify cod mutants that could not slow degradation of Hmg2p when degradation signals from the mevalonate pathway were low. Specifically, we isolated mutants that failed to stabilize Hmg2p upon treatment with lovastatin, an inhibitor of HMG-CoA reductase that lowers these signals (
The optical reporter protein Hmg2p-GFP undergoes normal, regulated degradation that can be observed by examining cellular fluorescence by microscopy or flow microfluorimetry (
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 (
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 (
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The cod1-1 mutant was hypersensitive to lovastatin, indicating that the coexpressed 1myc-Hmg2p was also misregulated. cod1-1 rendered cells 10 times more sensitive to lovastatin than the isogenic wild-type cells (Fig 2 C). In contrast, the growth sensitivity of cod1-1 mutants to inhibitors of other pathway enzymes including HMG-CoA synthase (L659,699), squalene synthase (zaragozic acid), or oxidosqualene-lanosterol cyclase (Ro48-8071) remained unchanged (Fig 1 A and 2 C, and data not shown). These results suggested that cod1-1 hypersensitivity to lovastatin was due to misregulation of Hmg2p rather than any general effects on pathway enzymes or other pleiotropic actions of the cod1-1 mutation.
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 (
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COD1 Was Required for Regulation of Hmg2p Ubiquitination
Ubiquitination is required for Hmg2p degradation and is regulated in response to the same stimuli that control Hmg2p stability. For example, treatment with zaragozic acid increases Hmg2p ubiquitination and this effect is blocked by simultaneous treatment with inhibitors of upstream pathway enzymes such as HMG-CoA synthase (
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ER Degradation Mutants Stabilized Hmg2p in cod1-1
We have proposed a model in which the COD genes regulating Hmg2p degradation are distinct from the genes encoding the ER degradation machinery (Fig 1). This model predicts that the unregulated degradation of Hmg2p in a cod1-1 mutant would still be blocked in hrd mutants, which are deficient in Hmg2p degradation. To test this model, we constructed strains with the cod1-1 mutation and null mutations in HRD1 or UBC7, each are essential for Hmg2p degradation ( 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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Degradation of Other ER Proteins in cod1-1
The cod1-1 mutation removed regulation of Hmg2p, rendering its degradation constitutive. We wanted to determine if cod1-1 mutation generally altered the stability of ER proteins. Accordingly, we examined the effect of the cod1-1 mutation on the stable, ER localized Hmg1p-GFP reporter protein derived from the HMGR isozyme Hmg1p (
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We also tested the effect of COD1 mutation on the stability of a short-lived, unregulated variant of Hmg2p, 6myc-Hmg2p-GFP ( 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 (
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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COD1 belongs to a large family of genes encoding P-type ATPases that actively transport various ions across membranes (
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 ( 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 (
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We also tested the effect of Ca2+ depletion on regulation of Hmg2p stability in wild-type cells by treatment with EGTA, a chelator of divalent ions with high Ca2+ specificity (Fig 9). Overnight treatment with a sub-lethal concentration of EGTA blunted the regulatory responses to both lovastatin and zaragozic acid when compared with untreated cells. These effects of EGTA treatment were overcome by addition of CaCl2, but not MgCl2. These experiments with EGTA were consistent with a role for CaCl2 in the regulation of Hmg2p degradation. However, EGTA treatment of wild-type cultures did not fully mimic the Cod- phenotype and significantly reduced growth (data not shown) indicating that the effects of COD1 were more specific for Hmg2p degradation than those caused by gross Ca2+ depletion.
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Discussion |
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The striking cis and trans specificity of Hmg2p stability regulation led us to posit that this process involves a separate set of genes referred to as COD genes. In this work, we have isolated the first member of this class of genes. We focused our search on cod mutants that always degrade Hmg2p even when signals for degradation are low. By our model (Fig 1), Hmg2p degradation in such a cod mutant would still be halted by mutations in genes encoding the degradation machinery, such as a hrd1 mutant.
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 (
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 (
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 (
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 (
The Golgi-localized P-type ATPase Pmr1p is thought to play a major role in maintaining Ca2+ levels in the secretory pathway ( 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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Footnotes |
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1 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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Acknowledgements |
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The authors thank Dr. Karen Berger (University of California San Diego, Department of Biology) for plasmids, Dr. Chise Suzuki (National Food Research Institute, Tsukuba, Japan) for providing information, strains, and plasmids before publication, and Dr. Robert Rickert (University of California San Diego, Department of Biology) for use of the FACScalibur® flow microfluorimeter and software.
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
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References |
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