A Highly Conserved Signal Controls Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) Reductase in Eukaryotes
1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês
10.1074/jbc.274.44.31671
ISSN1083-351X
AutoresRichard G. Gardner, Randolph Y. Hampton,
Tópico(s)RNA and protein synthesis mechanisms
ResumoSterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation. To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast. We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast. This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general. Rather, the FPP-derived signal specifically modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation. Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes. Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation. To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast. We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast. This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general. Rather, the FPP-derived signal specifically modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation. Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes. 3-hydroxy-3-methylglutaryl-coenzyme A reductase farnesyl diphosphate endoplasmic reticulum zaragozic acid lovastatin glyceraldehyde-3-phosphate dehydrogenase polymerase chain reaction kilobase pair herpes simplex virus fluorescence-activated cell sorter wild type hemagglutinin green fluorescent protein The mevalonate pathway is responsible for the biosynthesis of numerous essential molecules including prenyl groups, coenzyme Q, dolichol, and sterols such as cholesterol (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4700) Google Scholar). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR)1 is a key enzyme of the pathway and is rate-determining for cholesterol synthesis in mammals (2Siperstein M.D. Fagan V.M. J. Biol. Chem. 1966; 241: 602-609Abstract Full Text PDF PubMed Google Scholar, 3Dietschy J.M. Brown M.S. J. Lipid Res. 1974; 15: 508-516Abstract Full Text PDF PubMed Google Scholar). The mevalonate pathway is modulated in large part by feedback control of the amount of HMGR protein (1Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Crossref PubMed Scopus (4700) Google Scholar), and a significant portion of HMGR feedback control occurs through regulation of HMGR degradation (4Nakanishi M. Goldstein J.L. Brown M.S. J. Biol. Chem. 1988; 263: 8929-8937Abstract Full Text PDF PubMed Google Scholar, 5Chun K.T. Bar-Nun S. Simoni R.D. J. Biol. Chem. 1990; 265: 22004-22010Abstract Full Text PDF PubMed Google Scholar, 6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar). HMGR is an integral endoplasmic reticulum (ER) membrane protein, and its degradation occurs without exit from the ER (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar, 7Lecureux L.W. Wattenberg B.W. J. Cell Sci. 1994; 107: 2635-2642Crossref PubMed Google Scholar, 8Gil G. Faust J.R. Chin D.J. Goldstein J.L. Brown M.S. Cell. 1985; 41: 249-258Abstract Full Text PDF PubMed Scopus (275) Google Scholar). The non-catalytic, N-terminal transmembrane anchor of HMGR is both necessary and sufficient for regulated degradation (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar, 8Gil G. Faust J.R. Chin D.J. Goldstein J.L. Brown M.S. Cell. 1985; 41: 249-258Abstract Full Text PDF PubMed Scopus (275) Google Scholar, 9Jingami H. Brown M.S. Goldstein J.L. Anderson R.G. Luskey K.L. J. Cell Biol. 1987; 104: 1693-1704Crossref PubMed Scopus (89) Google Scholar, 10Skalnik D.G. Narita H. Kent C. Simoni R.D. J. Biol. Chem. 1988; 263: 6836-6841Abstract Full Text PDF PubMed Google Scholar). When there is abundant synthesis of pathway products, HMGR degradation is fast, and steady-state levels of the protein tend to be low. Conversely, when synthesis of pathway products is reduced, for instance when a patient is given the HMGR inhibitor lovastatin, HMGR degradation is slowed, and steady-state levels of the protein tend to increase. However, neither the identity of the mevalonate-derived signal nor the mechanism by which this signal is coupled to HMGR degradation is known. In order to discover and understand the mechanisms of HMGR-regulated degradation, we have been studying the process in the yeastSaccharomyces cerevisiae (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar, 11Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (477) Google Scholar, 12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar). Our earlier work has shown that the yeast HMGR isozyme Hmg2p is degraded in a regulated manner with many similarities to the analogous process in mammals. Through the use of genetic selections and screens, we have been able to identify genes required for the degradation of Hmg2p, calledHRD genes (11Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (477) Google Scholar), and genes required for normal regulation of Hmg2p degradation. 2S. Cronin and R. Hampton, manuscript in preparation. Concurrently with these screens, we have been studying the nature of the mevalonate-derived signals that control Hmg2p stability. Hmg2p degradation is regulated by an unknown signal from the mevalonate pathway (Fig. 1 a). Inhibiting early pathway enzymes, such as HMGR itself or HMG-CoA synthase, decreases the rate of Hmg2p degradation (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar). These early pathway blocks decrease the availability of a downstream signal for degradation. Conversely, inhibiting the enzyme squalene synthase, which is downstream of HMGR, stimulates degradation and ubiquitination of Hmg2p (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar). Furthermore, the degradation-enhancing effect of squalene synthase inhibition is abolished by simultaneous inhibition of HMG-CoA synthase or HMGR. These pharmacological studies imply that the signal for Hmg2p degradation is a pathway molecule between mevalonate and squalene (Fig.1 a). The most reasonable candidate for this signal is farnesyl diphosphate (FPP) or an off-pathway FPP derivative. The idea that FPP, or a derivative, is a positive signal for Hmg2p degradation is particularly interesting since there is accumulating evidence fromin vitro and in vivo studies that farnesol, an FPP-derivative, is a signal for regulation of mammalian HMGR stability (13Bradfute D.L. Simoni R.D. J. Biol. Chem. 1994; 269: 6645-6650Abstract Full Text PDF PubMed Google Scholar, 14Correll C.C. Ng L. Edwards P.A. J. Biol. Chem. 1994; 269: 17390-17393Abstract Full Text PDF PubMed Google Scholar, 15Meigs T.E. Roseman D.S. Simoni R.D. J. Biol. Chem. 1996; 271: 7916-7922Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 16Lopez D. Chambers C.M. Ness G.C. Arch. Biochem. Biophys. 1997; 343: 118-122Crossref PubMed Scopus (21) Google Scholar, 17Meigs T.E. Simoni R.D. Arch. Biochem. Biophys. 1997; 345: 1-9Crossref PubMed Scopus (104) Google Scholar). We have now tested the hypothesis that FPP provides a molecular signal for control of Hmg2p stability using unique genetic opportunities available in yeast. In conjunction with pharmacological and biochemical approaches, we have constructed yeast strains that allowed either overexpression or down-regulation of specific mevalonate pathway genes (Fig. 1 b). Our results indicated that FPP was indeed a source of a signal for Hmg2p degradation in yeast, demonstrating that there is striking conservation for this mode of HMGR regulation among eukaryotes. All enzymes were obtained from New England Biolabs (Beverly, MA). Chemical reagents were obtained from Sigma. Lovastatin and zaragozic acid were generously donated by Merck. Terbinafine was commercially obtained as a 1% Lamisil® solution from Novartis (East Hanover, NJ). ECLTM chemiluminescence immunodetection reagents were from Amersham Pharmacia Biotech. The anti-Myc 9E10 antibody was used as a cell culture supernatant obtained by growing the 9E10 hybridoma (ATCC CRL 1729) in RPMI 1640 culture medium (Life Technologies, Inc.) with 10% fetal calf serum. The anti-HA antibody was an ascites fluid obtained from Babco (Berkeley, CA). The anti-HSV-Tag antibody was obtained from Novagen (Madison, WI). Affinity-purified goat anti-mouse horseradish peroxidase-conjugated antiserum was obtained from Sigma. PCR was performed as described previously (18Gardner R. Cronin S. Leader B. Rine J. Hampton R. Mol. Biol. Cell. 1998; 9: 2611-2626Crossref PubMed Scopus (63) Google Scholar). The genes encoding squalene synthase (ERG9), farnesyl-diphosphate synthase (ERG20), and squalene epoxidase (ERG1) were PCR-amplified from yeast strain RHY623 genomic DNA (18Gardner R. Cronin S. Leader B. Rine J. Hampton R. Mol. Biol. Cell. 1998; 9: 2611-2626Crossref PubMed Scopus (63) Google Scholar), isolated by the Winston method (19Hoffman C.S. Winston F. Gene ( Amst. ). 1987; 57: 267-272Crossref PubMed Scopus (2127) Google Scholar), using separate primers that contained PstI andBamHI sites in the upstream primers and NheI andSalI sites in the downstream primers. The amplifiedERG9 and ERG20 genes were cloned between thePstI and SalI sites in pRH423 (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar), thereby placed under control of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) promoter (PGAPDH ) (20Bitter G.A. Egan K.M. Gene ( Amst. ). 1984; 32: 263-274Crossref PubMed Scopus (142) Google Scholar). The squalene synthase-containing plasmid was named pRH440, and the farnesyl-diphosphate synthase-containing plasmid was named pRH830. Squalene synthase (ERG9) was tagged at the C terminus with the HSV epitope sequence (21Isola V.J. Eisenberg R.J. Siebert G.R. Heilman C.J. Wilcox W.C. Cohen G.H. J. Virol. 1989; 63: 2325-2334Crossref PubMed Google Scholar). The plasmid containing an HSV-tagged squalene synthase expressed from the GAPDH promoter was made by annealing primers that encoded the HSV epitope sequence (QPELAPEDPED) and cloning the resulting DNA fragment between the NheI andSalI sites in pRH440 to yield pRH442 (ERG9-HSV). The plasmid to tag ERG9 at its genomic locus with HSV was made by digesting pRH442 with MunI, and the 5.4-kb vector fragment was reclosed to yield pRH885. The remaining portion oferg9 included codons 208–446 and the 3′ HSV sequence. Plasmids that allowed expression of the genomic copy of eitherERG9, ERG20, or ERG1 from theMET3 promoter (PMET3 ) (22Cherest H. Nguyen N.T. Surdin-Kerjan Y. Gene ( Amst. ). 1985; 34: 269-281Crossref PubMed Scopus (69) Google Scholar, 23Mountain H.A. Byström A.S. Larsen J.T. Korch C. Yeast. 1991; 7: 781-803Crossref PubMed Scopus (89) Google Scholar) were constructed as follows: pRH442 was digested with EcoRI, and pRH448 was digested with KpnI. Each vector was reclosed with the insert removed. The erg9 vector was named pRH948, and theerg20 vector was named pRH950. The MET3 promoter was cloned into pRH948 and pRH950 by digesting each plasmid withSspI and PstI and replacing the insert with theMET3 promoter containing SspI-PstI fragment from pHAM8 obtained from Dr. Harry Mountain (Staffordshire, UK). The PMET3 -erg9 plasmid was named pRH973 and the PMET3 -erg20 plasmid was named pRH975. A PCR product containing the ERG1 coding region was digested withBamHI and PvuII. The 840-base pair fragment was then cloned between the BamHI and HpaI sites in pRH973, and the resulting plasmid was named pRH1204. The plasmid to delete HRD1 was constructed as follows. A 1.45-kb BglII-XhoI fragment from pUG6 (24Güldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1390) Google Scholar), which contained the kanMX expression module (25Wach A. Brachat A. Poehlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2258) Google Scholar), replaced the 1.43-kb BglII-SalI fragment in pRH507, which contained the HRD1 gene (11Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (477) Google Scholar). The resulting plasmid was named pRH1122. Escherichia coli DH5α strains were grown at 37 °C in LB + amp (100 μg/ml). Yeast strains were grown at 30 °C in minimal medium supplemented with glucose and the appropriate amino acids, as described (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar). The lithium acetate method was used to transform yeast with plasmid DNA (26Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Yeast strain RHY871 (a his3Δ200 lys2-801 ade2-101 leu2 ura3-52::LEU2::hmg2-GFP met2 hmg1::LYS2 hmg2::HIS3::1MYC- HMG2) was the parent strain for transformation of plasmids containing GAPDH-expressed mevalonate pathway genes. RHY871 co-expressed 1Myc-Hmg2p (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar) and the autofluorescent Hmg2p-GFP (27Hampton R.Y. Koning A. Wright R. Rine J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 828-833Crossref PubMed Scopus (106) Google Scholar). Each integrating plasmid was introduced by targeted integration at the StuI site of theura3-52 genomic locus. Yeast transformants were selected for Ura+ prototrophy. Yeast strain RHY1326 (a his3Δ200 lys2-801 ade2-101 leu2 ura3-52::LEU2::hmg2-GFP MET2 hmg1::LYS2 hmg2::HIS3::1MYC- HMG2) was made by transforming RHY871 with a functional MET2 gene fragment from pGMET (28Baroni M. Livian S. Martegani E. Alberghina L. Gene ( Amst. ). 1986; 46: 71-78Crossref PubMed Scopus (24) Google Scholar), followed by selection for Met+prototrophy. RHY1326 and RHY1462 (a his3Δ200 lys2-801 ade2-101 leu2::6myc-hmg2-GFP::LEU2 ura3-52::6MYC-HMG2 MET2 hmg1::LYS2 hmg2::HIS3) were the parent strains for transformation of all plasmids containing PMET3 -expressed mevalonate pathway genes. Plasmid pRH973 (PMET3 -erg9) was introduced by targeted integration at the HpaI site of ERG9. Plasmid pRH975 (PMET3 -erg20) was introduced by targeted integration at the HindIII site of ERG20. Plasmid pRH1204 (PMET3 -erg1) was introduced by targeted integration at the AgeI site of ERG1. Yeast transformants were selected for Ura+ prototrophy. Cycloheximide chase assays and log phase steady-state assays were performed as described previously (18Gardner R. Cronin S. Leader B. Rine J. Hampton R. Mol. Biol. Cell. 1998; 9: 2611-2626Crossref PubMed Scopus (63) Google Scholar). Methionine chase assays were performed as follows. Cells were grown to early log phase with an absorbance (A 600) of 0.01. Methionine was added to a final concentration of 2 mm, and the cells grown at 30 °C for 15 h. Cells were then either used for the cycloheximide chase assay as described above or for the FACS analysis described below. Hmg2p ubiquitination assays were performed as described previously. 3Gardner, R. G., and Hampton, R. Y. (1999)EMBO J., in press. Strains were transformed with pRH1100,3 which expressed a triple HA epitope-tagged ubiquitin from the constitutive GAPDH promoter. Transformants were selected for Ade+ prototrophy. Hmg2p ubiquitination was assayed by immunoprecipitation of 1Myc-Hmg2p and then immunoblotting the precipitate for covalently linked HA-ubiquitin. FACS analysis was performed as described previously (18Gardner R. Cronin S. Leader B. Rine J. Hampton R. Mol. Biol. Cell. 1998; 9: 2611-2626Crossref PubMed Scopus (63) Google Scholar). Living cells were analyzed by flow microfluorimetry using a FACScaliburTM (Becton Dickinson, Palo Alto, CA) flow microfluorimeter with settings for fluorescein-labeled antibody analysis. Histograms were produced from 10,000 individual cells and were plotted with log fluorescence (arbitrary units) on the horizontal axis and cell number on the vertical axis. We previously discovered that addition of zaragozic acid (ZA), a potent inhibitor of squalene synthase (30Bergstrom J.D. Kurtz K.K. Rew D.J. Amend A.M. Karkas J.D. Bostedor R.G. Bansal V.S. Dufresne C. VanMiddlesworth F.L. Hensens O.D. Liesch J.M. Zink D.L. Wilson K.E. Onishi J. Milligan J.A. Bills G. Kaplan L. Nallin Omstead M. Jenkins R.G. Huang L. Meinz M.S. Quinn L. Burg R.W. Kong Y.L. Mochales S. Mojena M. Martin I. Pelaez F. Diez M.T. Alberts A.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 80-84Crossref PubMed Scopus (354) Google Scholar) (Fig.1 a), to yeast cells stimulated degradation of Hmg2p (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar). Squalene synthase inhibition would be expected to cause a build-up of farnesyl diphosphate (FPP), the substrate of the enzyme, implicating FPP as a source of the positive signal for Hmg2p degradation. However, it was possible that the effect of ZA was the result of some action of the drug. We addressed this by testing the effect of ZA on Hmg2p degradation in a strain that overexpressed squalene synthase. If ZA was inducing Hmg2p degradation through squalene synthase inhibition, then cells expressing increased levels of squalene synthase should require increased amounts of ZA to cause the same degree of degradation. We placed the squalene synthase gene (ERG9) under control of the constitutive GAPDH promoter, PGAPDH (20Bitter G.A. Egan K.M. Gene ( Amst. ). 1984; 32: 263-274Crossref PubMed Scopus (142) Google Scholar), and transformed the PGAPDH -ERG9 construct into yeast cells. PGAPDH -ERG9 was present as a single integrated copy through targeted insertion of an integrating vector. The strain expressing a single, integrated copy of PGAPDH -ERG9was 8-fold more resistant to the growth-slowing effect of ZA than a wild-type strain, consistent with approximately 8-fold higher squalene synthase levels in the PGAPDH -ERG9 strain than the wild-type strain, as measured by Western blot of HSV-tagged versions of squalene synthase. 4R. Gardner and R. Hampton, unpublished observations. We then tested if the Hmg2p degradation-enhancing effect of ZA required higher doses upon squalene synthase overexpression. The yeast strains used above also co-expressed two versions of Hmg2p, 1Myc-Hmg2p and the fluorescence reporter protein Hmg2p-GFP which have identical degradation behaviors as normal Hmg2p (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar, 18Gardner R. Cronin S. Leader B. Rine J. Hampton R. Mol. Biol. Cell. 1998; 9: 2611-2626Crossref PubMed Scopus (63) Google Scholar). Cells containing a single, integrated copy of PGAPDH -ERG9 required 8-fold more ZA to decrease 1Myc-Hmg2p and Hmg2p-GFP steady-state levels as was required for wild-type cells (Fig.2, a and b, 40 μg/ml for PGAPDH -ERG9 versus 5 μg/ml for wt), consistent with the 8-fold overexpression of squalene synthase. These results suggested that the mechanism for ZA-induced degradation of Hmg2p was through squalene synthase inhibition. The above results indicated that ZA altered Hmg2p degradation by decreasing squalene synthase activity. In that case, genetic down-regulation of the squalene synthase gene (ERG9) should also increase Hmg2p degradation. Because mevalonate pathway enzymes are essential for yeast viability, a null allele ofERG9 in yeast results in cell death (32Fegueur M. Richard L. Charles A.D. Karst F. Curr. Genet. 1991; 20: 365-372Crossref PubMed Scopus (49) Google Scholar, 33Jennings S.M. Tsay Y.H. Fisch T.M. Robinson G.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6038-6042Crossref PubMed Scopus (148) Google Scholar). Therefore, we made a conditional allele of squalene synthase by placing the wild-typeERG9 gene under control of the MET3 promoter (22Cherest H. Nguyen N.T. Surdin-Kerjan Y. Gene ( Amst. ). 1985; 34: 269-281Crossref PubMed Scopus (69) Google Scholar), which is repressed by the presence of high extracellular concentrations (>0.5 mm) of methionine (23Mountain H.A. Byström A.S. Larsen J.T. Korch C. Yeast. 1991; 7: 781-803Crossref PubMed Scopus (89) Google Scholar). We constructed a "promoter-switch" plasmid that contained a truncated version of erg9 placed behind the MET3 promoter. Targeted integration of this plasmid into the ERG9 locus resulted in the creation of a single, functional copy ofERG9 under control of the regulated MET3 promoter (PMET3 ) (Fig. 1 b). This plasmid was used to transform a methionine prototroph yeast strain to allow growth in any concentration of methionine. The strain also co-expressed 1Myc-Hmg2p and Hmg2p-GFP allowing a complete characterization of Hmg2p degradation. When grown in the absence of methionine, normal regulated Hmg2p degradation was observed in the yeast strain that expressed squalene synthase from the MET3 promoter (PMET3 -ERG9).4 Genetic down-regulation of squalene synthase enhanced Hmg2p degradation in a manner identical to inhibition with ZA. After 15 h growth in 2 mm methionine, Hmg2p degradation in the PMET3 -ERG9 strain was increased. This was indicated by a lower steady-state level of Hmg2p-GFP and 1Myc-Hmg2p in the PMET3 -ERG9 strain compared with the wild-type strain (Fig. 3, a and b, PMET3 -ERG9 versus wt). The effect of down-regulation was similar to that in the wild-type strain after 15 h incubation in the presence of ZA (PMET3 -ERG9 compared with wt ,+ ZA). Hmg2p-GFP in the PMET3 -ERG9 was stabilized by the addition of lovastatin to a similar degree as the stabilization of Hmg2p-GFP by lovastatin addition in the wild-type strain preincubated with ZA (PMET3 -ERG9, + Lov compared withwt ,+ZA+ Lov), indicating that enhanced Hmg2p degradation caused by squalene synthase down-regulation or ZA addition was still regulated by the mevalonate pathway. It was possible that enhanced Hmg2p degradation was the result of cell death inadvertently caused by squalene synthase down-regulation rather than the build-up of a positive signal for degradation. Cells containing the PMET3 -ERG9 allele ceased to grow after 15 h incubation in 2 mm methionine (6 doublings) (see below, Fig. 8 b). However, when the PMET3 -ERG9 cells from this 15-h time point were transferred to media without methionine, they retained the same plating efficiency4 and growth curve as identically treated wild-type ERG9 cells (see below, Fig. 8 b), indicating that these cells were still viable. The enhanced Hmg2p degradation by ZA addition or squalene synthase down-regulation required the HRD gene-encoded proteins. Enhanced degradation by squalene synthase or ZA addition was completely eliminated by the presence of the hrd1Δ allele (Fig.3 b, PMET3 -ERG9, hrd1Δ, and wt + ZA , hrd1Δ), which normally stabilizes Hmg2p (11Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Crossref PubMed Scopus (477) Google Scholar). This indicated that the degradation-enhancing effect of squalene synthase down-regulation or ZA addition required a functionalHRD pathway and was not due to aberrant degradation by an alternate pathway. Furthermore, identical steady-state levels of 1Myc-Hmg2p in the wild-type, hrd1Δ strain and the PMET3 -ERG9, hrd1Δ strain indicated that the lower steady-state levels of Hmg2p in the PMET3 -ERG9strain were due only to enhanced degradation and not reduced translation efficiency. Additionally, we observed that overexpression of squalene synthase stabilized Hmg2p. In the strains previously described in Fig. 2, which overexpressed squalene synthase by the presence of the PGAPDH -ERG9 allele, both versions of Hmg2p were significantly stabilized. This was observed as both an increase in the steady-state level of 1Myc-Hmg2p (Fig. 4, PGAPDH -ERG9 versus wt) and Hmg2p-GFP,4 and as a decrease in 1Myc-Hmg2p degradation when squalene synthase was overexpressed (Fig. 4, PGAPDH -ERG9 versus wt). Thus, squalene synthase overexpression had the opposite effect on Hmg2p degradation as squalene synthase down-regulation. The above studies implicated the substrate of squalene synthase, FPP, as a central molecule in the regulation of Hmg2p stability. Manipulation of squalene synthase predicted to increase FPP levels hastened Hmg2p degradation, whereas manipulation of squalene synthase predicted to decrease FPP levels slowed Hmg2p degradation. We wanted to test further the hypothesis that FPP was the source of the positive signal for Hmg2p degradation, by eliminating FPP production. This could be accomplished by inhibition of farnesyl-diphosphate synthase (FPP synthase), which generates FPP as a product. Unfortunately, no drugs are currently available that inhibit yeast FPP synthase in vivo. Therefore, we again used a genetic approach to lower FPP synthase production. As with the other enzymes of the mevalonate pathway, yeast cells that contain a null allele of FPP synthase are not viable (34Chambon C. Ladeveze V. Oulmouden A. Servouse M. Karst F. Curr. Genet. 1990; 18: 41-46Crossref PubMed Scopus (82) Google Scholar), so we generated a conditional allele of the FPP synthase coding region (ERG20) that resulted in the wild-type ERG20 gene placed under control of theMET3 promoter, similar to ERG9 described above. When grown in the absence of methionine, normal regulated Hmg2p degradation was observed in the yeast strain that expressed FPP synthase from the MET3 promoter (PMET3 -ERG20).4 In contrast to enhanced Hmg2p degradation caused by squalene synthase down-regulation, FPP synthase down-regulation resulted in stabilization of Hmg2p. When the PMET3 -ERG20 strain was grown 15 h in 2 mm methionine, Hmg2p was exceedingly stable, as indicated by both a higher steady-state level and decreased degradation of 1Myc-Hmg2p in the PMET3 -ERG20 strain compared with the wild-type strain (Fig.5 a,PMET3 -ERG20 versus wt). Similarly, Hmg2p-GFP steady-state levels were increased dramatically in the PMET3 -ERG20 strain (Fig. 5 b, PMET3 -ERG20 versus wt), and this effect was mimicked by growth of the wild-type strain in the presence of lovastatin (PMET3 -ERG20 compared withwt ,+ Lov), which acts to slow Hmg2p degradation (6Hampton R.Y. Rine J. J. Cell Biol. 1994; 125: 299-312Crossref PubMed Scopus (186) Google Scholar). The effect of FPP synthase down-regulation was reversed by the presence of ZA in the degradation assay (Fig. 5 a, PMET3 -ERG20, 4ZA lane), and in Hmg2p-GFP steady-state fluorescence (Fig. 5 b, PMET3 -ERG20,+ ZA compared withwt,+ ZA), indicating that regulated degradation of Hmg2p was still operative. Although the methionine treatment halted the growth of the PMET3 -ERG20 strain after 15 h (see below, Fig. 8 b), if these cells were then transferred to media that did not contain methionine, they retained a similar plating efficiency4 and growth curve as the wild-typeERG20 control cells (see below, Fig. 8 b), indicating that Hmg2p stabilization was not a result of inadvertent cell death. Thus, Hmg2p stabilization was caused by FPP synthase down-regulation, further strengthening the model that FPP was the source of a positive signal for Hmg2p degradation. The covalent attachment of ubiquitin is a critical and regulated step in Hmg2p degradation (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar). Because FPP synthase down-regulation stabilized Hmg2p, we also determined its effect on Hmg2p ubiquitination. To assay Hmg2p ubiquitination, strains containing the appropriate wild-type or regulated alleles of the FPP synthase gene were transformed with plasmids that expressed HA epitope-tagged ubiquitin.3 Hmg2p ubiquitination was assayed by immunoprecipitation of 1Myc-Hmg2p, followed by anti-HA immunoblotting to detect covalently attached HA-ubiquitin.3 Regulation of Hmg2p ubiquitination was assessed by performing each assay in the presence of lovastatin (Lov), which decreases ubiquitination, or zaragozic acid (ZA), which increases ubiquitination (12Hampton R.Y. Bhakta H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12944-12948Crossref PubMed Scopus (123) Google Scholar) (Fig.6, wt). Down-regulation of FPP synthase caused a drastic decrease in the level of Hmg2p ubiquitination (Fig. 6, PMET3 -ERG20 versus wt, no drug lanes). This effect was similar to the addition of lovastatin to the wild-type strain during the ubiquitination assay (wt , Lov lane). Furthermore, the addition of ZA for 10 min during the ubiquitination assay, which normally increases Hmg2p ubiquitination, had no effect on Hmg2p ubiquitination in the FPP synthase down-regulated strain (wt ZA lane versus PMET3 -ERG20, ZA10 lane). This effect of FPP synthase down-regulation on ZA action was identical to addition of lovastatin to the wild-type cells (PMET3 -ERG20, ZA10 lane compared withwt , ZA+ Lov lane). However, addition of ZA for 1 h did increase Hmg2p ubiquitination in the FPP synthase down-r
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