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THEJOURNAL OF BIOLOGICAL CHEMISTRY 01985 by The American Society of Biological Chemists, Inc. Val. 260, No. 1, Issue of January 10, pp. 522-530,1985 Printed in U.S.A. Domain Structure of 3-Hydroxy-3-methylglutarylCoenzyme A Reductase, a Glycoprotein of the Endoplasmic Reticulum* (Received for publication, June 26, 1984) Laura LiscumSQ, Janet Finer-Moorell, RobertM. Stroudv, Kenneth L. LuskeySII, MichaelS. Brown$, and Joseph L. Goldstein$ From the $Departments of Molecular Geneticsand InternalMedicine, University of Texas Health Science Center a t Dallas, Southwestern Medical School, Dallas, Texas 75235 and the llDepartment of Biochemistry and Biophysics, Schoolof Medicine, University of California, San Francisco, San Francisco, California 94143 We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NHz-terminal membrane-bounddomainof 339 residues and a COOHterminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NHz-terminal domain contains 7 membranespanning regions. Analysis of the gene structure reveals that each proposed membrane-spanningregion is encoded in a separate exon and is separated from the adjacent membrane-spanningregion by an intron. The COOH-terminal domain of the reductase is predicted to contain two @-structuresflanked by a series of amphipathic helices, which together may constitute the activesite. The NH2-terminal membrane-bounddomain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known. findings suggest that the active siteof the reductase is contained within a water-soluble 53-kDa domain that isexposed to proteases and thus mustproject into the cytoplasm. This domain is presumablycontiguous with a hydrophobic domain that fixes the reductase to the ER membrane. The nucleotide sequence of a cloned cDNA for hamster reductase was recently established, and the complete amino acid sequence of the enzyme was deduced (3). This sequence revealed that the NH2-terminal one-third of the 97-kDa reductase is extremely hydrophobic, while the COOH-terminal two-thirds is more hydrophilic and typical of a water-soluble protein (3). These findings suggest that thehydrophobic NH2terminal end of the reductase binds the molecule to the ER membrane. The hydrophilic COOH-terminal portion would thencorrespondtothe 53-kDawater-solublecatalytically active domain thatprojects into the cytoplasm. In the current paper, we test this hypothesis through structural analysis of the reductase protein. For this purpose, we have prepared antibodies against syntheticpeptides that correspond to specific regions of the reductase sequence, using the techniques pioneered by Lerner (6) and by Walter and Doolittle (7). These antibodies have been used to mapregions of the reductase that are releasedfrom the membrane by proteases. The informationderived from these studies isused in conjunction with computer-based analytic techniques (810) to develop a model for the secondary structure and membrane orientation of the reductase. EXPERIMENTALPROCEDURES HMG-CoA reductase,’ the rate-controlling enzyme of cholesterol biosynthesis (l),is a 97-kDa transmembrane glycoprotein that resides in the ER of animal cells (2-4). An enzymatically active 62-kDa fragment can be released from ER membrane vesicles by cleavage with an endogenous protease (45). This 62-kDa fragment can be reduced to a soluble 53-kDafragment by further digestion with a n exogenous proteasewithout loss of enzymaticactivity(4,5).These * This research was supported by Grants HL20948 and GM 24485 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of Postdoctoral Fellowship HL 06533 from the National Institutes of Health. I( Established Investigator of the American Heart Association. The abbreviations used are: HMG-CoA reductase, 3-hydroxy-3methylglutaryl coenzyme A reductase; ER, endoplasmic reticulum; kDa, kilodalton; KLH, keyhole limpet hemocyanin; EGTA, ethylene glycol bis(6-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; NaDodSO,, sodium dodecyl sulfate. ‘ Materials-Purified Ca2+-activatedproteasefrom bovineheart (11) was kindly provided by George N. DeMartino of the Department of Physiology, University of Texas Health Science Center at Dallas. Compactin was kindly provided by Akira Endo, Tokyo Noko University, Tokyo, Japan. A polyclonal antibody directed against the 53kDa water-soluble fragment of rat liver HMG-CoA reductase was prepared in rabbits as previously described (12).Other materials were obtained from previously described sources (4, 12). Antibodies against Synthetic Peptides-Peptides corresponding to amino acids (354-368), (379-393), and (874-887)of thehamster reductase (see Fig. 1A) were synthesized by solid-phase chemical methods (13)in the laboratory of Richard Lerner, Department of Molecular Biology, Scripps Clinic and Research Foundation, La Jolla, CA. Peptide (379-393)had a cysteine residue added to the COOHterminal end. The composition of each peptide was confirmed by amino acid analysis. Each peptide was coupled to KLH with maleimidobenzoyl-N-hydroxysuccinimideester (14).New Zealand White rabbits were injected subcutaneously on days 0 and 14 with synthetic peptide (0.2mg) coupledto KLH. The peptide-KLH conjugates were emulsified with Freund’s complete adjuvant (day 0) or incomplete adjuvant (day 14) in a total of 1.5 ml. Intraperitoneal injections of synthetic peptide (0.2mg) coupled to KLH were given on days 28 and 35 in a total of 1 nil containing 4 mg of alum. Rabbits were bled 522 Domains of HMG-CoA Reductase 523 first on day 35 and then 6-9 days after each booster injection. y20 @ 30 50 40 60 0 In Globulin fractions of the immune sera were prepared by precipitation I V G T V T L T I CW S M N M F T G NN K I C G Y N Y E CP K F E E O V L S SO l l l L T l T R C MLSRLFRllHGLNASHPUEV with 50% ammonium sulfate (15). 0 110 100 @ 140 90 80 130 120 Cells and Microsomes-UT-1 cells, a compactin-resistant clone of I A I L Y I Y F Q F QNLRQLGSKY I L G I A G L F TFI S S N F S T V VI H F L D K E L T GL N E A L P F F L L I D L S R A S A L Chinese hamster ovary cells, were grown in monolayer in Ham's F160 150 200 190 180 @ 170 @ 210 12 medium supplemented with 25 mM N'-2-hydroxyethylpiperazineAKFALSSNSQOEVRENIARGMAILGPTFTLOALVECLVIGVGTMSQVRQLEIMCCFCMSVLANVFVNT N'-2-ethanesulfonic acid (pH 7.4), 10% (v/v) newborn calf lipopro280 270 260 250 240 230 220 tein-deficient serum, and 40 PM compactin (12). Cells were seeded in FFPACVSLVLELSRESREGRPIYQLSHFARVLEEEENKPNPVTQRVMIMSLGLVLVHAHSRYIMPSPQ Petri dishes (60 X 15 mm) or roller bottles and harvested on day 6 290 300320 310 340@ 330 350 (12). Cells were washed a t 4 "C with Dulbecco's phosphate-buffered NSTTEHSKVSLGLOEOVSKRIEPSVSLYQFVLSMISMOIEQVVTLSLAFLLAVKYIFFEQAETESTLSL saline, scraped, collected by centrifugation (1000 X g for 10 min a t 4 "C), and disrupted inone of three ways: Method 1 , addition of buffer A (15% NaDodSO,, 8 M urea, 10% sucrose, 62.5 mM Tris-chloride, 490 480 470 460 450 440 430 100 mM dithiothreitol at pH 6.8); Method 2, hypotonic incubation (15 GTSPPVMRTQELEIELPSEPRPNEECLQILESAEKGAKFLSOAElIQLVNAKHIPAYKLETLMETHERG min at 24 "C) in buffer B (10 mM sodium phosphate, 5 mM EGTA, 560 550 540 530 5 00 520 510 10 mM dithiothreitol, 0.1 mM leupeptin, 0.2 mM phenylmethanesulVSIRRQLLSTKLPEPSSLQYLPYROVNYSLWGACCENVIGYMPIPVGVAGPLCLOGKEYQVWATTEGC fonyl fluoride at pH 6.5) followed by incubation (15 min at 24 "C) in 570 630 620 600 610 buffer B containing1%(w/v) Zwittergent 3-14; or Method 3,590 nitrogen 580 LVASTNRGCR AIGLGGGASSRVLAOUITRGPVVRLPRACOSAEVKAMLETPEGFAVIKOAFOSTSRFARL cavitation (4) in buffer C (0.15 M NaC1, 50 mM potassium phosphate, 670 660 650 640 680 7 690 00 5 mM EGTA, 10 mM dithiothreitol, 0.1 mM leupeptin, 0.2 mM phenQKLHVTWAGRNLYIRFQSKT GOACIGMNMIS KGTEKALLKLQEFFPEMQILAVSGNYCTOKKPAAINYIEG ylmethanesulfonyl fluoride at pH 7.5). When cells were disrupted by 750 740 730 720 710 760 770 Method 3, a 1000 X g supernatant fraction was centrifuged at 100,000 PGKTVVCEAVIPAKVVREVLKTTTEAMIOVNINKNLVGSAMAGSIGGYNAHAANIVTAIYIACGQOARQN X g for 60 min at 4 "C to obtain a microsomal pellet. 790 780 800 810 840 820 830 Immunoblotting and Other Assays-Samplesfor electrophoresis VGSSNCITLMEASGPTNEOLYISCTRPSIEIGTVGGGTNLLPQQACLPL GVQGACKONP GENARQLARI were prepared in buffer A. NaDodS04-polyacrylamide gel electropho880 870 850 860 resis and immunoblotting on nitrocellulose were performed as deVCGTVMAGELSLMAALMGHLVRSHMVHKI: SKIFWCEL~~-I~LKKJ~~ scribed (4) except that 2% (w/v) hemoglobin was used in place of 5% (w/v) bovine serum albumin. Gels were calibrated with M, standards obtained from Bethesda Research Laboratories. Protein was meaSOLUBLE W M A I N c sured by a modification of the method of Lowryet al. (16). Enzymatic 53kDa MEMBRANE BOUND c DOMAIN -662 kDa activity of reductase was measured as described (12). One unit of reductase activity represents the formation of 1 nmol of meValonate/ NH 2 COOH min at 37 "C. CHO Methods for Analysis of the Secondary Structure of HMG-GOA Reductase-Hydrophobicity plots of the amino acid sequence were FIG. 1. Amino acid sequence and domain map of hamster generated by the method of Kyte and Doolittle (10). The hydrophoHMG-CoA reductase. Panel A , amino acid sequence. Amino acid bicity values used for each amino acid were those of Eisenberg et al. residues are shown in the single-letter code, which translates to the (17). The secondary structure of the extra-membrane regions of three-letter code as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; reductase was analyzed by two procedures: 1) a secondary structure G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q , Gln; prediction scheme described by Garnier et al. (9); and 2) a Fourier R, Arg; S, Ser; T, Thr;V, Val; W, Trp; and Y, Tyr. The 7 postulated transform analysis of amino acid hydrophobicity described by Finer- membrane-spanning regions are numbered and indicated bysolid Moore and Stroud (8). The secondary structure prediction scheme lines above and below the sequence. The regions of the protein analyzes the amino acid sequence by taking into account 8 flanking sequence selected for synthesis of peptides (354-368), (379-393), and residues on either side of the subject residue and relies on a data base (874-887) are indicated by dashed underlines. Four pairs of closely of amino acids as they occur in x-ray crystal structures of soluble spaced dibasic amino acids between residues 360 and 400 are indicated proteins. The Fourier transform analysis was carried out as previously by brackets. The assignment of phenylalanine as the amino acid at described ( 8 ) , using a variable window size (number of successive residue 673, which was previously ambiguous on the basis of cDNA residues in each linear transform) of from 11 to 25 residues. The sequencing (3), has now been established from the DNA sequence of variable window size allows for a determination of the length of a a genomic clone that encodes this region (31). Panel B , linear map of given feature, such as an amphipathic a-helix or an amphipathic p- HMG-CoA reductase protein showing structural features in relation sheet in the structure, since the signal will be highest if the window to proteolytic fragments. The holoenzyme has a molecular weight of size exactly matches the size of the periodic feature. The transforms, 97,000. The cross-hatched areas in the membrane-bound domain called amphipathic Fourier transforms, were plotted as two-dimen- represent the 7 putative membrane-spanning regions. The presumed sional contour maps with frequency (l/periodicity) on the vertical site of carbohydrate addition (Asn 281) is indicated by CHO. The two axis and residue number on the horizontal axis. This procedure postulated sites at which proteolytic clips generate soluble fragments detects periodicity in hydrophobicity and, therefore, assays an overall of62 and 53 kDa are indicated. The location of the two synthetic peptides (residues 354-368 and 379-393) used to define the sites of property of secondary structures (and tertiary folding patterns) as they evolved in the extra-membrane folded structure. A repeat fre- proteolysis are indicated by asterisks. quency of 1/3.6 residues" is characteristic of an amphipathic a-helix, and a frequency of 1/2 residues" suggests an amphipathic 0-pleated protein) as a result of amplification and enhanced transcripsheet structure. Twisted tertiary structures alter these periodicities tion of the reductase gene (18). In previous studies we showed somewhat, and peaks can, therefore, be diagnostic of packing arrangethat a fully active fragment of the reductase could be released ment. - - RESULTS AND DISCUSSION Identification of the 53-kDa Fragment as the COOH-terminal Domain of HMG-GOAReductase Fig. 1A shows the amino acid sequence of HMG-CoA reductase as determined from the nucleotide sequence of cDNA clones isolated from UT-1 cells (3). UT-1cells are a clone of compactin-resistant Chinese hamster ovary cells (12) that express large amounts of HMG-CoA reductase (2% of total from UT-1 cell microsomes as a 53-kDa water-soluble protein after digestion with a Ca2+-activatedprotease (4).This 53kDa fragment is equivalent to the fragment that was initially purified fromratliver microsomes and used to generate a polyclonal anti-reductase antibody (12). To determine the portion of the intact 97-kDa reductase that is represented by the 53-kDa water-soluble fragment, we obtained a synthetic peptide corresponding to amino acids 874-887 at the extreme COOH terminus of the protein (Fig. 1A). After coupling this peptide to keyhole limpet hemocy- Domains of HMG-CoA Reductase 524 anin, we injected it into rabbits, and the resultant antibody was used as a probe to identify the COOH terminus of the reductase. This COOH-terminal peptide antibody was used in conjunction with the polyclonal antibody to perform the immunoblotting experiments shown in Fig. 2. The left panel of Fig. 2 shows a series of immunoblots using theantibodydirectedagainsttheentire53-kDafragment (anti-reductase IgG) (Lanes 1-6). When intact UT-1 microsomes were subjected to NaDodS04-gel electrophoresis and blotted against the anti-reductase IgG, a predominant 97-kDa band was seen (Lane I ). (The less prominent bands a t 62 and 53 kDa are proteolytic fragments that are produced during solubilization of the cells (Refs. 4 and 5 andsee below).) After centrifugation of the microsomes a t 100,000 x g, nearly all of the immunoreactive 97-kDa reductase was found in the membrane pellet (Lane 3 ) and very little was found in the supernatant (Lane 2).When the microsomes were treated with a Ca'+-activated protease, the predominant immunoreactive 97kDa reductase was reduced to 53kDa (Lane 4 ) . After centrifugation a t 100,000 x g, most of this fragment remainedin the supernatant (Lane 5 ) , and little was found in the pellet (Lane 6). The right panel of Fig. 2 shows aliquots of the same incubation mixtures that were subjected to electrophoresis and immunohlotted against the anti-COOH-terminal peptide antibody (Lanes 7-12). This antibody reacted with the intact 97-kDa protein (Lane 7), which was found in the pellet after centrifugation (Lane 9).After treatment with the Ca'+-activatedprotease,theanti-COOH-terminalantibody reacted with the 53-kDa fragment (Lane IO),which remained in the supernatant after centrifugation (Lane I I ). In the same experiment, we measured the reductase activity in the 100,000 x g supernatant and pellet fractions. After proteolysis, of the microsomal reductaseactivity was recovered in the 5.7-kDa 100,000 X g supernatant (data not shown). Thus, the water-soluhlecatalyticallyactivefragmentrepresentsthe COOH-terminal portionof t,he reductase. Since this fragment can he releasedfrommicrosomes by proteolysis, it must project to the exterior of the microsomes, i.c. into the c-ytoplasm. Identification of the Membrane-boundFragmcnt and Localization of Proteolytic Clcavagc Sites Reductasecontains a cluster of four pairs of positively charged amino acids located in a proline-rich region between residues 360 and 400 (Fig. 1 A ) . These basicresidues were deemed to be likely sites for the proteolytic cleavage that gives rise totheCOOH-terminal62-kDafragmentduring incubation of the cells in hypotonic buffer. Multiple attempts todeterminetheamino acidsequence of theproteolytic fragments after labeling of the cells with radioactive amino acidsfailed. This failure has occurred underconditions in which we can easily obtain sequence information from the 97kDa holoprotein (3). We believe that our failure to ohtain sequence information on the53- and 62-kDa fragmentsis due to the fact that the proteolytic clip is not a clean one and that Anti-Reductase Anti -Peptide (874-887) multiple amino termini around the area of the proteol-ytic ' 1 2 3 4 5 6"7 8 9 10 1 1 1 2 ' cleavage are formed. We, therefore, decided to localize the sites of proteolytic rr) I cleavage by probing with anti-peptide antibodies.Accordingly, 9 we prepared antibodies against synthetic peptides correspondx 97 c (379-293). ing toamino acids (354-368) andaminoacids which are twoneighboring stretches on the molecule that might flank the predicted protease cleavage site (Fig. 1A ). These two antibodies were used to localize the proteol-ytic -m fragments of the reductase after incubation of intact cells in P a hypotonic buffer, which increases the formation of the 62kDa fragment (4, 5), and after treatment in oitro with the S P M M S PS MP 1 Ca'+-activated protease, which reduces the 62-kDa fragment to 53 kDa. The proposed sites of proteolysis based on the experiments described below are shown schematically in Fig. PROTEOLYSIS 1R. FIG. 2. NaDodS0.-polyacrylamide gel electrophoresis and 3 (Lanes 1 - 4 ) shows a series of immunoblotting analysisof soluble and membrane-bound pro- The left panel ofFig. were immunoblots performedwith the anti-reductase antibody( i x . teolyticfragments of HMG-CoA reductase.UT-1cells disrupted by Method 3 , and a microsomal pellet was isolated as the antibody directed against the entire COOH-terminal5.7described under "Experimental Procedures." Aliquots of total micro- kDa fragment). When UT-1 cells were harvested in the ahsomes (300 pg of protein; 22-36 units of reductase activity) were sence of hypotonic incubation, the antihody stained the intact incuhated in buffer D with proteolytic inhibitors (50 mM Tris-chloride, 5 mM dithiothreitol, 5 mM EGTA, 0.1 mM leupeptin, 0.1 mM 97-kDa protein (Lane 1 ) . When cells were suhjected to hyphenylmethanesulfonyl fluoride at pH 7.5) at 4 "C for 30 min (Lanes potonic incubation prior tosolubilization, much o f the reductase was reduced to the 62-kDa form ( I m w 2)as a result of 1-3 and 7-9)or in buffer E without proteolytic inhibitors (50 mM Tris-chloride, 5 mM dithiothreitol, 5 mM CaCI2 at pH 7.5) supplecleavage by the endogenous protease (4, 5 ) . Previous studies mented with 15 pg of Ca2+-activatedprotease at 24 "C for 30 min have shown that this 62-kDa fragment contains virtually all (Lanes 4-6 and 10-12). After incubation, Na2C0,was added to 0.1 M of the catalytic activity of the intact 97-kDa enzyme (4, 5). to remove loosely adherent proteins, and EGTA and leupeptin were adjusted to 5 and 0.1 mM, respectively, in all tuhes. An aliquot from When microsomes were isolated and incubated with the exeach tube (125 pg of protein) was centrifuged at 100,000 x g for 20 ogenous Ca2+-activated protease, the immunoreactive reducmin at 24 'C to obtain a supernatant fraction (S)and a pellet ( P ) . tase was cleaved to the 5.7-kDa fragment, which remained in The pellet wasresuspended to thevolume of the supernatant fraction. the supernatant after a 100,000 X g centrifugation ( I m w .?). Aliquots (40 pl, 20 pg of protein) of microsomes before centrifugation Very little of the 53-kDa fragment was found in the pellet ( M )and aliquots (40p l ) of the supernatant fractions and pelletswere (Lane 4 ) . solubilized in buffer A and subjected to electrophoresis and immuThe middlepanel of Fig. 3 shows aliquots of the same noblotting with polyclonal rabbit anti-reductase IgC at 10 pg/ml or incubation mixtures that were subjected to electrophoresis anti-peptide (874-887) antihody (y-globulin fraction) at 100 pg/ml, followed by '2LI-labeledgoat anti-rabbit IgG (10' cpm/ml) and auto- and immunoblottedwith the antibody directed against peptide radiography for 18 h at -70 "C. (354-368). Again, this antibody identified the intact 97-kDa L d 1 + - + Domains of HMG-CoA Reductase Anti-Peptide Anti-Peptide 354 - 368 379 - 393 . 2i .i (379-393) are present on the 62-kDa fragment but are removed when this fragment is clipped further to the X-kDa I form. 10 The above data suggest that the endogenous protease that 9 liberates the 62-kDa fragment after h.ypotonic incubation cuts X the reductase in the region between peptides (354-368) and (379-393) (see Fig. 1, A and H ) . This region, from residues 368 to 379, contains two pairs of basic residues (Fig. 1A ). If thecleavageoccurredint.hisregion,thereleasedsoluble fragment would have a mass of 56 kDa, which agrees reasonably well with the 62 kDa that was estimated bv NaDodS0,gel electrophoresis.Most of the62-kDafragmentremains bound to the UT-1 membranes at normal ionic strength ( 5 ) . However, much of it can be released into the supernatant if the microsomes are washed with a solution containing 0.1 M Na,CO:I, suggesting that the 62-kDa fragment is soluble and (data not bound to themembranebyionicinteractions shown). Fraction I C C S P C C S P I C C S q , 35The other fragment produced by the cleavage ( i . ~the kDa fragment) remains totally memhrane hound after proteFIG. 3. NaDodS0.-polyacrylamide gel electrophoresis and olysis, even after washing in Na,CO, buffer (see Fig. 3, 1anr.s immunoblotting analysis of proteolytic fragments of HMG7 a n d 8 ) . This fragment is known to contain cart)ohydrate CoA reductase. UT-1 cells were disrupted by Methods 1. 2 , o r :{ a s it is labeled with [:'H]glucosamine ( 4 ) . If proteolysis because indicated helow. Cell extracts ( C ) ohtained hy Methods 1 and 2 were occurs between residues 368 and 380 as discussed above, the soluhilized in hufferAwithoutfurthertreatment.Microsomesohtained hv Method :% (225 p g of protein) were incuhated in huffer E membrane-bound NH,-terminal fragment should have an M, without proteolytic inhihitors and supplemented with 4.5 pg of Ca2+- of 42,000, as compared with a value of 35,000 estimated by activated protease at 24 "C for 30 min. After incuhation, Na2COn was electrophoresis. It is possible that the hydrophobic fragment added, EGTA and leupeptin were adjusted to 5 and 0.1 mM, respecis really 42 kDa, but it binds more NaDodSO, than the M, tively, and the sample was centrifuged to obtain a supernatant fraction ( S ) and pellet (1') a s descrihed in Fig. 2 . Aliquots (as in Fig. 2) standard proteins and, therefore, has an electrophoretic mobilitygreaterthanexpectedonthebasis of its true M,. of soluhilized cell extracts, supernatant t'ractions, and pellets were suhjected to electrophoresisandimmunohlottingwithpolvclonal Alternatively, it is possible that a protease clips the protein rahhit anti-reductase lgG at 10 pglml. anti-peptide (354-368) antiat a n exposed site near the NHZ-terminal end, reducing the 30 pg/ml, or anti-peptide (379-393) hodv(7-glohulinfraction)at 42 to 35 kDa. mass of the major membrane fragment from antihody (y-globulin fraction) at 200 pg/ml followed hy ""I-laheled We have attempted to test this possibility hv preparing an goat anti-rahhit IgC ( 1 0 " cpm/ml) and autoradiography for 9 h ( I x n m antibody against the extreme NH,-terminal peptide. Unfor1-4) or 24 h ( I x n r s -5-12) at -70 "C. 1mw.q I , 5 , 9, cells ( C )prepared hy rapid soluhilization (Method 1 ) ; l a n e s 2, 6. 1 0 , cells ( C )suhjected tunately, multiple immunizations have failed to yield antihodto h.ypotonic incuhationandsoluhilizationwithZwittergent3-14 ies that react with the intact protein, perhaps because there (Method 2); 1 m w . s .'I, 7, 11, supernatant fraction ( S ) of microsomes are only 9 hydrophilic amino acids at the NH, terminus (see prepared hv Method 3 and suhjected to proteolysis and centrifugation; below). 1.anr.s 4. 8. 12, pellet (1') of microsomes prepared hy Method 3 and From the dataof Fig. 3, we can estimate the pointat which suhiected to proteolysisandcentrifugation. (No proteinhandsare thecleavagetothe53-kDafragmenttakesplace. If t h e visihle in Ixnc 11; the smudge in this lane is a hlotting artifact due NaDodSO, gel reflects a true difference of 9 kDa hetween the to an imperfection in the gel.) 62- and 53-kDa fragments, then the cleavage that gives rise prot.ein (I,anc ,5). When the cells were subjectedto hypotonic to the 53-kDa fragment must occur in the region of residues incubation, the 62-kDa form of the reductase that was visu- 4.50-470 (Fig. 1, A and R ) . Since the 53-kDa fraffment retains full enzymatic activity, this would place the active site of the (Lane 2 ) wasnot alizedwiththeanti-reductaseantibody of residue 470. visualized wit.h the anti-peptide (354-368) antibody (Lane 6). enzyme on the COOH-terminal side Instead,thisanti-peptideantibodyreactedwith a 35-kDa fragment. When isolated microsomes were incubated with the Model for Secondap Structurc of HMC-C'oA Rrductnsc exogenous Ca"-activated protease, this 3.5-kDa fragment was The above proteolysis experiments reveal that the NH,also formed. After centrifugation the 35-kDa fragment was terminal one-third of the reductase is associated with the Eli found in the membrane pellet (Lane 8 ) and not in the super- membrane and that the COOH-terminal two-thirds, which natant ( I h e 7 ) . (The broad 70-kDa band Lane8 in is believed contains the catalytic site, projects into the cvtoplasm. To to he a dimer of the 35-kDa fragment.) Thus, the proteases learnmoreaboutthestructures of thesetworegions, we must. be cutting the reductase on the COOH-terminal side of turnedtocomputer-modelingschemesthatareknown to peptide (354-368), leaving this peptide (or at least the major predict, with reasonable accuracy, the secondary structuresof part of it) on a 35-kDa fragment that remains bound to t h e proteins based on characteristicsof the amino acid sequence. membrane. To locate hydrophobic regions of the sequence that span the T o localize the cleavage site more precisely, we used the ER membrane, we used the method of Kyteand1)oolittle antibody against peptide (379-393). This antibody also iden- (10). This method involves calculation of the relative hydrotified the int,act 97-kDa protein ( I h e 9 ) . After hypotonic phobicity at each amino acid residue in the reductase sequence incubation, it recognizedthe62-kDafragment ( I m w 10). as averaged over the adjacent 21 residues. In our model we When the microsomes were digested with the Ca2+-activated have drawn these putative membrane-spanning regions in t h e protease and then subject.ed to electrophoresis, the anti-pep- n-helical confimration because thevpreof the correct length, tide (379-393) antibody no longer reacted with any of t h e about 26 amino acids, to span a 40-A bilayer, and because in fragments (1,nnc.s 1 1 a n d 12).Thus, the amino acids in peptide for which the structureof bacteriorhodopsin, the only protein Anti -Reductase I I 526 Domains of HMG-CoA Reductase 263G J / FIG. 4 Domains of HMG-CoA Reductase a 527 postulated membrane-spanning regions in this protein (Table I). This membrane-spanning region is structurally similar to membrane-spanning region 7 of bacteriorhodopsin, according to the criteriaof McLachlan (27). 3 0.2 ALPFFLLLIDLSkASALAkFALSS (HMG-CoA reductase) 0.0 H IETLLFMVLDVSAkVGFGLILLkS (bacteriorhodopsin) (Ref.21) -0.2 Thismembrane-spanning region of thereductase shows strong peaks in the Fourier transforms at 1/3.6 residues", suggesting that it has an amphipathic a-helical configuration (Fig. 7). This structuremay be similar to the amphipathica- 0.64 I I I helix that spans thebilayer in the acetylcholine receptor (8). 0 200 400 600 800 Several other predicted membrane-spanning segments of the reductase contain amino acids that are usually charged. Residue These residues tend to cluster on faces of the putativehelices, FIG. 5. Hydrophobicity ( H ) plots of the amino acid sequence making ion pairing within orbetween packed helices possible of HMG-CoA reductase. Residue hydrophobicitiesh, were averaged (Fig. 4).There are approximately equal numbers of positively over windows of 21 residues. The uertical scale denotes the average free energy in kilocalories/mol/aminoacid for transfer from a hydro- charged and negatively charged amino acids in the predicted intramembrane regions of the reductase. phobic to a hydrophilic environment. The residue number is plotted on the horizontal scale. The 7 predicted membrane-spanning regions Each of the 7 membrane-spanning regions in the reductase are numbered 1-7. The two regions that are predicted to have 0is separated from the next by a hydrophilic linker.As depicted structure and that contain hydrophobic stretches are labeled bl and in Fig. 4, the linkers contain numerous chargedresidues and b2 (see Fig. 4). are predicted tocomprise extended 6-structure orloops. The longest linker region extends from residues 221 to 314. It is the structure of membrane-spanning regions is known (19postulated to lie on the luminal side of the ER membrane 23), the a-helices are oriented perpendicular to the membrane between the sixth and seventh membrane-spanning regions. plane (20). residues 221 t o 246 within To identify regions of secondary structure in the reductase The Fourier transform predicts that this linker form an amphipathic helix, whereas residues 247 protein, we employed a Fourier transform analysis to search to 307 have an amphipathic extended @-structure (Fig. 7). A for periodicities in hydrophobicity that would suggest the short stretch of hydrophobic residues (258-267) is present but presence of amphipathic a-helixor 6-pleated sheet structures either inside or outside the hydrophobic segments (8). The is not long enough to span the membrane. Residues 221-314 includea potentialsite for N-linked secondary structure prediction algorithmof Garnier et al. (9) glycosylation at asparagine 281, the only N-linked glycosylawas also used t o help predict the secondary structure of these regions. This program utilizes a data base of amino acids as tion signal (i.e. asparagine-X-serine or asparagine-x-threothey occur in x-ray crystal structures. proposed The secondary nine) in the NH2-terminal 35-kDa membrane-bound portion structure based on combined the useof these analytic methods of the protein. Since the NH,-terminal 35-kDa fragment is known to contain N-linked carbohydrate (4), this constitutes is presented in Fig. 4. supportive evidence thatasparagine 281 is locatedin the NH2-terminal Domain (Residues1 to 339)"The Kyte-Doolittle plot of amino acid hydrophobicities revealed that the lumen of the ER. COOH-terminal Domain (Residues 340-887)"As shown by NH2-terminal domain of the reductase (residues 1-339) is in the Kyte-Doolittle plot inFig. 5, the COOH-terminal domain generalmuch more hydrophobic than the COOH-terminal general hydrophilic.This region does contain domain (Fig. 5). Within the first339 residues, seven peaks of of reductase is in hydrophobicity are noted, each of which extends over a dis- a few short hydrophobic segments and a long one between tance large enough to span a membrane bilayer (Fig. 5, regions residues 520 and 545 labeled bl in Fig. 5. None of these 1-7). The boundaries andoverall hydrophobicities of each of segments can be membrane-spanning regions since each is these putative membrane-spanning regions are listed in Table contained in the water-soluble 53-kDa proteolytic fragment of the reductase. They probably form a hydrophobic core in I. Fig. 6 shows a detailed diagramof the predicted orientation this globular domain. The COOH-terminal domain includes of the amino acids in the NH2-terminal domain of the reduc- 7 long amphipathic regions with the periodicity of a-helices (2.6 to 4 residues) interspersedwith turns, random coil structase. The seven putative membrane-spanning segments are tures, and extended @-structures depicted as in Fig. 4. in generallesshydrophobic thantheputativemembraneResidues 351 to 365, which are just outside the seventh spanning regions inretinalrhodopsin (19, 24) and in the membrane-spanning region, have the unusual sequence ( x - y acetylcholine receptorsubunits (8, 25) butaresimilarin hydrophobicity to those in bacteriorhodopsin (21-23) and the z-proline)4.The amphipathic Fourier transformsshow a peak lac carrier protein of Escherichia coli (26). of periodicity at 1/6.5 residues-' (Fig. 7), which is exactly the The fourthhydrophobic stretch of reductase, which extends frequency found for the proline-richsequence in avian panfromresidue 124to 149, isthemost hydrophilic of the creatic polypeptide, whose crystal structure is known(28). I I 1 1 FIG. 4. Schematic model for the predicted secondary structure of HMG-CoA reductase. All charged residues are marked with a + or -. Cysteine residues are indicated by S. * indicates a potential N-linked glycosylation site at residue 281. G, glycine; N, asparagine; P, proline; Q, glutamine; R, arginine. The 40-A lipid bilayer boundaries are drawn on either side of the predicted membrane-spanning regions. Residues 516-586 and 734-830, enclosed by the clotted lines, each contain 8 t o 11 stretches of extended structure and are rich in cysteine, proline, and glycine residues. L)ornains o ~ ~ ~ G - Reductase C O A 528 TABLE I Hydrophobicity values for the me~~rane-spanning regions of the ~ H ~ - t e rdomain m i ~ ofH ~ e - e o Areductase " Membranespanning region Residues ____ Length Average hydrophobicity" no. residues 1 2 3 4 5 6 7 25 29 10-39 57-78 90-114 124-149 160-187 192-220 315-339 30 22 25 26 28 0.21 0.20 0.36 0.15 0.25 0.27 0.19 The value for each region represents the average hydrophobicity/ amino acid (kcai/mol) for the most hydrophobic set of 20 contiguous residues. The Fourier plots would, therefore, suggest that these residues form a p o l ~ r o ~ i n e - l i khelix e similar to that found in the proline-rich region of pancreatic polypeptide. This region is adjacent to the paired basic residues that appear to be the site where the active fragment of the reductase is cleaved proteolytically from the membrane-spanning domain (see above). Two regions of extended @-structureare predicted within the COOH-terminal domain between residues 495-595 and between 735-825 (Fig.4). Both of these regions are quite bland in the amphipathic transformand have several stretches of hydrophobic extended sequences interspersed with short hydrophilic stretches and other characteristic turnforming residues. Both regions are rich in glycines, prolines, and cysteines. Together, residues 516-586 and 734-830 con- stitute only about 19% of the sequence, yet they include 10 out of 27 cysteines in theprotein as well as nearly one-half of the glycines. Each of these two regionshas a central relatively hydrophobic core (bl and b2 in Fig. 5 ) that is almost symmetrically flanked by sequences with very strong amphipathic a-helical periodicity in the Fourier transform. The amphipathic structures undoubtedly interface between the hydrophobic core and the solvent, although we cannot predict the detailed arrangement of secondary structure elements into a three-~mensionalstructure from results of our analysis. It seems likely that residues from the putative @-domains form at least part of the active site for the reductase. In this regard, it is of interest that thereductase is known to require high concentrations of thiol-reducing agents for activity (29) and that there are large numbers of cysteine residues in the predicted @-domains.The enzyme reaction by whichreductase catalyzes the conversion of HMG-CoA to mevalonate is believed to involve thetransient protonation of a histidine residue (30). In this regard, 7 histidines are located at the periphery of the @-domainsat residues 474,487,634,751,860, 865, and 868. Relation between E x ~ n - ~ n t r o~n ~ rof the ~ Gene ~ and ~ r Domain Structure of the Protein The reductase is encoded by a 25-kilobasegene,which contains 20 exons that are separatedby 19 introns (31). Eight of these introns occur within the NH2-terminal hydrophobic domain. Fig. 8 (upper panel)relates the positions of these 8 introns to the 7 membrane-spanning regions of the reductase protein. As indicated by the arrows, 7 of theintronsare located near the junction of a membrane-spanning region and pGV%[4% Amino AcidsI@SA-COOH Lumen of ER N P V T Q FIG.6. Model for the possible orientation of the hydropho~icdomain of HMG-GOAreductase in the membrane of the endoplasmic reticulum. Amino acid residues are shown in the singie-letter code (see legend to Fig. I). Amino acids with positively charged side chains are shown in circks. Amino acids with negatively charged side chains are shown in squares. The N-linked carbohydrate chain a t residue 281 is indicated between the sixth and seventh membrane-spanningregion. e Domains of HMG-CoA Reductase 529 * * c m H M G CoA Reductase cytop1a.m ER Crystalloid ER iumm of ER Rhodopsin of D8.k Rod Outer Segment FIG. 7. Amphipathic Fourier transforms between frequencies 0 and 112 residues" for the sequence of HMG-CoA reductase. The transforms were calculated with a moving window of 20 residues and were not normalized (8).The central residue number is plottedonthe horizontalanis;uerticallines aredrawn every 10 residues on the plots. The frequency is plotted on thevertical anis. A frequency of 1/2 corresponds to the characteristic amphipathic pstructure frequency of 1/2 residues". A horizontal line on the plot is drawn at the characteristic amphipathic a-helix frequency of 1/3.6 residues". Seven hydrophobic segments between residues 1 and 339 are predicted to span the membrane as a-helices; their positions are denoted with open cylinders above the horizontal axis and are numbered 2-7. The hydrophilic sequence 220-310 is predicted to contain an amphipathic a-helix(shaded cylinder)and a (?-strand(dotted line). Residues 495-595 and 735-825, marked by dottedlines along the horizontal anis, are predicted to have a P-structure. On either side of these regions, the predicted amphipathic a-helices are marked with shaded cylinders. The hydrophobic sequence 846-862 is predicted to have an a-helical structure (open cylinder). FIG. 8. Similarities in the membrane organization of HMGCoA reductase (upper panel) and rhodopsin (lower panel). The position in the protein sequence where an intron interrupts the coding region of the gene for each protein is indicated by an arrow. The DNA sequence data for exon-intron junctions for the reductase and rhodopsingenes were obtained from Refs. 31 and 36, respectively. Of the 19 introns in the reductasegene, one occurs in the 5' untranslated region and 18 occur within the coding region (31). These 18 intronsinterruptthe gene atthepositionscorrespondingtothe following amino acid residues: 55, 93, 122, 150, 186, 221, 260, 314, 397,455,520, 573, 626, 661,718, 765,818, and870 (31). only two intracellular glycoproteins whose amino acid sequences are known. They show asurprisingsimilarity in several respects including mode of biosynthesis, post-translational glycosylation and phosphorylation, location in membranes of intracellular organelles, organization of 7 membrane-spanning regions, and domain structures of their genes. These similarities are discussed below and illustrated schematically in Fig. 8. Rhodopsin consists of an apoprotein, opsin, of 348 amino acids that bears a covalently attached polyisoprenoid chromophore, 11-cis-retinal (24).Rhodopsin is inserted cotranslationally into the rough ER even though it has no cleaved signal sequence (33, 34). The protein is then transported to an external loop. These introns divide the coding region of the rod-outer-segment disk where it remains embedded as an the gene in such a way that each of the first 6 membraneinternal membrane protein (reviewed in Ref. 34). Hydrophospanning regions is separated from the adjacent membrane- bicity plots of the amino acid sequence of rhodopsin suggest spanning region by a single intron. The coding region for the that the protein crosses the membrane 7 times (24,35). relatively large hydrophilic domain between the sixth and "High mannose" carbohydratechainsare added to two seventh membrane-spanning region is split by 1 intron. asparagine residues near the NH2 terminus during synthesis In theCOOH-terminal domain of the reductase, the introns of rhodopsin in the ER (34). Rhodopsin also undergoes phosgenerally fall on thehydrophilic side of predicted amphipathic phorylation on serine and threonineresidues near the COOHhelices (4 introns occur at residues 455,626, 661, and 718) or terminal endof the molecule (35). The gene encoding rhodopin predicted @-turns(4 introns occur at 573, 765, 818, and sin contains 4 introns, 3 of which interrupt the coding se870) (31). One intron occurs in the center of a predicted p- quence at points corresponding to the interface between a strand (residue 520), and anotheroccurs in the vicinity of the postulated membrane-spanningregion and anexternal hydroproteolytic site that generates the 62-kDa fragment of the philic loop (36) (Fig. 8). reductase (residue 397). These findings are consistent with Like rhodopsin, the amino acid sequence of HMG-CoA the hypothesis that introns usually occur at the surface of reductase suggests a protein with 7 membrane-spanning reproteins (32). gions and a hydrophilic NH, terminus that projects into the lumen of the ER(Fig. 8).Like rhodopsin, reductase is believed Similarities between HMG-CoA Reductase and Rhodopsin to be inserted into the ERcotranslationally (37),even though The 339-residue membrane domain of HMG-CoA reductase it lacks a cleavable signal sequence (3, 37). bearsa strong resemblance to rhodopsin, the 348-residue Like rhodopsin, reductase has a high-mannose N-linked membrane proteinof the retinalphotoreceptor that is respon- oligosaccharide that is added cotranslationally and is subsesible for light reception (24). Reductase and rhodopsin are the quently trimmed but is never processed to the mature form 530 Domains of H M ~ - ~ Reductase oA (4). Unlike rhodopsin, the carbohydrate of reductase is not near the NH2terminus but appears to be located between the sixth and seventh membrane-spanning regions. The assignment of this location is based on two observations: 1) the carbohydrate remains with the 35-kDa membrane-bound fragment after proteolysis (4); and 2) asparagine 281 is the only asparagine in this 35-kDa fragment that is followed by the sequence X-serine or X-threonine (Fig. 1).Reductase has two other potential N-linked glycosylation signals, both of which are found on the water-soluble 53-kDa fragment(3).However, this fragment contains no carbohydrate (4), presumably because the asparagine residues at these two glycosylation signals are never exposed to the lumen of the ER. Like rhodopsin, reductase is phosphorylated on two or more serine residues in its COOH-terminal domain (38). Like rhodopsin, the reductase is targeted to a specialized intracellular membraneafter synthesis(Fig. 8). In UT-1cells, the reductase is localized in an extensive network of smoothsurfaced tubules known as the crystalloid ER (12, 39). Both the crystalloid ER (39) and therod-outer-segment disk membrane (34) are extremely poor in cholesterol. It is attractive to speculate that the 7 membrane-spanning regions of the reductase and rhodopsin may play a role in stabilizing the membranes of these cholest~roi-poor organelles. The tendency of introns to be inserted into the genome between exons that encode membrane-spanning regions, which was first noted by Nathans and Hogness in rhodopsin (36), is illustrated much more dramaticallyin HMG-CoA reductase (Fig. 8). This finding suggests that each membranespanning region represents a separate evolutionarily distinct domain. The amino acid sequence of each membrane-spanning region is unique and, therefore, these regions are not repetitions of the sameexon. No homology between these and other transmembrane sequences in rhodopsin or other proteins hasbeen identified. One other relation between reductase and rhodopsin is intriguing. The covalently bound 11-&-retinal, which forms the functional unit of animal-cell rhodopsin, is a polyisoprenoid compound that is synthesized in plantsfrom mevalonate, which is produced by HMG-CoA reductase. The similarity between rhodopsin, which uses a polyisoprenoid, and reductase, which synthesizes the precursor of isoprenoid compounds, raises the possib~lity that the reductase might also contain a mevalonate-derived isoprenoid bound covalently or noncovalently to its hydrophobic domain. It is conceivable that such a substance might function to regulate the rate of degradation of the reductase, a reaction that i s known to be accelerated by a nonsterol product derived from mevalonate (1, 40). Ackno~~edgments-Weare grateful to Richard Lerner for advice on the strategy for preparing synthetic peptides and for generously providing us with the peptides used in this study. Clarice Grimes, Deborah Thompson, and Claudia Stewart provided excellent technical assistance. REFERENCES 1. Brown, M. S., and Goldstein, J. L. (1980) J. Lipid Res. 21,505- 517 2. Chin, D. J., Luskey, K. L., Faust, J. R., MacDonald, R. J., Brown, M. S.. and Goldstein. J. L. 11982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 7704-7708 3. Chin. D. J.. Gil.G.. Russell, D. W.. Liscum. L.. Luskev. K. L.. Basu, S. K., Okayama, H., Berg,P.1 Goldstein, J. L., anhBrown; M. S. (1984) Nature (Lond.)308,613-617 ' 4. Liscum, L., Cummings, R. D., Anderson, R.G. W . , DeMartino, G. N., Goldstein, J. L., and Brown, M. S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,7165-7169 5 . Faust, J. R., Luskey, K. L., Chin, D. J., Goldstein, J. L., and Brown, M. S. (1982) Proc. Natl. Acad. Sci. U. S. A . 79,52055209 6. Lerner, R. A. (1982) Nature (Lond.)299,592-596 7. Walter, G., and DoolittIe, R. F. (1983) in Genetic ~ ~ i ~ e r i ~ : Principles and M e ~ (Setlow, ~ s J. K., and Hollaender, A., eds) Vol. 5, pp. 61-91, Plenum Press, New York 8. Finer-Moore, J., and Stroud, R. M. (1984) Proc. Natl. Acad. Sci. U. S. A . 8 1 , 155-159 9. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 1 2 0 , 97-120 10. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Bwl. 157, 105-132 11. DeMartino, G.N., and Croall, D. E. (1983) Biochemistry 2 2 , 6287-6291 12. Chin, D. J., Luskey, K. L., Anderson, R. G.W., Faust, J. R., Goldstein, J. L., and Brown, M. S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,1185-1189 13. MargIin, A., and Merrifield, R.B. (1970) Annu. Rev. Biochem. 3 9 , 739-866 14. Russell, D. W . , Schneider, W. J., Yamamoto, T., Luskey, K. L., Brown, M. S., and Goldstein, J. L. (1984) Cell 37, 577-585 15. Garvey, J. S., Cremer, N. E., and Sussdorf, D. H. (eds) (1977) in Methods inImmunology, 3rd Ed., pp. 218-219, W. A. Benjamin, Inc., Reading, MA 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 17. Eisenberg, D., Weiss, P., Terwilliger, T., and Wilcox, W. (1982) Faraday Symp. Chem. SOC.17,109-120 18. Luskey, K. L.,Faust, J. R., Chin, D. J., Brown, M. S., and Goldstein, J. L. (1983) J. Biol. Chem. 258,8462-8469 19. Ovchinnikov, Y. A. (1982) FEBS Lett. 148, 179-191 20. Unwin, N., and Henderson, R. (1984) Sci. Am. 250, 78-93 21. Seehra, J. S., and Khorana, H.G. (1984) J. Biol. Chem. 2 5 9 , 4187-4193 22. Ovchinnikov, Y. A.,. Abdulaev, N. G., Feigina, M. Y., Kiselev, A. V., and Lobanov, N. A. (1979) FEBS Lett. 100,219-224 23. Agard, D. A., and Stroud, R. M. (1982) B~ophys.J. 37,589-602 24. Hargrave, P. A.,McDowell, J. H., Curtis, D.R., Wang, J. K., Juszczak, E., Fong, S.-L., Rao, J. K. M., and Argos, P. (1983) Biophys. Struct. Mech. 9 , 235-244 25. Claudio, T., Ballivet, M., Patrick, J., and Heinemann, S. (1983) Proc. Natl. Acad. Sci. U. S. A . 80,1111-1115 26. Foster, D. L., Boublik, M., and Kaback, H. R. (1983) J. Biol. Chem. 258,31-34 27. McLachlan, A. D. (1971) J. Mol. Biol. 61,409-424 28. Blundell, T. L.,,Pitts, J. E., Tickle, I. J., Wood, S. P., and W u , C. W. (1981) Proc. NatL Acad. Sci. U. 5'. A. 78,4175-4179 29. Roitelman, J., and Shechter I. (1984) J. Biol. Chem. 2 5 9 , 870877 30. Rogers, D. H., Panini, S. R., and Rudney, H. (1983) in 3-Hydroxy3-Methylglutaryl Coenzyme AReductase (Sabine, J. R., ed) pp. 58-75, CRC Press, Inc., Boca Raton, FL 31. Reynolds, G., Basu, S. K., Osborne, T. F., Chin, D. J., Gil, G., Brown, M. S., Goldstein, J. L., and Luskey, K. L. (1984) Cell 38,275-286 32. Craik, C. S., Sprang, S., Fletterick, R., and Rutter, W. J. (1982) Nature (Lond.)299,180-182 33. Goldman, B. M., and Blobel, G. (1981) J. Cell Bioi. 90, 236-242 34. Papermaster, D. S., and Schneider, B. G. (1982) in Cell Biology of the Eye (McDevitt, D. S.,ed) pp. 475-531, Academic Press, New York 35. Dratz, E. A., and Hargrave, P. A. (1983) Trends Biochem. Sci. 8 , 128-131 36. Nathans, J., and Hogness, D. S. (1983) Cell 3 4 , 807-814 37. Brown, D. A., and Simoni, R. D. (1984) Proc. Natl. Acad. Sci. U. S. A . 81,1674-1678 38. Keith, M.L., Kennelly, P. J., and Rodwell, V. W. (1983) J. Protein Chem. 2,209-220 39. Orci, L., Brown, M. S., Goldstein, J. L., Garcia-Segura, L.M., and Anderson, R. G. W . (1984) Cell 36,335-345 40. Edwards, P. A., Lan, S.-F., Tanaka, R. D., and Fogelman, A. M. (1983) J. Biol. Chem. 2 5 8 , 7272-7275