The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34) ()catalyzes the synthesis of mevalonic acid, a major rate-limiting step in the isoprenoid biosynthetic pathway(1) . This enzyme has been extensively studied in mammalian systems due to its critical role in mediating cholesterol biosynthesis. In mammals, HMGR is a 97-kDa transmembrane glycoprotein of the endoplasmic reticulum (ER), is encoded by a single gene, and functions in the production of sterols, dolichol, prenyl modifications of proteins, steroid hormones, and several other non-sterol isoprenoid compounds(2) . As shown in Fig. 1, plants synthesize a significantly greater and more diverse array of isoprenoids (in excess of 22,000 different compounds), which function in many aspects of plant growth, development, reproduction, and disease resistance(1, 3) . Consistent with this complexity, plant HMGRs exist as multiple isozymes and are encoded by small gene families. Specific members of the HMGR gene families are differentially expressed during development or in response to environmental factors, and distinct HMGR isoforms may be critical in directing the flux of pathway intermediates into specific isoprenoid compounds ( (3, 4, 5, 6) and references therein). Although synthesis of many of these compounds occurs in the cytosol, several classes of isoprenoids are synthesized in plastids (e.g. carotenoids, plastoquinone, phylloquinone), mitochondria (e.g. ubiquinone), or specialized vesicles (e.g. rubber). HMGR activity has been associated with mitochondria and chloroplasts in addition to microsomes although the organellar location of plant HMGRs remains controversial(1) .

Figure 1: A simplified biosynthetic pathway for plant isoprenoids highlighting important end products. PP, pyrophosphate; PGR, plant growth regulator.

The domain structure of plant, animal, and fungal HMGRs can be roughly divided into three regions: the C-terminal catalytic domain, the N-terminal transmembrane domain, and a divergent linker or hinge region. The catalytic domain, consisting of about 400 amino acids at the C terminus of the protein, is highly conserved between organisms (7, 8, 9, 10) . The N-terminal membrane domain shows greater divergence between organisms and is significantly smaller in plant species compared with yeast or animal HMGRs(10, 11) . In mammalian systems, the N-terminal domain of HMGR contains a highly hydrophobic region of about 340 amino acids, which lacks a typical N-terminal signal peptide or signal peptide cleavage site. This domain is necessary and sufficient to confer localization to ER (microsomal) membranes (12, 13, 14) and has been implicated in the rapid degradation of HMGR triggered by increases in serum cholesterol or low density lipoproteins(15, 16) . Initial models proposed for mammalian HMGR secondary structure and membrane orientation, based on limited proteolysis and hydropathy plot analyses, suggested a transmembrane domain of seven membrane-spanning regions (9) . More recent evidence using specific antibodies raised against synthetic peptides from the HMGR membrane domain supports a model with eight membrane-spanning regions and both the C and N termini localized on the cytosolic face(13, 17) . The HMGR membrane domains of yeast, Drosophila, sea urchin, and Xenopus show a similar transmembrane organization of 7-8 membrane spans based on hydropathy and sequence comparisons(7, 8, 18, 19) . In contrast, the N-terminal hydrophobic domain of plant HMGRs is less than 120 amino acids and has been predicted to contain one (20, 21) or two(11, 22) transmembrane spans. The significance of this reduced membrane domain is unknown although plants presumably lack the requirement for rapid down-regulation of sterol biosynthesis since no dietary source is utilized.

Because the membrane domain of plant HMGRs differs so markedly from that of animal and yeast HMGRs, we are interested in experimentally determining the transmembrane structure of plant HMGRs and the role of this simplified domain in subcellular localization. As a first step toward this goal, we initiated experiments to analyze microsomal targeting and transmembrane orientation of two distinct tomato HMGR isoforms. Tomato contains at least 4 genes encoding HMGR(20, 22) . The current study uses HMG1 and HMG2 N-terminal peptides generated from the differentially regulated tomato HMGR isogenes, hmg1 and hmg2. Tomato hmg1 expression is elevated in rapidly growing cells consistent with a role in sterol biosynthesis (23, 24) . In contrast, hmg2 is induced in response to pathogens and wounding associated with host defense responses(4, 22, 25) . Defense-related induction is correlated with increases in microsomal HMGR activity and production of isoprenoid phytoalexin antibiotics(6, 26, 27, 28) . Microsomal targeting and protease protection assays were performed on in vitro synthesized N-terminal domains of both HMG1 and HMG2 to establish the membrane topography of these distinct isoforms.

EXPERIMENTAL PROCEDURES

cDNA Amplification and Cloning

The N-terminal domain of hmg2 was generated from a tomato hmg2 genomic clone, pTH295(22) , by PCR amplification using primers 23 (5`-CTTTGTCGACCATGGACGTTCGCCGGAGAT) and 5 (5`-CGCAAGCTTGTTCATTCCCATTGCATCACCAGT) as described above. The resulting hmg2-specific fragment, which contains exon I starting at the translation start site, intron I, and part of exon II, was inserted into the SmaI site of pBluescript SK to yield plasmid pXY12. A c-myc epitope (31) was incorporated into a BglII site of pXY12 just downstream of the translation start site. The resulting plasmid, pXY12(c-myc), encodes a polypeptide (HMG2(myc)) that begins MDVRRRSEQKLISEEDLLGSEEP . . . (c-myc epitope is underlined).

In Vitro Transcription

In Vitro Translation, Microsomal Insertion, and Protease Protection Analyses

Translation products, including those subsequently fractionated or protease-treated, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(33) . Ten µl of the original volume of the translation mixture (25 µl) were added to 20 µl of gel loading buffer (10% v/v glycerol, 0.01% bromphenol blue, 1% SDS, 1% -mercaptoethanol in 60 mM Tris-HCl, pH 6.8) and heated (95 °C, 5 min). Five µl of radioactively labeled translation samples were loaded per lane on SDS-PAGE gels (12 or 18% separating; 5% stacking gel). After electrophoresis, gels were fixed (50% methanol, 10% acetic acid, 1 h), treated for 1 h with Enlightning (DuPont NEN), dried, and exposed to XAR film (Kodak Co., Rochester, NY). Both low (Amersham Corp.) and high (Life Technologies, Inc.) molecular weight C-labeled protein standards were included on the gels.

Western Blotting and Immunodetection

RESULTS

Sequence Analysis of the N Termini of Tomato HMG1 and HMG2

Figure 2: A, sequence comparison of plant HMGR N termini. Sequences include tomato (L. esculentum) HMG1 and HMG2 (Tom1, Tom2(22) ), potato (Solanum tuberosum) HMG1 and partial HMG3 (Pot1, Pot3(36) ), wood tobacco (Nicotiana sylvestris) HMGR (Nico(37) ), periwinkle (Catharanthus roseus) HMGR (Peri(38) ), Arabidopsis thaliana HMG1 and HMG2 (Ara1, Ara2(5, 11) ), radish (Raphanus sativa) HMG1 and HMG2 (Rad1, Rad2(34) ), and rubber tree (Hevea brasiliensis) HMG1 and HMG3 (Hev1, Hev3(39) ). Putative membrane domains are boxed, and N-linked glycosylation sites within the putative lumenal domain are underlined. B, hydropathy plot of the N-terminal domains of tomato HMG1 and HMG2 as predicted by Kyte and Doolittle(40) .

The regions preceding the first hydrophobic sequences and those linking the second hydrophobic sequences to the catalytic domains are variable both in amino acid sequence and in length. In contrast, the hydrophilic region between the two putative transmembrane spans is highly conserved among plant HMGRs. Interestingly, this conserved region, which is predicted to lie within the ER lumen, varies with respect to a potential asparagine-linked glycosylation site (N-X-S/T; Fig. 2A).

Insertion of HMG1 and HMG2 into Microsomal Membranes

Figure 3: Sizes of transmembrane and lumenal peptides predicted to be protected from proteinase K digestion, comparing models having one versus two membrane-spanning regions and maintaining the location of the C terminus (catalytic domain in the full-length HMGR) in the cytosol.

The sizes of HMG1 and HMG2 N-terminal peptides resulting from rabbit reticulocyte lysate-mediated translation of in vitro synthesized mRNAs (Fig. 4) were consistent with those predicted from the derived amino acid sequences (37.5 kDa for HMG1- and 39.3 kDa for HMG2-exon I-encoded products). In vitro translation was enhanced in the presence of microsomes (Fig. 4, lanes 3). The peptides synthesized in the presence of microsomes were the same size as those synthesized without microsomes (Fig. 4, lanes 2 and 3), suggesting either that microsomal insertion was unsuccessful or that no signal peptide was cleaved during synthesis and insertion. That HMGR peptides were in fact integrally associated with microsomal membranes was demonstrated by co-sedimentation of labeled HMGR peptides with microsomal membranes upon re-isolation by centrifugation (Fig. 4, lanes 4). Membrane insertion was further confirmed by the inability of a high salt wash to remove the protein from the microsomal pellet (data not shown) and the detection of protease-insensitive fragments (Fig. 4, lanes 6). Proteinase K treatment of peptides translated in the absence of microsomes resulted in complete loss of the protein (Fig. 4, lanes 5), whereas small protected fragments were evident following digestion of peptides synthesized in the presence of microsomes (Fig. 4, lanes 6). The protease-insensitive fragments were not detected if protease treatment was carried out in the presence of 1% Triton X-100, which should liberate peptides from the membranes (data not shown). In order to address whether HMGR microsomal insertion occurred co- or post-translationally, microsomes were added to the translation mix following peptide synthesis (in vitro translation was carried out for 1 h and stopped by addition of 2 mM cycloheximide). In this case, labeled HMGR peptides were not detected in microsomal pellets (data not shown), suggesting co-translational insertion. We also tested the efficiency of in vitro translation of tomato HMG2 using a wheat germ translation system (lacking soluble signal recognition particle(12) ). The wheat germ system was less efficient in synthesizing HMG2 (HMG1 was not tested), and translation was not enhanced by microsomes (data not shown). These data are consistent with co-translational, signal recognition particle-dependent insertion into ER membranes analogous to that determined for mammalian HMGRs(12) .

Figure 4: Insertion of HMG1 and HMG2 into microsomal membranes. In vitro synthesized mRNAs encoding HMG1 and HMG2 peptides were translated in the presence of [S]methionine. Translation was carried out in the presence of dog pancreatic microsomes (M) and/or followed by treatment with proteinase K (P) as indicated prior to separation by SDS-PAGE. The positions of molecular mass markers are indicated. Bands corresponding to HMGR products and protected fragments (PF) are indicated by arrows. Lane 1, no RNA control; lane 2, translation of mRNA in absence of microsomes; lane 3, translation of mRNA in the presence of microsomes; lane 4, microsomal pellet; lane 5, translation in absence of microsomes and followed by proteinase K treatment; lane 6, translation in the presence of microsomes and followed by proteinase K treatment.

The size of the protease-protected fragments for both HMG1 and HMG2 was estimated at 6-8 kDa (e.g. Fig. 4, lanes 6) using several different acrylamide concentrations (data not shown). The signal for the HMG2 proteinase K-insensitive fragment was weaker than that for HMG1 due to decreased translational efficiency of the HMG2 vector (which lacks a poly(A) tail) and fewer methionine residues within the protected region. The protease-insensitive peptides for HMG1 and HMG2 were reproducibly smaller than that predicted for a single transmembrane span (Fig. 3, model B). These data are consistent with predictions based on two transmembrane spans linked by a short lumenal domain as shown in model A of Fig. 3.

Localization of the N Terminus to the Cytosol

Figure 5: Immunodetection of HMG2 peptide containing an epitope tag at the N terminus. In vitro synthesized mRNA encoding HMG1 and HMG2-myc peptides were translated in the presence of dog pancreatic microsomes. Microsomally targeted translation products were separated by centrifugation, treated with proteinase K as indicated, and detected by Western immunoblotting using c-myc monoclonal antibody. Protein standards (Amersham Rainbow protein markers) are indicated. Lanes 1 and 2 contained untagged HMG1 microsomal pellets, plus or minus proteinase K, as controls. Lane 3 contained the HMG2-myc microsomal pellet (cross-reactive band of expected size denoted by arrow). Lane 4 contained the HMG2-myc microsomal pellet treated with proteinase K showing loss of the cross-reacting material. Additional gels (not shown) were run to ensure that protease-protected fragments had not run off the gel.

Tomato HMG2 Is Glycosylated in Vitro

Figure 6: Effect of Endo H on gel mobility of HMG1 and HMG2 protease-insensitive microsomal fragments. Tomato HMGR peptides were synthesized in vitro in the presence of microsomes and treated with proteinase K, and microsomes were isolated by centrifugation. S-Labeled microsomal peptides were deglycosylated by treatment with Endo H in the presence of Triton X-100 (see text), separated by SDS-PAGE, and detected by fluorography. Lane 1, HMG1 without Endo H treatment; lane 2, HMG1 with Endo H treatment; lane 3, HMG2 without Endo H treatment; lane 4, HMG2 with Endo H treatment.

DISCUSSION

In mammals, HMGR is an integral membrane glycoprotein localized to the endoplasmic reticulum and is encoded by a single gene(2, 9, 14) . In contrast, plant HMGRs are encoded by small gene families, and HMGR enzyme activity has been associated with plastid, mitochondrial, and ER membranes. We have shown that two tomato HMGR isoforms that are differentially regulated during development and in response to stress are both targeted to microsomal membranes in vitro. Attempts to demonstrate post-translational insertion of tomato HMG1 peptides into intact chloroplasts were unsuccessful (data not shown). Our results are in agreement with those of Enjuto et al.(5) in which they reported that both Arabidopsis HMGR isoforms are co-translationally targeted to microsomal membranes. These findings do not preclude the possibility that other plant HMGR isoforms may be targeted to plastids or mitochondria, although it is unlikely that those HMGRs that are highly conserved in the membrane-spanning domain (e.g. those listed in Fig. 2) are targeted to these organelles.

Mechanisms mediating ER targeting and insertion of integral membrane proteins of the endomembrane system have been reviewed(42, 43) . For mammalian HMGR, the N-terminal 39 residues, which includes the first transmembrane domain, function as a permanent uncleaved signal for targeting mammalian HMGR to the ER membrane(12, 13) . Orientation of the protein within the ER membrane is determined by a total of eight transmembrane spans such that both N and C termini are cytosolic(13, 17) . Based on our results we propose a similar model for the membrane topology of plant HMGRs localized to the endomembrane system. Tomato HMG1 and HMG2 appear to be targeted to and co-translationally inserted into the ER by a permanent, uncleaved signal. The proteins span the membrane twice such that both the N and C termini are located in the cytoplasm and thus are classified as group III transmembrane proteins (transmembrane protein classifications as reviewed in (43) and (44) ). Three direct experimental approaches were used to assess orientation within the membrane. First, tomato HMGR peptides, synthesized and inserted into microsomes in vitro, were treated with proteinase K. Those amino acids embedded in the membrane or within the lumen are protected from proteolysis. The sizes of the protected fragments for both HMG1 and HMG2 are consistent with digestion of the C and N termini and protection of peptides consisting of the two hydrophobic helices and the hydrophilic residues in between (e.g. residues 48-117 of tomato HMG2; see Fig. 4). Second, cytosolic localization of the N terminus of tomato HMG2 was demonstrated by proteinase K digestion of a c-myc epitope engineered between residues 6 and 7. In addition, immunodetection of the c-myc epitope in the full-length product further confirms that no signal peptide is cleaved during microsome-associated translation. Finally, our experiments indicate that an asparagine within the predicted lumenal domain of tomato HMG2 is glycosylated. The HMG1 sequence used for these experiments contains no N-linked glycosylation sites. The tomato HMG2 peptide contains two potential sites, one within the stretch of 14-22 amino acids predicted to be within the ER lumen and one just downstream of the second transmembrane span and predicted to be cytosolic. Our proteinase K digestion experiments indicate that this second site is cytosolic and thus should not be glycosylated and would be absent from the peptides used for Endo H treatment. As predicted by our model, treatment of protease-protected fragments of in vitro synthesized and microsomal targeted HMGRs with Endo H resulted in a shift of molecular weight of HMG2 but not HMG1.

We find it intriguing that only a subset of plant HMGRs has an N-linked glycosylation site within the lumenal segment (see Fig. 2A). Of plant HMGRs currently in the data bases, those HMGRs associated with constitutive expression, rapid growth involving membrane biogenesis, or showing activity levels coordinated with squalene synthase activity (sterol branch(3, 6) ) (e.g. tomato HMG1, potato HMG1, Arabidopsis HMG1, Hevea HMG3) lack this glycosylation site. All HMGRs in dicots that have expression patterns associated with production of defense compounds or other secondary products such as rubber or coordinated with expression of sesquiterpene cyclase (key branch pathway enzyme for phytoalexin synthesis in the Solanaceae) contain the glycosylation site (e.g. tomato HMG2, Hevea HMG1, the Nicotiana sylvestris and Catharanthus roseus HMGRs). The one exception is an elicitor-inducible rice HMGR gene, the first HMGR membrane domain sequence reported for a monocot species, which is highly divergent in the transmembrane and lumenal region and lacks the lumenal glycosylation site(45) . In the dicot systems, both defense responses and rubber biosynthesis are associated with specific endomembrane differentiation(39, 46) . Although N-linked glycans have not been shown to function as sorting signals in plants (42, 47) , the conservation of the glycosylation site in a specific and highly specialized subset of plant HMGRs suggests that the glycan may play an important biological role. We are currently designing experiments to differentially tag tomato HMG1 and HMG2 to address whether these two isoforms localize to distinct compartments within the plant endomembrane system. Future experiments will address the importance of HMGR isoform-specific expression, subcellular localization, and protein-protein interaction in directing the production of specific isoprenoid compounds and thus mediating key physiological functions involving plant isoprenoids.

FOOTNOTES

*

This research was supported by National Institutes of Health Grant R29-GM39549 (to C. L. C.), Cooperative State Research Service USDA Project 135-265 (to C. L. C.), and Academy of Finland Scholarship 7517 (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Fralin Biotechnology Center, Virginia Polytechnic Inst. and State University, Blacksburg, VA 24061-0346. Tel.: 540-231-6757; Fax: 540-231-7126.

()

The abbreviations used are: HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; ER, endoplasmic reticulum; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Endo H, endo--N-acetylglucosaminidase H.

ACKNOWLEDGEMENTS

Note Added in Proof-During review of this article, related work analyzing the membrane topology of Arabidopsis HMGR was reported (Campos, H., and Boronat, A.(1995) Plant Cell7, 2163-2174).

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