Abstract
The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, considered the rate-limiting step in isoprenoid biosynthesis. In plants, isoprenoid compounds play important roles in mediating plant growth and development, electron transport, photosynthesis, and disease resistance. Sequence comparisons of plant HMGR proteins with those from yeast and mammalian systems reveal high levels of sequence identity within the catalytic domain but significant divergence in the membrane domain. Mammalian HMGRs are integral membrane proteins of the endoplasmic reticulum with eight membrane-spanning regions. In contrast, the membrane domain of plant HMGRs is predicted to contain only one to two transmembrane spans. We have isolated and sequenced a clone (pCD4) encoding exon 1 of tomato hmg1. The membrane domain structures of two differentially regulated tomato HMGR isoforms, HMG1 and HMG2, were analyzed using in vitro transcription and translation systems. Microsomal membrane insertion of the tomato HMGRs is co-translational and does not involve cleavage of an N-terminal targeting peptide. HMGR membrane topography was established by protease protection studies of the HMG1 membrane domain and an analogous region of HMG2 engineered to contain a c-myc epitope tag. The data indicate that both tomato HMGRs span the membrane two times with both the C and N termini located in the cytosol. Lumenal localization of the short peptide predicted to lie within the endoplasmic reticulum was further confirmed by in vitro glycosylation of an asparagine-linked glycosylation site present in HMG2.
Highlights
The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of 3-hydroxy-3methylglutaryl-CoA to mevalonic acid, considered the rate-limiting step in isoprenoid biosynthesis
The most highly conserved region contains two hydrophobic segments (Fig. 2B), each of which extends over 20 amino acids, a distance sufficient to span a membrane bilayer
HMGR is an integral membrane glycoprotein localized to the endoplasmic reticulum and is encoded by a single gene [2, 9, 14]
Summary
CDNA Amplification and Cloning—Tomato hmg sequences encoding the putative membrane domain were generated by reverse transcription PCR using DNA synthesized from poly(Aϩ) RNA [29, 30] from immature tomato fruit (2–5 mm, Lycopersicon esculentum cultivar EP7) using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) and oligo(dT) primers (Promega Corp., Madison, WI). For experiments designed to assess the presence of N-linked glycans, 10 l of the protease-protected microsomal fragments were treated with 1 milliunit/l endo--N-acetylglucosaminidase H (Endo H, Boehringer Mannheim) and 1% Triton X-100 (Sigma) overnight at 37 °C. Translation products, including those subsequently fractionated or protease-treated, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [33]. 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 14C-labeled protein standards were included on the gels. The second antibody, horseradish peroxidase-conjugated sheep anti-mouse Ig antibody (Amersham Corp.) was diluted from 1:3,000 to 1:20,000 and detection was done using an ECL Western blotting detection system (Amersham Corp.) according to the manufacturer’s protocols except that additional blocking agent (5% dry milk) was included in the antibody incubation steps
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