The N-terminal Domain of Tomato 3-Hydroxy-3-methylglutaryl-CoA Reductases

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.

Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase

“…The three Arabidopsis HMGR isoforms insert in vitro into dog pancreas endoplasmic reticulum (ER)-derived vesicles (Enjuto et al, 1994;Lumbreras et al, 1995). The generally accepted hypothesis is that they are first targeted to the ER , which is in agreement with results showing insertion of tomato HMGR into microsomal membranes (Denbow et al, 1996) and localization of yeast (Saccharomyces cerevisiae) HMGR in ER (Hampton et al, 1996).…”

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

“…Although details concerning the structure and function of the HMG-CoA reductase catalytic domain are beginning to be solved (Darnay et al, 1992;Lawrence et al, 1995;Rogers et al, 1997;Tabernero et al, 1999), the HMG-CoA reductase membrane domain remains much more cryptic. In plants, the membrane domain contains two transmembrane helices (Denbow et al, 1996;Re et al, 1997), whereas in animals and fungi, it contains of a series of seven or eight transmembrane helices (Liscum et al, 1985;Basson et al, 1988;Sengstag et al, 1990;Olender and Simon, 1992;Roitelman et al, 1992). Although there is little obvious primary sequence conservation, the structural conservation of this complex membrane domain over the 1 billion years of evolution since divergence of fungi and animals (Doolittle et al, 1996;Lum et al, 1996;Feng et al, 1997) suggests that the structure has an important functional role.…”

The STT3a Subunit Isoform of the Arabidopsis Oligosaccharyltransferase Controls Adaptive Responses to Salt/Osmotic Stress

“…In plants, the glycosylated form of the ER-localized 3-hydroxy-3-methylglutaryl-CoA reductases appears to produce phytoalexin, whereas the unglycosylated form produces sterols (Denbow et al, 1996). In view of these important functions of glycosylation (A) Glycosylated proteins in crude extracts of wild-type (C24) and stt3a-1 plants were resolved by SDS-PAGE and detected by Con A lectin blot analysis in total proteins (left gel) and Con A binding proteins (CBPs; right gel).…”