THE N-TERMINAL DOMAIN OF TOMATO 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASES - SEQUENCE, MICROSOMAL TARGETING, AND GLYCOSYLATION

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes t he conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, con sidered the rate-limiting step in isoprenoid biosynthesis. In plants, isoprenoid compounds play important roles in mediating plant growth an d development, electron transport, photosynthesis, and disease resista nce. 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 re ticulum with eight membrane-spanning regions. In contrast, the membran e domain of plant HMGRs is predicted to contain only one to two transm embrane spans. We have isolated and sequenced a clone (pCD4) encoding exon 1 of tomato hmg1. The membrane domain structures of two different ially regulated tomato HMGR isoforms, HMG1 and HMG2, were analyzed usi ng in vitro transcription and translation systems. Microsomal membrane insertion of the tomato HMGRs is co-translational and does not involv e cleavage of an N-terminal targeting peptide, HMGR membrane topograph y was established by protease protection studies of the HMG1 membrane domain and an analogous region of HMG2 engineered to contain a c-myc e pitope tag, The data indicate that both tomato HMGRs span the membrane two times with both the C and N termini located in the cytosol, Lumen al localization of the short peptide predicted to lie within the endop lasmic reticulum was further confirmed by in vitro glycosylation of an asparagine-linked glycosylation site present in HMG2.