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 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.
Denbow
C J
CJ
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute, Blacksburg, Virginia 24061, USA.
Lång
S
S
Cramer
C L
CL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006031:Glycosylation; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D018551:Solanum lycopersicum / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D006903:Hydroxymethylglutaryl CoA Reductases