title: A two-component enzyme complex is required for dolichol biosynthesis in tomato.
abstract: Dolichol plays an indispensable role in the N-glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a 'glycan', which is preassembled onto a membrane-anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis-prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi-mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a ~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT-binding protein (SlCPTBP), which is a close homolog of the human Nogo-B receptor. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact in vivo and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2∆. Co-expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the ER, the site of dolichol accumulation and synthesis in eukaryotes.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Megan I
authors.initials: MI
authors.lastname: Brasher
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego Street, 02-106, Warsaw, Poland.
authors.forename: Liliana
authors.initials: L
authors.lastname: Surmacz
authors.affiliation: Department of Molecular and Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Leong
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Pitcher
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego Street, 02-106, Warsaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Swiezewska
authors.affiliation: Department of Molecular and Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Eran
authors.initials: E
authors.lastname: Pichersky
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
authors.forename: Tariq A
authors.initials: TA
authors.lastname: Akhtar
mesh_terms: D004122:Dimethylallyltranstransferase / Q000235:genetics; D004286:Dolichols / Q000096:biosynthesis*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D005816:Genetic Complementation Test; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014166:Transferases / Q000235:genetics / Q000378:metabolism
chemical_list: D004286:Dolichols; D009097:Multienzyme Complexes; C512614:NUS1 protein, human; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; C106821:cis-prenyl transferase; D004122:Dimethylallyltranstransferase; C485360:RER2 protein, S cerevisiae
keywords: Nogo-B receptor; Solanum lycopersicum; cis-prenyltransferase; endoplasmic reticulum; polyisoprenoid; polyprenol