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Plant J. 2015 Jun;82(6):903-14. doi: 10.1111/tpj.12859. Epub 2015 May 26.

A two-component enzyme complex is required for dolichol biosynthesis in tomato.

Author information

1
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
2
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego Street, 02-106, Warsaw, Poland.
3
Department of Molecular and Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

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.

KEYWORDS:

Nogo-B receptor; Solanum lycopersicum; cis-prenyltransferase; endoplasmic reticulum; polyisoprenoid; polyprenol

PMID:
25899081
DOI:
10.1111/tpj.12859
[Indexed for MEDLINE]
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