HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M800580-JLR200v1 50/8/1630    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gupta, S. D. Articles by Harmon, J. M. Search for Related Content PubMed PubMed Citation Articles by Gupta, S. D. Articles by Harmon, J. M. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 50, 1630-1640, August 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

Sita D. Gupta^*, Kenneth Gable^*, Gongshe Han^*, Anna Borovitskaya, Luke Selby^*, Teresa M. Dunn^* and Jeffrey M. Harmon^1,

^* Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20184-4799
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20184-4799

¹ To whom correspondence should be addressed. e-mail: jharmon{at}usuhs.edu

In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.

Supplementary key words sphingolipids • 3-ketososphinganine reductase • membrane protein topology

Abbreviations: CHO, Chinese hamster ovary; DHS, dihydrosphingosine; EndoH, endoglycosidase H; ER, endoplasmic reticulum; fXa, factor Xa; GC, glycosylation cassette; GFP, green fluorescent protein; HRP, horseradish peroxidase; 3-KDS, 3-ketosphinganine; β-ME, β-mercaptoethanol; PHS, phytosphingosine; QCM, Quikchange mutagenesis; SDR, short-chain reductase; siRNA, short interfering RNA; SMA, spinal muscular atrophy; SML, sucrose monolaurate; SPT, serine palmitoyltransferase

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?