Glycobiology Advance Access first published online on October 25, 2008
This version published online on October 31, 2008
Glycobiology, doi:10.1093/glycob/cwn118
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The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major
1 Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Regensburg, Germany
2 Medizinische Hochschule Hannover, Zentrum Biochemie, Hannover, Germany
Corresponding author: Ludwig Lehle Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany., Tel.: +49 941 943 3043, Fax: +49 941 943 3352, E-mail: ludwig.lehle{at}biologie.uni-regensburg.de
Received on September 25, 2008; accepted on October 21, 2008
The key step of protein N-glycosylation is catalyzed by the multimeric oligosaccharyltransferase complex (OST). Biochemical and genetic studies have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1, Swp1, Stt3, Ost1, Ost2, Ost3, Ost4, Ost5 and Ost6. With the exception of Stt3, assumed to contain the catalytic site, little is known about the function of other OST subunits. The existence of the OST complex is suggested to allow substrate specificity and efficient transfer, a close interaction with the translocon and the prevention of protein folding to ensure the efficient co-translational modification of proteins. However, in the recently completed genome of the trypanosomatid parasite Leishmania major STT3, of which four paralogs exist, STT3–1, STT3–2, STT3–3 and STT3–4, is the only OST subunit that can be identified. Here we report that L.m.STT3 proteins, except STT3–3, are able to complement stt3 deficiency in yeast during vegetative growth, but only poorly during sporulation. By blue native electrophoresis we demonstrate that the L.mSTT3 is active mainly as a free, monomeric enzyme. In cell free assays and also in vivo we find that L.mSTT3, expressed in yeast, has a broad specificity for non-glucosylated lipid-linked mannose-oligosaccharides, typical for several protists. But when incorporated into the OST complex, L.mSTT3 transfers also the common eukaryotic Glc3Man9GlcNAc2-PP-Dol donor. Finally, three L.m.STT3 paralogs were shown to complement not only stt3, but also ost1, ost2, wbp1 or swp1 mutants. Thus, STT3 from Leishmania can substitute for the whole OST complex.
Key words: oligosaccharyltransferase / lipid-linked oligosaccharide / N-glycosylation / Saccharomyces/Leishmania
Updated to include Table 1.