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Glycobiology. 2012 Apr;22(4):504-16. doi: 10.1093/glycob/cwr162. Epub 2011 Nov 7.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.

Author information

1
Graduate School of Life Science, Hokkaido University, N8, W5, Kita-Ku, Sapporo 060-0808, Japan.

Abstract

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

PMID:
22061998
DOI:
10.1093/glycob/cwr162
[Indexed for MEDLINE]

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