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

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 biosynthesisare catalyzed by a functional multienzyme complex com-prised of the Alg7 GlcNAc phosphotransferase and the het-erodimeric Alg13/Alg14 UDP-GlcNAc transferase on thecytosolic face of the ER. In the Alg13/14 glycosyltransfer-ase, Alg14 recruits cytosolic Alg13 to the ER membranethrough interaction between their C-termini. Bioinformaticanalysis revealed that eukaryotic Alg14 contains an evolvedN-terminal region that is missing in bacterial orthologs.Here, we show that this N-terminal region ofSaccharomyces cerevisiae Alg14 localize its green fluores-cent protein fusion to the ER membrane. Deletion of thisregion causes defective growth at 38.5°C that can be par-tially complemented by overexpression of Alg7.Coimmunoprecipitation demonstrated that the N-terminalregion of Alg14 is required for direct interaction with Alg7.Our data also show that Alg14 lacking the N-terminalregion remains on the ER membrane through a nonperiph-eral association, suggesting the existence of another mem-brane-binding site. Mutational studies guided by the 3Dstructure of Alg14 identified a conserved α-helix involvedin the second membrane association site that contributes toan integral interaction and protein stability. We propose amodel in which the N- and C-termini of Alg14 coordinaterecruitment of catalytic Alg7 and Alg13 to the ERmembrane for initiating LLO biosynthesis.Keywords: Alg glycosyltransferase/endoplasmic reticulum/lipid-linked oligosaccharide/multiglycosyltransferasecomplex/N-glycosylationIntroductionN-glycosylation is an essential posttranslational modificationof proteins occurring in eukaryotes. It begins with the multistepbiosynthesis of a highly conserved lipid-linked oligosaccharide(LLO), which is extended by a series of membrane-associatedAlg (asparagine-linked glycosylation) glycosyltransferases toproduce Glc