title: Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.
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.
authors: Graduate School of Life Science,  Hokkaido University, N8, W5, Kita-Ku, Sapporo 060-0808,  Japan.
authors: Jishun
authors: J
authors: Lu
authors: Tetsuo
authors: T
authors: Takahashi
authors: Atsuko
authors: A
authors: Ohoka
authors: Kei-ichi
authors: K
authors: Nakajima
authors: Ryo
authors: R
authors: Hashimoto
authors: Nobuaki
authors: N
authors: Miura
authors: Hiroyuki
authors: H
authors: Tachikawa
authors: Xiao-Dong
authors: XD
authors: Gao
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology; D004795:Enzyme Stability; D006017:Glycolipids / Q000096:biosynthesis*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000201:enzymology; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009844:Oligosaccharides / Q000096:biosynthesis*; D010641:Phenotype; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017384:Sequence Deletion
chemical_list: D006017:Glycolipids; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C506082:Alg13 protein, S cerevisiae; C506083:Alg14 protein, S cerevisiae; D017351:N-Acetylglucosaminyltransferases; C572901:Alg7 protein, S cerevisiae; D017856:Phosphotransferases (Phosphate Group Acceptor)