Kaempf, Michael and Absmanner, Birgit and Schwarz, Markus and Lehle, Ludwig (2009) Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional alpha 1,3-and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis. JOURNAL OF BIOLOGICAL CHEMISTRY, 284 (18). pp. 11900-11912. ISSN 0021-9258, 1083-351X
Full text not available from this repository. (Request a copy)Abstract
N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha 1,3- and the alpha 1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N-terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains ful-fill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX7E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | HAMSTER OVARY CELLS; FUNGUS RHIZOMUCOR-PUSILLUS; YEAST MUTANTS DEFICIENT; 2 ACTIVE-SITES; ENDOPLASMIC-RETICULUM; ESCHERICHIA-COLI; MOLECULAR-CLONING; PROTEIN TOPOLOGY; OLIGOSACCHARYLTRANSFERASE COMPLEX; TRANSMEMBRANE TOPOLOGY; |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 16 Sep 2020 11:45 |
| Last Modified: | 16 Sep 2020 11:45 |
| URI: | https://pred.uni-regensburg.de/id/eprint/29058 |
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