The Yeast ALG11 Gene Specifies Addition of the Terminal α1,2-Man to the Man5GlcNAc2-PP-dolicholN-Glycosylation Intermediate Formed on the Cytosolic Side of the Endoplasmic Reticulum

Abstract

The initial steps in N-linked glycosylation involve the synthesis of a lipid-linked core oligosaccharide followed by the transfer of the core glycan to nascent polypeptides in the endoplasmic reticulum (ER). Here, we describe alg11, a new yeast glycosylation mutant that is defective in the last step of the synthesis of the Man 5 GlcNAc 2 -PP-dolichol core oligosaccharide on the cytosolic face of the ER. A deletion of the ALG11 gene leads to poor growth and temperature-sensitive lethality. In an alg11 lesion, both Man 3 GlcNAc 2 -PP-dolichol and Man 4 GlcNAc 2 -PP-dolichol are translocated into the ER lumen as substrates for the Man-P-dolichol-dependent sugar transferases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm ␣1,2-linked Man residues. The former are elongated to mannan, whereas the latter are poor substrates for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y., Tanaka, A., Haga-Toda, Y., and Jigami, Y. (1997) FEBS Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 gene is predicted to encode a 63.1-kDa membrane protein that by indirect immunofluorescence resides in the ER. The Alg11 protein is highly conserved, with homologs in fission yeast, worms, flies, and plants. In addition to these Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. All of these Alg11-related proteins share a 23-amino acid sequence that is found in over 60 proteins from bacteria to man whose function is in sugar metabolism, implicating this sequence as a potential sugar nucleotide binding motif.

Specific transbilayer translocation of dolichol-linked oligosaccharides by an endoplasmic reticulum flippase

“…The yeast alg11 mutant cannot convert M3-to M5-DLO, but is able to elaborate M3-DLO to iM7-DLO in the ER lumen through the action of Man-P-dolichol-dependent glycosyltransferases (30). This indicates that M3-DLO is a substrate for the flippase in vivo.…”

“…This indicates that M3-DLO is a substrate for the flippase in vivo. Even though iM7-DLO is used as an oligosaccharide donor by OST, ⌬alg11 cells under-glycosylate proteins and display a severe growth defect (16,30). This phenotype has been attributed to inefficient flipping of M3-DLO, resulting in a reduced rate of iM7-DLO production and inadequate synthesis of glycoproteins.…”

Cloning and transcriptional expression of mouse mannosyltransferase IV/V cDNA, which is involved in the synthesis of lipid-linked oligosaccharides

“…The budding yeast S. cerevisiae mannosyltransferase IV/V gene disruptant (ALG11Δ strain JCY20-1c: ura3-52 his3-Δ200 trp1-Δ901 lys2-801 suc2-Δ9 leu2-3, 112 alg11Δ::URA3) (ALG11Δ) 16) was kindly provided by Prof. Neta Dean (State University of New York at Stonybrook). In most cases, ALG11Δ was grown in YPDA medium (polypeptone 20 g, yeast extract 10 g, glucose 20 g, and adenine sulfate 0.02 g in 1 L of double distilled water) supplemented with 0.5 M KCl (0.5 M KCl-YPDA) at 26°C, and grown on an agar plate containing 2% agar at 26°C.…”

“…We isolated human genes encoding GDP-Man-dependent mannosyltransferases (mannosyltransferase I-V), such as human mannosyltransferase-I (hALG1), 17) III (hALG2), 18) and V (hALG11) (data not shown), which are homologous to S. cerevisiae ALG1, 19) ALG2, 20) and ALG11, 16) respectively, by tBLASTn search. However, as the genes of mannosyltransferase II and IV had not been identified at that time, we tried to isolate the human mannosyltransferase IV gene.…”

Glycoengineering of yeasts from the perspective of glycosylation efficiency

“…This strategy stops LLO synthesis already during the cytoplasmic steps creating the Man3GlcNAc2. Deletion of alg11 precludes the formation of the A-branch of the LLO, which leads to a very strong hypoglycosylation [18]. In addition to the reduced transfer efficiency, inefficient flipping of the LLO into the ER lumen contributes to the hypoglycosylation phenotype observed in the Δalg11 strain.…”