The yeast ALG11 gene specifies addition of the terminal alpha 1,2-Man to the Man(5)GlcNAc(2)-PP-dolichol N-glycosylation intermediate formed on the cytosolic side of the endoplasmic reticulum

The initial steps in N-linked glycosylation involve the synthesis of a lipi d-linked core oligosaccharide followed by the transfer of the core glycan t o 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 o n 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 transfer ases in this compartment. This leads to a unique family of oligosaccharide structures lacking one or both of the lower arm alpha1,2-linked Man residue s. The former are elongated to mannan, whereas the latter are poor substrat es for outerchain initiation by Ochlp (Nakayama, K.-I., Nakanishi-Shindo, Y ., Tanaka, A, Haga-Toda, Y,, and Jigami, Y. (1997) FEES Lett. 412, 547-550) and accumulate largely as truncated biosynthetic end products. The ALG11 g ene is predicted to encode a 63.1-kDa membrane protein that by indirect imm unofluorescence resides in the ER. The Alg11 protein is highly conserved, w ith homologs in fission yeast, worms, flies, and plants. In addition to the se Alg11-related proteins, Alg11p is also similar to Alg2p, a protein that regulates the addition of the third mannose to the core oligosaccharide. Al l of these Alg11-related proteins share a 23-amino acid sequence that is fo und in over 60 proteins from bacteria to man whose function is in sugar met abolism, implicating this sequence as a potential sugar nucleotide binding motif.