Physical interactions between the Alg1, Alg2, and Alg11 mannosyltransferases of the endoplasmic reticulum.
The early steps of N-linked glycosylation involve the synthesis of a lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-PP-dolichol, on the endoplasmic reticulum (ER) membrane. Prior to its lumenal translocation and transfer to nascent glycoproteins, mannosylation of Man(5)GlcNAc(2)-PP-dolichol is catalyzed by the Alg1, Alg2, and Alg11 mannosyltransferases. We provide evidence for a physical interaction between these proteins. Using a combination of biochemical and genetic assays, two distinct complexes that contain multiple copies of Alg1 were identified. The two Alg1-containing complexes differ from one another in that one complex contains Alg2 and the other contains Alg11. Alg1 self-assembles through a C-terminal domain that is distinct from the region required for its association with Alg2 or Alg11. Missense mutations affecting catalysis but not Alg1 protein stability or assembly with Alg2 or Alg11 were also identified. Overexpression of these catalytically inactive alleles resulted in dominant negative phenotypes, providing genetic evidence for functional Alg1-containing complexes in vivo. These data suggest that an additional level of regulation that ensures the fidelity of complex oligosaccharide structures involves the physical association of the related catalytic enzymes in the ER membrane.
Gao
Xiao-Dong
XD
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1 Higashi, Tsukuba 305-8566, Japan.
Nishikawa
Akiko
A
Dean
Neta
N
D000595:Amino Acid Sequence; D001704:Biopolymers; D002384:Catalysis; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D008364:Mannosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D001704:Biopolymers; D029701:Saccharomyces cerevisiae Proteins; C083733:ALG2 protein, S cerevisiae; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase