Identification and functional characterization of the HpALG11 and the HpRFT1 genes involved in N-linked glycosylation in the methylotrophic yeast Hansenula polymorpha.
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). In this study, we have identified two genes, HpALG11and HpRFT1, in the metylotrophic yeast Hansenula polymorpha. Detailed analysis of the glycan structures of the N-linked glycans of secreted recombinant glucose oxidase in mutant strains Hpalg3Δ, Hpalg11Δ, and Hpalg3Δalg11Δ with the assistance of over-expression of RFT1 was performed by linkage-specific mannosidase digestion. The results suggest that HpALG11 and HpRFT1 were responsible for catalyzing the sequential transfer of terminal α-1,2-Man residues to form the Man(5)GlcNAc(2)-PP-Dol intermediate at the cytosolic side of the ER before flipping to the luminal side and encoding an evolutionarily conserved protein required for the translocation of Man(5)GlcNAc(2)-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane, respectively. Deletion of the HpALG11 gene leads to poor growth and temperature-sensitive lethality, whereas over-expression of HpRft1p can improve growth of the Hpalg11Δ and Hpalg3Δalg11Δ strains. Furthermore, deletion of the HpALG11 gene in the Hpalg3Δ strain resulted in the secretion of glycoproteins with a predicted structure mainly containing trimannosyl core N-linked glycans (Man(3)GlcNAc(2)).
Song
Haolei
H
Institute of Microbiology Chinese Academy of Sciences, NO 1 Beichen West Road, Chaoyang District, Beijing 100101, China.
Qian
Weidong
W
Wang
Hui
H
Qiu
Bingsheng
B
D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal / Q000502:physiology; D006031:Glycosylation; D007425:Intracellular Membranes / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D046188:Protein Modification, Translational / Q000502:physiology*
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D008364:Mannosyltransferases