Identification and characterization of two α-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica

In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae alpha-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains DeltaYlmnn9, DeltaYlanl1, DeltaYloch1, DeltaYlanl1DeltaYlmnn9 and DeltaYlmnn9DeltaYloch1 was performed. Disruption of the YIMNN9, YIANL1 and YIOCH1 genes caused an increased sensitivity to SIDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YIMnn9p and YIAnl1p in the N-glycosylation process. These mutant strains, DeltaYlmnn9, DeltaYlanl1, DeltaYloch1, DeltaYlanl1DeltaYlmnn9 and DeltaYlmnn9DeltaYloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.