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

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Abstract
In this study, the identification and characterization of theYarrowia lipolyticahomologues ofSaccharomyces cerevisiae α-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of theY. lipolyticaproteins, including the previously isolated YlMnn9p, in theN-glycosylation pathway, a phenotypic analysis of the disrupted strains ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9and ΔYlmnn9ΔYloch1was performed. Disruption of theYlMNN9,YlANL1andYlOCH1genes caused an increased sensitivity to SDS, 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 YlMnn9p and YlAnl1p in theN-glycosylation process. These mutant strains, ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9and ΔYlmnn9ΔYloch1may thus be used to establish a model for theY. lipolytica N-linked glycosylation pathway.