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

Laboratoire de Microbiologie et Génétique Moléculaire, CNRS-Institut National Agronomique Paris-Grignon-INRA, 78850 Thiverval-Grignon, France Correspondence Stéphanie Barnay-Verdier barnay{at}grignon.inra.fr In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae -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 Ylmnn9 , Ylanl1 , Yloch1 , Ylanl1 Ylmnn9 and Ylmnn9 Yloch1 was performed. Disruption of the YlMNN9 , YlANL1 and YlOCH1 genes 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 the N -glycosylation process. These mutant strains, Ylmnn9 , Ylanl1 , Yloch1 , Ylanl1 Ylmnn9 and Ylmnn9 Yloch1 may thus be used to establish a model for the Y. lipolytica N -linked glycosylation pathway. Abbreviations: ER, endoplasmic reticulum; RST, random sequence tag The GenBank/EMBL/DDBJ accession numbers for the YlANL1 and YlOCH1 sequences reported in this paper are DS55218 /138556 and DS55232 /138580, respectively.