Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae

The incorporation of radioactive orthophosphate into the cell walls of Sacc haromyces cerevisiae was studied. P-33-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase, This radioa ctive material stayed in the stacking gel during SDS-polyacrylamide gel ele ctrophoresis but entered the separating gel upon treatment with N-glycosida se F, indicating that phosphate was linked directly or indirectly to N-mann osylated glycoproteins. The phosphate was bound to covalently linked cell w all proteins as mannose-6-phosphate, the same type of linkage shown previou sly for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phos phate-labeled glycoprotein fraction released by laminarinase, three cell wa ll mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified b y N-terminal sequencing, For Ccw13p (encoded by DAN1 [also called TIR3]) an d Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A . Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radi oactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a sh ift of the P-33-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensiti vity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination .