Identification of two mannoproteins released from cell walls of a Saccharomyces cerevisiae mnn1 mnn9 double mutant by reducing agents

In this report, we present the identification of the main polypeptides that are extracted from purified cell wails of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of thi s strain with P-mercaptoethanol releases several mannoproteins, of which th ree, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Ana lysis of the amino-terminal sequences revealed that the 120-kDa mannoprotei n is Bar1p, the protease involved in the so-called barrier activity in yeas t cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) Y JL158c, an ORF that belongs to the PIR (protein with internal repeats) fami ly of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3, Accordingly, ve have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed fo rm of the mannoprotein. We have characterized Pir4 and have shown the feasi bility of using it as a fusion partner for the targeting of recombinant pro teins to the cell wall.