SHARED FUNCTIONS IN-VIVO OF A GLYCOSYL-PHOSPHATIDYLINOSITOL-LINKED ASPARTYL PROTEASE, MKC7, AND THE PROPROTEIN PROCESSING PROTEASE KEX2 IN YEAST

The MKC7 gene was isolated as a multicopy suppressor of the cold-sensi tive growth phenotype of a yeast kex2 mutant, which lacks the protease that cleaves pro-alpha-factor and other secretory proproteins at pair s of basic residues in a late Golgi compartment in yeast. MKC7 encodes an aspartyl protease most closely related to product of the YAP3 gene , a previously isolated multicopy suppressor of the pro-alpha-factor p rocessing defect of a kex2 null. Multicopy MKC7 suppressed the alpha-s pecific mating defect of a ken2 null as well as multicopy YAP3 did, bu t multicopy YAP3 was a relatively weak suppressor of kex2 cold sensiti vity. Overexpression of MKC7 resulted in production of a membrane-asso ciated proteolytic activity that cleaved an internally quenched fluoro genic peptide substrate on the carboxyl side of a Lys-Arg site. Treatm ent with phosphatidylinositol-specific phospholipase C shifted Mkc7 ac tivity from the detergent to the aqueous phase in a Triton X-114 phase separation, indicating that membrane attachment of Mkc7 is mediated b y a glycosylphosphatidylinositol anchor. Although disruption of MKC7 o r YAP3 alone resulted in no observable phenotype, mkc7 yap3 double dis ruptants exhibited impaired growth at 37 degrees C. Disruption of MKC7 and YAP3 in a kex2 null mutant resulted in profound temperature sensi tivity and more generalized cold sensitivity. The synergism of mkc7, y ap3, and ken:! null mutations argues that Mkc7 and Yap3 are authentic processing enzymes whose functions overlap those of Kex2 in vivo.