TRACING INTRACELLULAR PROTEOLYTIC PATHWAYS - PROTEOLYSIS OF FATTY-ACID SYNTHASE AND OTHER CYTOPLASMIC PROTEINS IN THE YEAST SACCHAROMYCES-CEREVISIAE

Yeast fatty acid synthase consists of two independent polypeptide stra ins, alpha and beta. The functional multienzyme complex, composed of s ix alpha- and six beta-subunits, is rather stable against proteolysis in vivo. Mutations in one of the subunits or deletion of one subunit l ead to degradation of the nonmutated remaining fatty acid synthase pro tein. We show that the unassembled alpha-subunit of this enzyme is sho rt-lived, and degradation depends on the presence of active cytoplasmi c proteinase yscE, the yeast proteasome. The unassembled beta-subunit is degraded by a nonvacuolar proteolytic system under vegetative growt h conditions. However, starvation of a vacuolar proteinase mutant stra in, which lacks the alpha-subunit of fatty acid synthase, leads to app earance of the unassembled beta-subunit in isolated vacuoles. This ind icates that the major vacuolar peptidases proteinase yscA and yscB are at least partly involved in degradation of the beta-subunit of fatty acid synthase. In a proteinase yscA and yscB double mutant strain wild type for fatty acid synthase both subunits of fatty acid synthase, al pha and beta, are detectable in vacuoles. In addition, under the same starvation conditions other cytoplasmic proteins are found in the vacu ole of a proteinase yscA and yscB double mutant strain. The experiment s in conjunction with the previous finding of the appearance of vesicl es in vacuoles of starved cells (Simeon, A., van der Klei, I. J., Veen huis, M., and Wolf, D. H. (1992) FEBS Lett. 301, 231-235) indicate tha t transport of these tested cytoplasmic proteins into the vacuole is a n unselective bulk process induced by nutritional stress.