ROLE FOR THE UBIQUITIN-PROTEASOME SYSTEM IN THE VACUOLAR DEGRADATION OF STE6P, THE A-FACTOR TRANSPORTER IN SACCHAROMYCES-CEREVISIAE

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a mult ispanning membrane protein with 12 transmembrane spans and two cytosol ic ATP binding domains, Ste6p belongs to the ATP binding cassette (ABC ) superfamily and provides an excellent model for examining the intrac ellular trafficking of a complex polytopic membrane protein in yeast, Previous studies have shown that Ste6p undergoes constitutive endocyto sis from the plasma membrane, followed by delivery to the vacuole, whe re it is degraded in a Pep4p-dependent manner, even though only a smal l portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-de pendent proteases reside, Ste6p is known to be ubiquitinated, a modifi cation that may facilitate its endocytosis, In the present study, we f urther investigated the intracellular trafficking of Ste6p, focusing o n the role of the ubiquitin-proteasome machinery in the metabolic degr adation of Ste6p, We demonstrate by pulse-chase analysis that the degr adation of Ste6p is impaired in mutants that exhibit defects in the ac tivity of the proteasome (deal and pre1,2), Likewise, by immunofluores cence, we observe that Ste6p accumulates in the vacuole in the deal mu tant, as it does in the vacuolar protease-deficient pep4 mutant, One m odel consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of bot h the cytosolic proteasomal degradative machinery and the vacuolar lum enal proteases, acting in a synergistic fashion, Alternatively, we dis cuss a second model whereby the ubiquitin-proteasome system may indire ctly influence the Pep4p-dependent vacuolar degradation of Ste6p, This study establishes that Ste6p is distinctive in that two independent d egradative systems (the vacuolar Pep4p-dependent proteases and the cyt osolic proteasome) are both involved, either directly or indirectly, i n the metabolic degradation of a single substrate.