METABOLIC INSTABILITY AND CONSTITUTIVE ENDOCYTOSIS OF STE6, THE A-FACTOR TRANSPORTER OF SACCHAROMYCES-CEREVISIAE

STE6, a member of the ATP binding cassette (ABC) transporter superfami ly, is a membrane protein required for the export of the a-factor mati ng pheromone in Saccharomyces cerevisiae. To initiate a study of the i ntracellular trafficking of STE6, we have examined its half-life and l ocalization. We report here that STE6 is metabolically unstable in a w ild-type strain, and that this instability is blocked in a pep4 mutant , suggesting that degradation of STE6 occurs in the vacuole and is dep endent upon vacuolar proteases. In agreement with a model whereby STE6 is routed to the vacuole via endocytosis from the plasma membrane, we show that degradation of STE6 is substantially reduced at nonpermissi ve temperature in mutants defective in delivery of proteins to the pla sma membrane (sec6) or in endocytosis (end3 and end4). Whereas STE6 ap pears to undergo constitutive internalization from the plasma membrane , as do the pheromone receptors STE2 and STE3, we show that two other proteins, the plasma membrane ATPase (PMA1) and the general amino acid permease (GAP1), are significantly more stable than STE6, indicating that rapid turnover in the vacuole is not a fate common to all plasma membrane proteins in yeast. Investigation of STE6 partial molecules (h alf-and quarter-molecules) indicates that both halves of STE6 contain sufficient information to mediate internalization. Examination of STE6 localization by indirect immunofluorescence indicates that STE6 is fo und in a punctate, possibly vesicular, intracellular pattern, distinct from the rim-staining pattern characteristic of PMA1. The punctate pa ttern is consistent with the view that most of the STE6 molecules pres ent in a cell at any given moment could be en route either to or from the plasma membrane. In a pep4 mutant, STE6 is concentrated in the vac uole, providing further evidence that the vacuole is the site of STE6 degradation, while in an end4 mutant STE6 exhibits rim-staining, indic ating that it can accumulate in the plasma membrane when internalizati on is blocked. Taken together, the results presented here suggest that STE6 first travels to the plasma membrane and subsequently undergoes endocytosis and degradation in the vacuole, with perhaps only a transi ent residence at the plasma membrane; an alternative model, in which S TE6 circumvents the plasma membrane, is also discussed.