ENDOCYTOSIS AND VACUOLAR DEGRADATION OF THE PLASMA MEMBRANE-LOCALIZEDPDR5 ATP-BINDING CASSETTE MULTIDRUG TRANSPORTER IN SACCHAROMYCES-CEREVISIAE

Multidrug resistance (MDR) to different cytotoxic compounds in the yea st Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter . We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mecha nisms underlying MDR development. Subcellular fractionation and indire ct immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane prot ein with a half-life of about 60 to 90 min. A dramatic metabolic stabi lization of Pdr5 was observed in Delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase Delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. Howeve r, Pdr5 turnover does not require a functional proteasome, since the h alf-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 muta nts defective in the multicatalytic cytoplasmic proteasome that is ess ential for cytoplasmic protein degradation. Immunofluorescence analysi s revealed that vacuolar delivery of Pdr5 is blocked in conditional en d4 endocytosis mutants at the restrictive temperature, showing that en docytosis delivers Pdr5 from the plasma membrane to the vacuole.