Kranz, A. and Kölling, R (Februar 1997), Isolation of Ste6-stabilizing mutants,
FEBS Advanced Lecture Course 97/02: "ATP Binding Cassette (ABC) Transporters: From Multidrug Resistance to Genetic Disease"

 The ABC-Transporter Ste6 mediates the secretion of the mating pheromone a-factor in the yeast Saccharomyces cerevisiae. A Mata ste6 deletion strain produces mature a-factor, but is not able to secrete the peptide.
Ste6 is an extremely short-lived protein. We are interested in analyzing the mechanisms underlying the fast turnover of Ste6 by isolating Ste6-stabilizing mutants. Since it is difficult to directly assay the stabilization of Ste6 we fused it with LacZ as a reporter protein. The fusion protein behaves like wildtype Ste6:

  • it is fully functional
  • it fractionates with wildtype Ste6 (Fig. 1)
  • it accumulates at the plasma membrane in an endocytois mutant (not shown)
  • it is stabilized in a pep4 mutant (Fig. 2B)

After EMS mutagenesis about 30 mutants with increased b-galactosidase activity could be identified in a lacZ-filter test. The five mutants with the highest Ste6-LacZ activity were characterized further (Fig. 2). It could be demonstrated by pulse chase experiments, that wildtype Ste6 was stabilized in these mutants (Fig. 3). A screen with a cDNA library and backcross experiments prooved, that PEP4 was affected in mutant #81 (Fig. 4). The identification of a function which has already been shown to be involved in the degradation of Ste6 prooved the efficiency of the screen. Other mutants, in which Ste6 is even more stabilized than in pep4 cells, and in which the mutation has no effect in the proteolytic function of the vacuole, are currently being characterized.

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