Control of expression of the ABC-transporter
encoding genes PDR5 and YOR1 by the transcription factor Pdr1p is
required for resistance to the cytotoxic agents cycloheximide and
oligomycin in S. cerevisiae. PDR13 encodes an Hsp70 molecule most
similar to the yeast SSA chaperones. High copy and mutant (S295F) forms
of Pdr13p elevate cycloheximide and oligomycin resistance by increasing
Pdr1p dependent expression of PDR5 and YOR1. Neither high copy nor S295F
Pdr13p function by increasing steady state Pdr1p levels suggesting that
Pdr13p activates Pdr1p post-translationally. We are interested in
determining how Pdr1p function is positively regulated by Pdr13p.
Several stuctural changes in Pdr1p, including single amino acid
substitutions and an internal deletion which removes amino acids 205-963
(out of 1063), result in hyperactive forms of Pdr1p which do not require
activation by Pdr13p. This is consistent with the idea that Pdr13p
delivers a necessary positive regulatory signal to wild-type Pdr1p which
is not required for the hyperactive forms of Pdr1p. Internal deletions
in Pdr1p are being used to precisely identify the domains required for
regulation by Pdr13p. Because Hsp70 proteins can be found in many
compartments in the cell, we identified the subcellular localization of
Pdr13p. Wild-type and mutant Pdr13p are both present in the P100
fraction of whole cell extracts and are released into the S100 fraction
after treatment with sodium carbonate but not by Triton-X-100. Indirect
immunofluorescence indicates that both wild-type and mutant Pdr13p are
present in the cytoplasm. These data suggest that Pdr13 is associated
with a large complex located in the cytoplasm.
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