Figure 1.
Prm1p is incorporated into a reduction-sensitive high molecular weight complex.
(A) Anti-GFP Western blot on whole cell lysate of PRM1-GFP MATa cells induced with 10 µg/ml α-factor for 90 min. (B) Anti-HA Western blot on whole cell lysate after galactose induction of HA-PRM1. (C) Prm1p was purified from a population of mating cells by α-myc immunoprecipitation, eluted with 2% SDS, run on a 10% bis-Tris polyacrylamide gel, and visualized by colloidal blue staining. Protein samples were reduced with 100 mM DTT.
Figure 2.
Prm1p forms covalent homodimers.
MATa and MATα cells expressing epitope tagged PRM1 as indicated were mated on YPD plates at 30°C for 3 h. After cell disruption, membrane proteins were solubilized in 1% Triton X-100 and immunoprecipitated using α-myc-Agarose. Western blotting with α-myc and α-GFP antibodies was used to assay pull-down efficiency and co-immunoprecipitation.
Figure 3.
Two cysteines are required for formation of functional covalent Prm1p dimers.
(A) Anti-GFP Western blot on whole cell lysate after expression of PRM1-GFP or cysteine-substituted mutants was induced with 10 µg/ml α-factor for 90 min. (B) prm1Δ MATα cells expressing cytoplasmic GFP and prm1Δ MATa cells bearing the indicated PRM1 alleles on low copy plasmids were mated on EGTA-containing plates at 30°C for 3 h. Mating pairs were fixed and mating pair fate was scored microscopically by observing diffusion of cytoplasm throughout the mating pair (fusion) or loss of GFP signal and abnormal morphology (lysis).
Figure 4.
Prm1p localization and self-association are not affected by cysteine substitution.
(A) Transmitted light and epifluorescence images of live mating pairs shortly after coupling. Prm1p and indicated alleles were C-terminally tagged with GFP. (B) MATa cells expressing epitope tagged PRM1 or mutant alleles as indicated were mated to wild-type MATα cells on YPD plates at 30°C for 3 h. Myc-tagged fusion proteins were immunoprecipitated using α-myc-Agarose.
Figure 5.
Charged residues within a conserved hydrophobic region adjacent to Cys-120 promote Prm1p covalent dimerization and polarized localization.
(A) ClustalW alignment of the Prm1p hydrophobic region adjacent to Cys-120. (B) Anti-GFP Western blot on whole cell lysate after expression of prm1-E104L-GFP or prm1-D112L-GFP was induced with 10 µg/ml α-factor for 90 min. (C) MATa cells coexpressing prm1-D112L-GFP and prm1-D112L-myc were mated to wild-type MATα cells on YPD plates at 30°C for 3 h. Myc-tagged fusion proteins were immunoprecipitated using α-myc-Agarose. (D) Transmitted light and epifluorescence images of prm1D112LGFP in a live mating pair.
Table 1.
Plasmids constructed for this study.