Fig 4. Fes1 NEF function, but not release domain, is dispensable for Fes1 functions in vivo.
(A) Serial dilutions of strains lacking Fes1 (SY346, Δ) or expressing chromosomally encoded Fes1 wild type (1075, wt), Fes1A79R,R195A (SKY207, RA) or Fes1ΔRD (1853–35, ΔRD) were plated on YPAD and grown as indicated. Each image is a single plate and the same cultures are on all plates. (B) Western analysis of Fes1 proteins from the same four strains in panel (A), as indicated, grown in liquid YPAD at the indicated temperature. For Fes1ΔRD lysates, 1/5 as much total protein was loaded on gels. Fes1 is expressed in both long (L) and short (S) forms, but the genomically expressed RA mutant is expressed only as the short form (see text). Image labeled "load" shows the blotted membrane stained by amido-black as loading and transfer controls. (C) As in panel (A), except plates contain 1 M sorbitol. (D) Left and center images show the same diluted strains plated in panel (A) grown 2 days at 30°C on YPAD without or with 0.0075% SDS as indicated. Image on right shows same strains grown 2 days on YPAD at 30°C and then overlaid with agar containing BCIP to detect leakage of alkaline phosphatase, which is seen as blue coloration. (E) Serial dilutions of fes1Δ cells with plasmids encoding Fes1 or indicated Sse1 proteins were plated on medium selecting for plasmid maintenance and grown at indicated temperatures for two days. Sse1-K69M is ATPase dead, but binds ATP and retains Hsp70 interaction and NEF function. Sse1-G233D does not bind ATP or act as a NEF {Shaner, 2004 #67}. Expressing Sse1 inhibits growth, but less so when its NEF activity is disrupted. (F) As in panel (A) except plates contain 5 mM H2O2 and 1 M sorbitol as indicated. (G) Five tetrads (1–5) each from sporulated diploids of parents indicated above were replica-plated onto -Ade medium, which selects for cells propagating [URE3] (see text), and medium selecting for cells expressing indicated FES1 allele. The fes1A79R,R195A strain (SKY207) is Ura+ (see Methods) and fes1ΔRD segregates with Lys+ (FES1 is near LYS2 on chromosome 2).