Figure 8.

C-terminal domain of Hsp70 governs its specificity for v-Src maturation. (A) Upper panel shows schematics of designed hybrid Hsp70 proteins. Amino acid sequences at hybrid junction in Ssa24 and Ssa42 are 537–538 of Ssa2 and 538–539 of Ssa4, respectively. Lower panel shows growth curve of indicated strains in liquid YPAD media. (B) Shown is the growth of indicated strains on solid SD or SGal media after 5 days of incubation at 30°. (C) Immunoblot showing steady-state expression of FLAG-tagged v-Src or Pgk1 (as control) in indicated strains. (D) The relative abundance of mRNA encoding v-Src in indicated strains as measured using qRT-PCR. The qRT-PCR was carried out using primers specific for v-SRC or ACT1 (as control). Lower panels (C and D) show relative quantification. Error bar represents SD from three different biological replicates. P-values were calculated using the Student’s t-test and A2 as a control. A.U. arbitrary units; CTD, C-terminal domain; EV, empty vector; mRNA, messenger RNA; NBD, nucleotide-binding domain; SBD, substrate binding domain; qRT-PCR, quantitative real-time PCR; SGal, SD media supplemented with 2% raffinose, 2% galactose and 2% dextrose respectively. YPAD, 1% yeast extract, 2% peptone, and 2% dextrose supplemented with 0.005% adenine.