PMC

SDS-PAGE analysis of His₆-TEV-Cak1 (also—at lower level—the His₆-TEV-Cak1/Cdc37-Strep complex) overexpressed in galactose-grown MGY140 cells. A total eluate of the IMAC-retained material (left) analysed by gel filtration (right), revealing both resin-included material (His₆-TEV-CAK1) and a 165-kDa peak formed substantially of His₆-TEV-Cak1 and Cdc37-Strep (also a little untagged Cdc37 derived from the chromosomal CDC37 gene of strain MGY140) and Cdc28 (asterisk)

a Analysis of Cak1 and Cdc28 in Cdc37 phosphorylation mutants. Protein extracts from 24 °C cultures of strains DH211-5 CAK1-myc were western blotted and probed using anti-HA, anti-myc, PSTAIRE and anti-actin (loading control) antisera. Two exposures of the actin blot are shown; a nonspecific band from the anti-myc probing is indicated with an asterisk. The control lane (C) is an identically treated extract from strain TM141 in which neither the Cdc37 nor the Cak1 is epitope tagged. b An analysis of Cdc37-HA levels in the original DH211-5 (Hawle et al. )

a Overexpression of an N-terminally GFP- or GST-tagged Cdc37 does not rescue the osmosensitivity of the hsp90-G81S mutant. Serial dilutions of untransformed wild-type (82a WT) or osmosensitive hsp90-G81S mutant cells, as well as hsp90-G81S cells transformed with the empty multicopy vector YEplac195 or YEplac195-based plasmids with genes either native Cdc37; N-terminally GFP- or GST-tagged Cdc37 were pinned on YPD, as well as YPD containing 2 M sorbitol. Plates were photographed after 3 days at 24 °C. b Growth (3 days at 30 °C) of Trp+ transformants of strain XX201 on FOA, cells containing either empty pBDC vector, or pBDC containing a Cdc37-BD or Cdc37(S14A)-BD gene insert. No growth is apparent with the empty vector pBDC

a Four sample plates of the 384-colony format 16-plate library array of PJ694a/α cells containing the Cdc37(S14A)-BD bait. Activation of the interaction-responsive HIS3 gene is monitored as 3-AT resistant growth (16 days at 30 °C) on SD medium without tryptophan, leucine and histidine containing 4 mM 3-AT; Cdc37(S14A)-BD interactions represented on these plates being: Plate 2: AD-Cak1; Plate 6: AD-Ndt80; Plate 7: AD-Vps71; Plate 15: AD-Nmd5. The colony size is an indicator of interaction strength. b Serial dilutions of PJ694a/α cells expressing Cdc37-BD or Cdc37(S14A)-BD baits and the indicated AD fusions after growth (10 days at 30 °C) on this same medium and either zero or 6 mM 3-AT. c Measurements of interaction-responsive LacZ reporter gene expression in PJ694a/α cells analysed after growth to mid-log phase at 24° (grey), or 1 h following a heat shock from 24 to 39 °C (black). LacZ expression is expressed as percentage increase relative to that of PJ694a/α control cells containing empty pBDC plasmid and the corresponding AD-protein fusion. d Total versus IMAC-retained His₆-Cak1, Cdc37-HA and Cdc37(S14A)-HA in extracts from 28 °C glucose and galactose-grown cultures of DH211 and DH212 containing pYES2-His₆-CAK1, a vector for GAL1 promoter-driven overexpression of His₆-Cak1. Blots were probed with anti-HA, anti-His and (as loading control) anti-GAPDH antisera