Figure 2
Construction and characterization of a strain expressing Cdc23-CFP with an internal TEV cleavage site (Cdc23 S424 ::Tcs). A. Regions of Cdc23 with attributed functions (see text for details), and location of Tcs in Cdc23S424::Tcs. The grey bar superimposed on the black Zn-finger region corresponds to the domain of S. cerevisiae Mcm10 required for homocomplex assembly [31]. "ts" indicates the location of cdc23-1E2 and -M36 ts mutations. B. Expression of TEV protease causes loss of viability of a cdc23 S424 ::Tcs strain (P1323) at 37°C. Serial dilutions of the P1323 strain were spotted onto minus or plus thiamine media and incubated at the temperatures shown. C. Western analysis of Cdc23 levels in cdc23 S424 ::Tcs strains in presence or absence of pTEV (pREP3X-TEV-NLS) plasmid, grown in the presence or absence of thiamine. The blot was probed with antibodies against Cdc23, and α-tubulin is shown as a loading control. Cells were first grown at 25°C in the presence of thiamine to repress TEV protease expression, then washed and grown either in the presence or absence of thiamine for 24 h at the indicated temperatures, before extracting protein for western analysis. Cdc23S424::Tcs indicates the position of full length protein, N & C Cdc23 indicate the fragments (49 kDa (N) and 46 kDa (C)) after cleavage by TEV protease. Comparison of Cdc23 levels in strains 3 and 4 (wt) shows that the modification of Cdc23 does not affect protein levels under conditions where TEV protease is absent. Strains used were: (1) P1323-1; (2) P1323-2; (3); P1322 (4) P138.