FIGURE 7.

Overproduction of Mcb1 causes dissociation of Mcm2 from other MCM proteins. A, nmt1-mcb1HA mcm2V5 mcm4GFP (FY4961) overnight culture grown in low thiamine was inoculated into −thiamine (−T) and +thiamine (+T) media and grown at 32 °C for 14 h. Cells were harvested and lysed in B88 buffer. Soluble lysates were immunoprecipitated (IP) with anti-GFP (lanes 3 and 4) and anti-V5 (lanes 5 and 6). Immunoprecipitated samples were separated by SDS-PAGE gel and blotted for Mcm2V5, Mcm4GFP, Mcm7, Mcm6, and Mcb1HA. B, wild-type (FY254), nmt1-mcm4HA (FY1602), and nmt1-mcm2HA (FY861) cells were transformed with plasmids overexpressing Mcb1 (pLD18 or pLD10) and empty vector (pSLF972 or pSGP72). Transformants (top, a–d, and bottom, a–d) were restreaked on −thiamine and +thiamine plates and incubated at 32 °C. C, mcm2V5 mcm4GFP cells carrying full-length mcb1 (FY4961 and FY5407) and mcb1 deletion mutants (FY5408–5415) at the leu1-32 locus were grown in thiamine-containing medium, then harvested, and lysed in B88 buffer. Soluble lysates were immunoprecipitated with anti-GFP. Immunoprecipitated samples were separated by 12% SDS-PAGE. We used an 8% gel to separate bigger Mcb1 truncations from IgG (bottom panel). We blotted for Mcm4GFP and Mcb1HA. The data are summarized in Fig. 3A. D, mcm2V5 mcm4GFP cells carrying six mcb1 deletion mutants at the leu1-32 locus (nmt1-mcb1D2HA (FY5408), nmt1-mcb1D5HA (FY5411), nmt1-mcb1D6HA (FY5412), nmt1-mcb1D78HA (FY5413), nmt1-mcb1D9HA (FY5414), and nmt1-mcb1D22HA (FY5415)) were grown in −thiamine medium, then harvested, and lysed in B88 buffer. Soluble lysates were immunoprecipitated with anti-GFP. Fifteen micrograms of soluble proteins (lanes 1-8) and immunoprecipitated samples (lanes 9-14) were separated by SDS-PAGE and blotted for Mcm4GFP, Mcm2V5, Mcm6, Mcm7, and Mcb1HA.