Figure 2.
The Mcm2 and Mcm3 NLSs are each required for nuclear localization of the Mcm2-7 complex. (A) The Mcm2 NLS is required for nuclear localization of Mcm2-GFP. YJL3265 (MCM2::{MCM2-GFP}), YJL1231 (MCM2::{mcm2-nls-GFP}), and YJL1228 (MCM2::{mcm2-nls-GFP-SVNLS2}) were arrested in G1 phase with α-factor then examined by fluorescence microscopy. (B) The Mcm3 NLS is required for nuclear localization of GFP-Mcm3. YJL2669 (MCM3::{GFP-MCM3}), YJL2675 (MCM3::{GFP-mcm3-nls}), and YJL2665 (MCM3::{SVNLS2-GFP-mcm3-nls}) were arrested in G1 phase with α-factor then examined by fluorescence microscopy. (C) The Mcm2 NLS is required for nuclear localization of Mcm7-GFP. Cultures of YJL3765 (MCM7-GFP mcm2-td MCM2), YJL3840 (MCM7-GFP mcm2-td mcm2-nls), and YJL3799 (MCM7-GFP mcm2-td mcm2-nls-SVNLS2) growing exponentially at 23°C were arrested in G2/M phase by addition of nocodazole for 3 h. Galactose was added and cultures were shifted to 37°C for 30 min to degrade Mcm2-td. Cells were then released from G2/M phase into a G1 phase arrest by shifting them to fresh medium containing α-factor for 2 h (still in the presence of galactose and at 37°C). Cells examined by fluorescence microscopy are shown just before the G2/M phase release (NOC arrest) and at the G1-phase block (α-factor arrest). (D) The Mcm3 NLS is required for nuclear localization of Mcm7-GFP. Cultures of YJL3464 (MCM7-GFP mcm3-td MCM3), YJL3469 (MCM7-GFP mcm3-td mcm3-nls), and YJL3474 (MCM7-GFP mcm3-td SVNLS2-mcm3-nls) were subjected to the same experimental protocol described for Figure 2C.