Figure 9.

Together the Mcm3 NES and the consensus CDK sites in Mcm3 NLS are required for nuclear export of GFP-Mcm3 and Mcm7-GFP. (A) GFP-Mcm3 is strongly nuclear throughout the cell cycle when both the leucine-rich motif of the Mcm3 NES and the four CDK consensus sites flanking the Mcm3 NLS are mutated. Exponentially growing cultures of YJL2162 (GFP-MCM3), YJL2714 (GFP-mcm3-cdk4A), and YJL5216 (GFP-mcm3-cdk4A-nes) were examined by fluorescence and DIC microscopy. Cells were categorized as unbudded (UB), small budded (SB), or uninucleate large budded (LB) based on their DIC image and DAPI fluorescence. Binucleate large budded cells, which comprise approximately half of all large budded cells, are unseparated postmitotic cells (many in G1 phase) and were thus not included in the analysis. Each bud category was further subclassified into cells with or without detectable nuclear GFP fluorescence above cytoplasmic levels. Bar graphs show the percent of cells with nuclear or nonnuclear GFP fluorescence and the total number of cells counted (in parentheses) for each bud category. (B) Mcm7-GFP is strongly nuclear throughout the cell cycle when both the leucine-rich motif of the Mcm3 NES and the four CDK consensus sites flanking the Mcm3 NLS are mutated. Exponentially growing cultures of YJL1979 (MCM3 MCM7-GFP), YJL5691 (mcm3-cdk4A mcm7::{MCM7-GFP}), and YJL5221 (mcm3-cdk4A-nes MCM7-GFP) were examined by fluorescence and DIC microscopy as described in Figure 9A.