Figure 5.

The Mcm3 NES is required for efficient nuclear export of Mcm3 and Mcm7 in cycling cells. (A) Mutation of the Mcm3 NES increases the population of cells containing nuclear GFP-Mcm3 during exponential growth. Exponentially growing cultures of YJL2162 (GFP-MCM3) (top) and YJL2741 (GFP-mcm3-nes) (bottom) were examined by fluorescence and DIC microscopy. Representative fluorescent fields are shown. 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 of these categories was further subclassified into cells with or without detectable nuclear GFP fluorescence above cytoplasmic levels as exemplified by the pictures of individual cells. 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 stage. (B) Mutation of the Mcm3 NES increases the population of cells containing nuclear Mcm7-GFP during exponential growth. The same experiment and analysis described in Figure 5A was performed for YJL1979 (MCM3 MCM7-GFP) and YJL5439 (mcm3-nes MCM7-GFP).