Figure 4.

Mcm3 contains an NES, which in combination with the Mcm2 NLS and Mcm3 NLS, directs the cell cycle-regulated localization of GFP₃. (A) The cytoplasmic localization of NLS2-NLS3NES3-GFP₃ in G2/M phase is dependent on the leucine-rich motif of the Mcm3 NES. Cultures of YJL4662 (crm1Δ trp1::{pGAL-NLS2-NLS3NES3-GFP₃),TRP1} [crm1-T539C]) and YJL4860 (crm1Δ trp1::{pGAL-NLS2-NLS3nes3-GFP₃),TRP1} [crm1-T539C]) constitutively expressing their GFP₃ fusion proteins in YEP-Gal medium were arrested for 90 min in either G1 phase with α-factor or G2/M phase with nocodazole before being examined by fluorescence microscopy. (B) NLS2-NLS3NES3-GFP₃ is exported from the nucleus by a Crm1-dependent mechanism. YJL4662 constitutively expressing NLS2-NLS3NES3-GFP₃ in YEPGal medium was arrested in G1 phase with α-factor. At time 0, cells were released from the arrest in the presence or absence of 100 ng/ml leptomycin B (LMB). Samples were collected at the indicated times for flow cytometry and fluorescence microscopy.