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. 2004 Apr;15(4):1711–1723. doi: 10.1091/mbc.E03-07-0461

Figure 7.

Figure 7.

Bbp1p243–385-dependent localization of Cdc5pΔC to the SPB remedies the cdc5Δ defect. (A) Bbp1p243–385-dependent targeting of the localization-defective Cdc5pΔC to the spindle poles. Wild-type strain KLY1546 transformed with control YCplac111 vector, GFP-CDC5 (pKL2701), GFP-CDC5ΔC-BBP1243–385 (pKL2078), GFP-CDC5ΔC-MPS2 (pKL2422), or GFP-CDC5ΔC (pCJ232) was grown in the presence of 15 μg/ml of nocodazole for 3 h at 23°C, fixed, and then subjected to fluorescent microscopy to examine the localization of the GFP-fused proteins. (B) To examine the ability of various constructs in A to complement the cdc5Δ defect, strain KLY3721 (cdc5Δ + GAL1-EGFP-PLK1) transformed with each plasmid was cultured overnight, serially diluted, and spotted onto either YEP-galactose (YEPG) or YEP-glucose (YEPD) plates, and then incubated at 30°C for 3 d. (C and D) To examine the ability of CDC5ΔC-BBP1, CDC5ΔC-BBP1243–385, or CDC5ΔC-MPS2 to complement the bbp1-1 or mps2-1 defect, strains KLY2761 (bbp1-1) or SMY6-4b (mps2-1) were transformed with the indicated plasmids. The resulting transformants were cultured overnight, serially diluted, spotted onto YEP-glucose plates, and then incubated at the indicated temperatures for 3 d. BBP1, pSK1878; CDC5ΔC-BBP1243–385, pKL2078; CDC5ΔC-BBP1, pKL2420; MPS2, pKL1135; and CDC5ΔC-MPS2, pKL2422. (E) To measure the kinase activities associated with Flag-YFP-Cdc5p, Flag-YFP-Cdc5pΔC-Bbp1p243–385, or Flag-YFP-Cdc5pΔC-Mps2p, a protease-negative JB811 strain transformed with pKL2772, pKL2078, or pKL2422, respectively, were arrested with nocodazole for 3 h, and then harvested. Equal amounts of protein prepared from each transformant were subjected to anti-FLAG immunocomplex kinase assays. Reaction mixtures were separated in SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and then exposed to detect the kinase activities (top). The same blot was subjected to immunoblotting with anti-GFP antibody to determine the amount of Flag-YFP-Cdc5p or the corresponding chimera proteins in each immunoprecipitate (bottom). Asterisks indicate nonspecific phosphorylation bands. Cs, casein.