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PNAS 101 (12): 4124-4129

Copyright © 2004 by the National Academy of Sciences.

Coupling morphogenesis to mitotic entry

Krisada Sakchaisri*,{dagger}, Satoshi Asano*, Li-Rong Yu{ddagger}, Mark J. Shulewitz§, Chong J. Park*, Jung-Eun Park*, Young-Wook Cho*, Timothy D. Veenstra{ddagger}, Jeremy Thorner§, and Kyung S. Lee*,

*Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; {ddagger}Mass Spectrometry Center, National Cancer Institute–Frederick, Frederick, MD 21702; §Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and {dagger}Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand


Figure 1
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Fig. 1. Cdc5 and Cla4 phosphorylate Swe1 in an additive manner. Recombinant HA-Cdc5-Flag, HA-Cdc5(N209A)-Flag, HA-Cla4-Flag, and HA-Cla4(K594A)-Flag were immunoprecipitated from Sf9 cells by using an anti-Flag antibody cross-linked to agarose beads (Sigma). The reactions were carried out with or without kinase-inactive GST-Swe1(K473A) in the presence of 5 µM ATP (10 µCi of [{gamma}-32P]ATP; 1 Ci = 37 GBq) for 30 min at 30°C, separated in 10% SDS/PAGE, stained with Coomassie blue (Lower), and then subjected to autoradiography (Upper). Asterisks indicate a contaminating protein that copurified with Cdc5 and Cla4.

 

Figure 2
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Fig. 2. Requirement of Cdc5 and Cla4 for proper phosphorylation and degradation of Swe1. (A) Strain KLY4864 (cdc5{Delta} HSL1-Myc13 + YCplac33-GAL1-cdc5-1) transformed with either a centromeric CDC5 (pCDC5) (KLY4900) or a control vector (KLY4899) was arrested in G1 by α-factor treatment immediately after transferring to yeast extract/peptone (YEP)-glucose medium, then released into nocodazole-containing medium. Total cellular proteins were prepared at the indicated times and separated by 8% SDS/PAGE (without SDS in the separation gel to detect the phosphorylated Swe1 isoforms). Immunoblotting analyses were carried out by using antibodies against Swe1, c-Myc epitope (for Hsl1-Myc13), and Hsl7. P-Hsl7, phosphorylated Hsl7. Asterisks in A, B, D, and E are the same nonspecific protein cross-reacting with anti-Swe1 antibody. (B) Strain KLY4497 (cdc5{Delta} HSL1-Myc13 + YCplac33-GAL1-PLK1) transformed with either a centromeric pCDC5 (KLY4532) or a centromeric pCDC5{Delta}C-CNM67 (KLY4533) were cultured overnight in YEP-glucose medium, arrested with α-factor, and then released into YEP-glucose medium containing nocodazole. Samples were analyzed as in A. (C) To determine the timing of Cdc5 localization to the bud-neck, asynchronously growing KLY4816 cells, which express EGFP-CDC5 under endogenous CDC5 promoter control, were cultured overnight, fixed, and subjected to immunostaining with anti-tubulin antibody. Arrows indicate the bud-neck localized Cdc5. (Scale bar, 5µm.) (D) Strains KLY4071 (CLA4) and KLY4536 (cla4{Delta}) were grown either asynchronously or arrested with hydroxyurea (HU) or nocodazole for 4 h before the preparation of total cellular lysates. (E) Strains KLY4913 (cla4{Delta} + pCLA4) and KLY4914 (cla4{Delta} + pcla4-as3) were arrested with either hydroxyurea or nocodazole for 2 h before the addition of either 25 µM of 1-NM-PP1 or control DMSO into the medium. Cells were cultured for additional 2 h before harvesting the samples.

 

Figure 3
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Fig. 3. Cdc5, Cla4, and Hsl1 function in pathways distinct from one another. (A) Strains KLY1546 (wild type), KLY4651 (cdc5-3 CDC14TAB6-1), KLY2869 (hsl1{Delta}), KLY4649 (cla4{Delta}), KLY4653 (cdc5-3 CDC14TAB6-1 cla4{Delta}), and KLY5098 (hsl1{Delta} cla4{Delta}) were cultured overnight. These cultures were serially diluted, spotted on YEP-glucose, and incubated at the indicated temperature. (B and C) Strain KLY5097 (hsl1{Delta} cla4{Delta} swe1{Delta}) (B) or strain KLY4756 (cdc5-3 CDC14TAB6-1 cla4{Delta} swe1{Delta}) (C) was transformed with either a centromeric SWE1 (pSWE1) or control vector. The resulting transformants were cultured overnight, serially diluted, and spotted on synthetic minimal plate (SDM) lacking histidine (B) or uracil (C) to select for the presence of the introduced plasmid. (D) To examine the morphologies of the strains used in AC, cells cultured overnight at 23°C were fixed and then examined by differential interference contrast (DIC) microscopy. The synthetically elongated bud morphologies were manifest among cdc5-3 CDC14TAB6-1, cla4{Delta}, and hsl1{Delta} mutants even at 23°C. (Scale bar, 5 µm.) (E) To examine whether Cdc5 and Cla4 cooperate to regulate the Swe1 stability, cla4{Delta} was introduced into strains KLY4714 (cdc5{Delta} + pGAL1-cdc5-1 + YCplac22) and KLY4715 (cdc5{Delta} + pGAL1-cdc5-1 + YCplac22-CDC5), generating KLY4811 and KLY4813, respectively. All four strains were cultured overnight in YEP-galactose medium, shifted to YEP-glucose medium containing either HU or nocodazole, and cultured for an additional 4 h. Longer exposure time was required to detect Swe1 from nocodazole-arrested samples (Right). Asterisks, a cross-reacting protein with the anti-Swe1 antibody.

 

Figure 4
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Fig. 4. Mutations in the Cdc5- and Cla4-dependent phosphorylation sites onto Swe1 induce elongated bud morphology as a result of Swe1 stabilization at the bud-neck. (A) An mih1{Delta} strain (M-600) was integrated with control vector or various forms of SWE1-Myc12 at the URA3 locus. The resulting strains (KLY4802–KLY4810) were cultured overnight, fixed, and subjected to DIC microscopy (A) to measure the cell size (Fig. 11). (Scale bar, 5 µm.) (B) To determine the steady-state level of various Swe1-Myc12 proteins, total cellular lysates were prepared from the asynchronously growing strains used in A. Samples were separated in 10% SDS/PAGE and then subjected to immunoblotting with anti-Swe1 antibody. Asterisk indicates a nonspecific band. (C)To examine the localization of various Swe1 proteins, the wild-type and mutant forms of SWE1 in the strains used in A were C-terminally tagged with three copies of GFP by replacing the Myc12 epitope. The resulting strains were then cultured at 23°C, arrested with hydroxyurea for 3 h to enrich the Swe1 protein, and subjected to confocal microscopy. (Scale bar, 5 µm.)

 

Figure 5
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Fig. 5. Model illustrating the Swe1 phosphorylation and degradation events during the cell cycle (see text for details).

 


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