1. Volume 6, Issue 5, Pages 1155-1167 (November 2000)
Phosphorylation of the Cdc42 Exchange Factor Cdc24 by the PAK-like Kinase Cla4 May Regulate Polarized Growth in Yeast 
Marie-Pierre Gulli, Malika Jaquenoud, Yukiko Shimada, Guy Niederhäuser, Philippe Wiget, Matthias Peter 
Molecular Cell 
Volume 6, Issue 5, Pages (November 2000)
DOI: /S (00)

2. Figure 1
Cdc28-Cln2 Triggers Actin Cytoskeleton Polarization by Activating Cdc42
(A) cln1,2,3Δ, pMETCLN2 (YMG258) cells expressing wild-type Gic2-GFP or Gic2-crib−-GFP from the GPD promoter were arrested in G1 by repression of Cln2 in medium containing methionine (−Cln2). Cells were quickly washed, and Cln2 was induced in medium lacking methionine but containing the actin polymerization inhibitor Lat-A (+Cln2 +Lat-A) to prevent bud emergence. Localization of the GFP-tagged proteins was analyzed by fluorescence microscopy.
(B) Protein levels and the phosphorylation state of Gic2 were determined by immunoblotting with anti-Gic2 antibodies from cells treated as described in (A). The arrowhead points to the position of endogenous Gic2, whereas the arrow marks GFP-tagged Gic2 or Gic2-crib−. Equal loading was confirmed by immunoblotting with antibodies against actin.
(C) cln1,2,3Δ, pMETCLN2 (YMG258) cells were grown at 30°C in raffinose medium and arrested by depletion of Cln2 as described in (A). Expression of the indicated proteins from the GAL1 promoter was induced by addition of 2% galactose for 3 hr at 30°C. The polarization state of the cells was assessed by staining F-actin with rhodamine-conjugated phalloidin.
(D) Extracts were prepared from cln1,2,3Δ, pMETCLN2 (YMG258) cells overexpressing the constitutively active (Cdc42-G12V) or the dominant-negative form of Cdc42 (Cdc42-D118A) from the GAL1 promoter, and incubated with glutathione-Sepharose beads coated as indicated with GST, GST-CRIBwt, or GST-crib− purified from E. coli. Bound Cdc42 was detected by immunoblotting. The asterisk marks the position of a protein that cross-reacted with Cdc42 antibodies.
(E) cln1,2,3Δ, pMETCLN2 (YMG258) cells were arrested as described in (A). GAL-expression of either no protein (crosses), Cln2 (diamonds), or Cdc42-G12V (circles) was induced at time 0, and samples were collected every hour. Polymerization of the actin cytoskeleton (continuous line) and cell lysis (dotted line) were determined by rhodamine-conjugated phalloidin staining and phase contrast microscopy, respectively.
(F) The indicated strains were tested for their ability to induce cytoskeletal polarization upon expression of Cdc42-GTP by staining the actin cytoskeleton with rhodamine-labeled phalloidin. The percentage of cells exhibiting a polarized actin cytoskeleton is shown on the right.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2
Cdc28-Cln2 Activation Triggers Redistribution of Cdc24 to the Plasma Membrane
(A) Cdc24-GFP and Cdc24-m1-GFP were expressed from the CYC1 promoter in cln1,2,3Δ, pMETCLN2 (YMG258) cells or cln1,2,3Δ, pMETCLN2, far1-c (YYS77) cells expressing a C-terminally truncated form of Far1. Their subcellular localization was determined by fluorescence microscopy in cells arrested in G1 (−Cln2) or released by expression of Cln2 for 60 min in the presence of Lat-A (+ Cln2 +Lat-A).
(B) Localization of Cdc24-GFP and Cdc24-m1-GFP was examined in G1 cyclin–depleted cells (YMG258) overexpressing Cdc42-GTP.
(C) Localization of Cdc24-GFP or Gic2-GFP was analyzed in cdc42-27 (MOSY0124) cells after 3 hr at 37°C.
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3
Bem1 Is Required to Maintain Cdc24 at the Site of Polarized Growth
(A) The subcellular distribution of Bem1-GFP (YMG687) and Cdc24-GFP (YMG697) was compared at different cell cycle stages of asynchronously growing wild-type cells. The arrows point to cells that have undergone the switch from polar to isotropic bud growth.
(B) Wild-type (YMP480, YMG697) and bem1Δ (YMP459, YMG698) cells expressing either Cdc24-GFP or Gic2-GFP were arrested at 25°C in stationary phase (G0), and released by resuspending the cells into fresh medium containing Lat-A. The localization of Cdc24-GFP and Gic2-GFP was examined by fluorescence microscopy after 3 hr.
(C and D) The subcellular localization of Cdc24-GFP in bem1Δ (YMG698) or wild-type cells (YMG697) was analyzed as they synchronously released from an arrest in stationary phase at 25°C. The arrows point to Cdc24-GFP at tips of small-budded cells and the mother bud-neck region of cells during mitosis. The experiment was quantified (D) by counting at least 400 cells every 30 min by fluorescence and phase contrast microscopy for localization of Cdc24-GFP at the cell cortex (thick lines) and the presence of buds (dotted lines). Circles, bem1Δ (YMG698); squares, wild-type (YMG697).
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Phosphorylation of Cdc24 Is Triggered by Activated Cdc42 and Requires Cla4 in a Complex with Bem1
(A) cdc15-2, CLN2-HA3 (YMP134) cells were released from their block in late telophase (0′). Samples were collected at 15 min intervals, and the protein levels of Cdc24 and Cln2-HA were determined by immunoblotting. Equal loading was confirmed by immunoblotting with antibodies against actin (bottom panel). The arrow indicates the timing of bud emergence.
(B) Wild-type (K699) and cdc42-17 (MOSY0122) cells were grown at 25°C to early log phase, at which time the cultures were shifted to 37°C for 3 hr. cln1,2,3Δ, pMETCLN2 cells (YMG258) expressing as indicated either wild-type or Cdc42 mutant proteins from the GAL1 promoter were arrested in G1 for 3 hr as described, at which time expression of the Cdc42 proteins was induced by addition of 2% galactose for 3 hr. Where indicated, 200 μM Lat-A was added to prevent actin polymerization. The phosphorylation state of Cdc24 was analyzed by immunoblotting.
(C) The indicated strains were transformed with an empty control vector (−) or a plasmid expressing Cdc42-GTP from the GAL1 promoter (+). Cells were grown to early log phase at 30°C in medium containing 2% raffinose, at which time expression of Cdc42-GTP was induced by addition of 2% galactose for 3 hr. The phosphorylation state of Cdc24 was analyzed by immunoblotting.
(D) Wild-type (K699) or cla4Δ (YMP203) cells were cotransformed with an empty vector (−) or a plasmid expressing Cdc42-GTP from the GAL1 promoter (+) together with either another control vector (vector) or plasmids allowing expression of wild-type or the indicated Cla4 mutant proteins from the endogenous promoter. Phosphorylation of Cdc24 and protein levels of Cla4-myc were determined by immunoblotting.
(E) Cla4 interacts with Bem1. Cla4-myc was immunoprecipitated with 9E10 antibodies from cla4Δ (YMP203) cells expressing either untagged Cla4 (−) or Cla4-myc (+) and harboring as indicated either an empty vector (−) or a plasmid expressing Cdc42-G12V (+) from the GAL1 promoter along with either another control vector (−) or a plasmid allowing the expression of Bem1-GFP (+) from the ADH promoter. The immunoprecipitates (IPs) were examined for the presence of Cla4-myc or Bem1-GFP. Total cell lysates prior to the immunoprecipitation are shown below.
(F) The indicated strains harboring either control plasmids (−) or vectors expressing from the GAL1 promoter Cdc42-GTP or Cla4 as indicated (+) were grown at the appropriate temperature (30°C or 25°C for ts alleles) in raffinose medium to early log phase. The cultures were then either maintained at 30°C or shifted to 37°C in the case of ts alleles, and expression of Cdc42-GTP and Cla4 was induced for 3 hr by addition of 2% galactose. The presence of phosphorylated Cdc24 was determined by immunoblotting.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
Overexpression of Cla4 Antagonizes Activation of Cdc42 In Vivo
(A) cln1,2,3Δ, pMETCLN2 (YMG258) cells expressing either no protein (vector), Cla4, or Cdc42-GTP were tested for their ability to induce actin polarization (actin) or membrane recruitment of Gic2-GFP.
(B) Wild-type (K699) cells expressing either no protein (vector), Cla4, or Cdc42-GTP from the GAL1 promoter were grown as described above, and extracts were analyzed for the levels and phosphorylation state of Gic2 by immunoblotting. The arrow marks unphosphorylated Gic2, while the arrowhead indicates the position of the phosphorylated species of Gic2. Equal loading of the gel was confirmed by immunoblotting with antibodies against actin.
(C) The polarization state of wild-type cells expressing either no protein (vector), Cla4 (YMG694), or Cdc42-GTP (YMG472) from the GAL1 promoter was assessed by staining F-actin with rhodamine-conjugated phalloidin (actin). Phase contrast images of the same cells are also shown (phase). The percentage of cells with an unpolarized actin cytoskeleton and unbudded cells is shown on the right.
(D) Wild-type (K699) cells harboring plasmids as indicated in the left panel were grown for 3 days at 30°C on selective media with ([Gal-on], Cdc42 fully expressed) or without ([Gal-off], Cdc42 weakly expressed) galactose to regulate expression of Cdc42, and with ([Met-off], Cla4 not expressed) or without ([Met-on], Cla4 expressed) methionine to regulate expression of Cla4. Note that low levels of Cdc42-GTP can restore growth of cells overexpressing Cla4 (arrows mark the relevant sectors). The expression of Cla4 (top panel) and Cdc42 (bottom panel) was verified by immunoblotting; the cells were grown in media containing galactose but lacking methionine where both Cdc42 and Cla4 were fully induced. The asterisk marks a cross-reacting band.
Molecular Cell 2000 6, DOI: ( /S (00) )

7. Figure 6
Cdc24 Phosphorylation Is Not Required to Activate Cdc42 but May Be Involved in Ending Polarized Growth
(A) ste20Δ, cla4Δ, pcla4-100 (YMG638) cells expressing Gic2-GFP were arrested at 25°C in stationary phase (G0) and released from the arrest by resuspending the cells into fresh medium with or without Lat-A at 37°C to inactivate Cla4. Polarized localization of Gic2-GFP was analyzed by fluorescence microscopy. (B) Wild-type (YMT263) or cla4K594A (YMG454) cells were arrested in G1 by α-factor treatment for 2 hr (block). Cells were then washed with prewarmed medium without α factor to resume cell cycle progression (0`). Samples were collected every 15 min, and the levels and phosphorylation state of Cdc24 and Cln2-HA were determined by immunoblotting.
(C) The percentage of small-budded cells was determined by phase contrast microscopy by counting at least 300 cells for each time point. Note that both wild-type and cla4K594A cells bud with virtually identical kinetics.
(D) The localization of GFP-tagged Cdc24, Bem1, or Gic2 was analyzed by fluorescence microscopy in wild-type (YMT263) or cla4K594A cells (YMG683 and DK281, respectively) as indicated.
(E) The localization of Cdc24-GFP and Gic2-GFP was examined by fluorescence microscopy in cdc42V44A cells (TRY5-3A) shifted to 37°C for 3 hr. The phosphorylation state of Cdc24p in wild-type (K699) and cdc42-V44A (TRY5-3A) cells shifted to 37°C for 3 hr was examined by immunoblotting.
Molecular Cell 2000 6, DOI: ( /S (00) )

8. Figure 7
Deletion of BEM1 or Overexpression of Cla4 Can Reverse the Long-Bud Phenotype of cdc34-2 Cells
(A) The localization of Cdc24-GFP (YMG696), Bem1-GFP (YMG695), or Gic2-GFP (YMT670) was examined in cdc34-2 cells grown at 37°C for 4 hr.
(B) The subcellular distribution of Cdc24-GFP expressed from the CYC1 promoter was analyzed after 4 hr at 37°C in cdc34-2 (YMG696), cdc34-2 bem1Δ (YMG693), and cdc34-2 cells overexpressing Cla4 from the GAL1 promoter (YMG699). The effect on cellular polarization and localization of Cdc24p was quantified. The numbers represent percentage (%) of cells in each category with standard deviations.
(C and D) Bem1-GFP was immunoprecipitated with monoclonal GFP antibodies from cla4Δ (YMP203, [C]) or wild-type cells (K699, [D]) harboring as indicated either an empty control vector (−) or a plasmid allowing the expression of Bem1-GFP (+) from the ADH promoter along with either another control vector (−) or a plasmid (+) expressing Cla4 (C) or Cdc42-G12V (D) from the GAL1 promoter. The immunoprecipitates (IP) were examined for the presence of Bem1-GFP and endogenous Cdc24. The presence of Bem1-GFP, Cdc24 and Cla4, or Cdc42 in cell lysates prior to the immunoprecipitation is shown in the right panels. Note that Bem1 interacts preferentially with unphosphorylated Cdc24 (arrowhead).
Molecular Cell 2000 6, DOI: ( /S (00) )