GTP-locked Rho1 fails to support CAR assembly because it fails to be targeted to the bud neck during anaphase. (A) GTP-locked Rho1 cannot bypass the requirement for Polo kinase to promote CAR assembly. Control cdc15 RHO1strains (telophase arrest, high Cdc5/Polo activity) or cdc5 RHO1 strains (telophase arrest, low Cdc5/Polo activity) bearing the indicated plasmids were released from a G1 block for 2 h at 34.5°C and CAR assembly was monitored with Tpm2-GFP. (B) RHO1-Q68H (GTP-locked allele) is a recessive loss-of-function allele with respect to CAR assembly. The indicated strains were synchronized in metaphase by Cdc20 depletion, released under conditions that inactivate endogenous Rho1, and CAR assembly was monitored at intervals by Tpm2-GFP. The RHO1 alleles are expressed from the endogenous promoter on low-copy-number plasmids. (C) GTP-locked Rho1 fails to concentrate at the bud neck during anaphase. Cells were arrested in telophase with high or low Cdc5/Polo activity as in A. The cells either expressed the wild-type Rho1 GEF Tus1 or a Tus1-Mlc2 fusion that forces Tus1 localization to the bud neck. The localization of HA-tagged wild-type Rho1 or the indicated Rho1 mutants was determined by immunofluorescence. (D) Nucleotide free Rho1-G22A localizes to the division site. Because Rho1-G22A acts as a dominant-negative mutant and is highly toxic when expressed even from the endogenous promoter (not shown), this experiment was performed in strains containing activated Pkc1, Pkc1-R398P (Nonaka et al. 1995), which we found rescued the near-lethal phenotype of Rho1-G22A expression. Arrows indicate the bud neck localization of Tpm2 (A, B) or Rho1 (C). Arrowheads in C indicate lack of the bud neck signal. Error bar is the SEM. Bar, 3 μm.

Creation of viable yeast strains lacking all Rho1 GEFs. (A) Outline of Rho1 regulation in budding yeast. See the text for details. (B) A plasmid shuffle assay demonstrates that the essential function of Rho1 GEFs requires their catalytic activity. The ΔGEF strain grows in the presence of a <ROM2 URA3 CEN> plasmid but fails to grow after counterselection with 5-FOA. A point mutation in a critical catalytic residue in the DH domain of Rom2 (T666A) fails to complement the lethality of the ΔGEF strain. The indicated strains were grown for 3 d at 24°C. (C) GAP gene deletions or rapid cycling Rho1 mutants restore viability to the ΔGEF strain. A plasmid shuffle was performed to assess viability as in B. (Top) Deletion of the Rho1 GAP genes LRG1 or SAC7 (but not BAG7) restored growth to the ΔGEF strain at 24°C. Deletion of the sole Rho1 GDI gene RDI1 had no effect on growth of the ΔGEF strain. (Bottom) Two predicted Rho1 “rapid cycling” mutations restore growth to the ΔGEF strain. (D) The ΔGEF ΔGAP strain has high levels of Rho1-GTP; deletion of Rho1 GEFs has only a subtle effect on the total cellular levels of Rho1-GTP. GTP-Rho1 was assayed in the indicated strains by a Rho-binding domain pull-down assay.

The catalytic activity of Rho1 GEFs is required for CAR assembly. (A) Defective CAR assembly in the absence of catalytically active Rho1 GEFs. (Top panel) Alexa 568-phalloidin labeling to visualize the CAR in wild-type and ΔGEF ΔGAP cells. (Bottom panel) Anaphase spindles visualized with GFP-tubulin. Arrows indicate the position of the CAR, and arrowheads indicate absence of the CAR. (B,C) Percentage of anaphase cells (fully elongated spindles) with detectable CARs in the indicated strains. (D) Localization of Rho1 in anaphase cells lacking Rho1 GEFs. Rho1 was detected by immunofluorescence using a rabbit anti-Rho1 antibody in the indicated strains. Microtubules were labeled with GFP-α-tubulin. Representative cells are shown in the left and the results are summarized in the right. The arrow indicates bud neck localizaton of Rho1, and the arrowhead indicates the absence of Rho1.

GEFs recruit Rho1 to the division site during anaphase; a GEF-independent mechanism recruits Rho1 after mitotic exit. (A) Anaphase localization of Rho1 variants. Rho1 mutants were expressed as GFP-fusions. Microtubules were labeled with CFP-α-tubulin. Representative images are shown in the left. Arrows indicates the bud neck localizaton of Rho1 in anaphase and arrowheads indicate the absence of Rho1. The results are summarized on the right. (B) Box plot distributions of the timing of the bud neck localization of GFP-Rho1 examined by time-lapsed imaging. CFP-tubulin (in green) was used to track cell cycle position. Time of mitotic exit is judged by spindle disassembly (time = 0). In these distributions, the maximum and minimum values, the interquartile range (marked by boxes), and the median value (marked by horizontal line) for the initial bud neck localization of GFP-Rho1 in each population are shown. CFP and GFP signal were imaged every 30 sec at 24°C. Examples are shown at the bottom. Arrows indicate the appearance of Rho1 signal at the bud neck.

The Rho1 PBS binds lipids and is a bud neck targeting signal. (A) PIP strip (Echelon) membranes were incubated with yeast cell lysates from the cells expressing endogenous-level GFP-RHO1 or GFP-RHO1-5KA. Membrane-bound proteins were detected with an anti-GFP antibody. (LPA) Lysophosphatidic acid; (LPC) lysophosphatidylcoline; (PI) phosphatidylinositol; (PE) phosphatidylethanolamine; (PC) phosphatidylcholine; (S1P) sphingosine-1-phosphate; (PA) phosphatidic acid; (PS) phosphatidylserine. (B) Amino acid sequence of the Rho1 tail and derivatives. Positively charged residues (K) are in capital letters. The CAAX sequence of Rho1 and plasma membrane targeting sequence of Ras2 (288–322) are in italics. Mutated residues are in bold face. (C) Localization of Rho1 tail mutants. GFP-tagged Rho1 mutants were localized in the presence (ΔGAP) or absence (ΔGEFΔGAP) of Rho1 GEFs. Log-phase cells at 24°C were imaged without fixation. Bar, 3 μm. (D) The level of GTP-Rho1 was measured in the indicated strains by a RBD pull-down assay as in Figure 2B. (E) Localization of GFP-Rho1tail and GFP-Ras2tail during cytokinesis. (F) GFP-Rho1tail was expressed in the strain expressing septin (Shs1)-mRFP. The Rho1 tail localized to the bud neck only after the septin rings split. The localization of GFP-Rho1tail in cdc15-2-arrested cells and after inactivation of Mss4 (3 h, 37°C) are shown. Arrows indicate accumulation to the bud neck.

The Rho1 PBS is necessary for normal Rho1 function including cytokinesis. (A) Amino acid sequence of Rho1 tail and its mutants as in Figure 5B. (B) The functional importance of the basic residues in the Rho1 tail for growth and cytokinesis. Strains of the indicated genotype were spotted onto plates in serial dilutions. Wild-type RHO1, expressed from the GAL1 promoter, was repressed by growth on medium containing glucose. Images were taken after 3 d growth at the indicated temperature. Cytokinesis failure (more than three connected cells, n = 200) was scored after depletion of wild-type Rho1 for 20 h in glucose-containing medium at 24°C. Cells were sonicated after fixation.

Rho1 promotes CAR-independent cytokinesis. (A) GTP-locked Rho1, an activated mutant of Pkc1, or overexpression of the PI(4)P 5-kinase Mss4 rescues growth defect of Δmyo1 at 37°C. (B) Neither GTP-locked Rho1 nor Mss4 restore CAR assembly in Δmyo1 strains. The indicated strains were fixed and stained with Alexa-568-conjugated phalloidin. The percentage of large budded cells with detectable CAR assembly was scored. (C) The bud neck localization of Chs3 requires Rho1 activity. Localization of Chs3-GFP was monitored after Rho1 inactivation (2 h at 37°C). Mitotic spindle visualized by GFP-tubulin is used as a cell cycle marker. The percentage of late mitotic cells with Chs3 accumulation to the neck was scored after fixation. (D) GTP-locked Rho1 requires CHS3 to suppress the growth defect of Δmyo1. A plasmid shuffle was performed as in A for 3 d at 37°C.

A model of Rho1 regulation and function in cytokinesis. (Left) CAR assembly in late anaphase. Polo-dependent recruitment of Tus1 promotes CAR assembly by recruiting Rho1 to the division site. After mitotic exit, Chs2 is transported to the bud neck and CAR constriction guides primary septum formation. (Right) After mitotic exit, Polo is degraded and PIP2 plays an important role in concentrating Rho1 activity at the bud neck. See the Discussion for details. (GS) Glucan synthase; (CS) chitin synthase.