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. 2013 Jun 13;8(6):e66321.
doi: 10.1371/journal.pone.0066321. Print 2013.

Msb1 interacts with Cdc42, Boi1, and Boi2 and may coordinate Cdc42 and Rho1 functions during early stage of bud development in budding yeast

Yuan Liao  1 , Fei He, Ting Gong, Erfei Bi, Xiang-Dong Gao
Affiliations

Msb1 interacts with Cdc42, Boi1, and Boi2 and may coordinate Cdc42 and Rho1 functions during early stage of bud development in budding yeast

Yuan Liao et al. PLoS One. .

Abstract

Msb1 is not essential for growth in the budding yeast Saccharomyces cerevisiae since msb1Δ cells do not display obvious phenotypes. Genetic studies suggest that Msb1 positively regulates Cdc42 function during bud development, since high-copy MSB1 suppressed the growth defect of temperature-sensitive cdc24 and cdc42 mutants at restrictive temperature, while deletion of MSB1 showed synthetic lethality with cdc24, bem1, and bem2 mutations. However, the mechanism of how Msb1 regulates Cdc42 function remains poorly understood. Here, we show that Msb1 localizes to sites of polarized growth during bud development and interacts with Cdc42 in the cells. In addition, Msb1 interacts with Boi1 and Boi2, two scaffold proteins that also interact with Cdc42 and Bem1. These findings suggest that Msb1 may positively regulate Cdc42 function by interacting with Cdc42, Boi1, and Boi2, which may promote the efficient assembly of Cdc42, Cdc24, and other proteins into a functional complex. We also show that Msb1 interacts with Rho1 in the cells and Msb1 overproduction inhibits the growth of rho1-104 and rho1-3 but not rho1-2 cells. The growth inhibition appears to result from the down-regulation of Rho1 function in glucan synthesis, specifically during early stage of bud development. These results suggest that Msb1 may coordinate Cdc42 and Rho1 functions during early stage of bud development by promoting Cdc42 function and inhibiting Rho1 function. Msb1 overproduction also affects cell morphology, septin organization, and causes increased, aberrant deposition of 1,3-β-glucan and chitin at the mother-bud neck. However, the stimulation of glucan synthesis mainly occurs during late, but not early, stage of bud development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Msb1 localizes to sites of polarized growth and interacts with Cdc42.
(A) GFP-Msb1 localization during bud development. Cells of yeast strain YEF1395 (msb1Δ) carrying plasmid pRS426-GFP-MSB1 were grown in SC-Ura medium and examined for GFP fluorescence. Bar, 5 µm. (B) Msb1 interacts with Cdc42 by GST pull-down assay. Cells of yeast strain YEF473A carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (GST-Cdc42, WT), pEGKT-CDC42Q61L (GST-Cdc42, Q61L), or pEGKT-CDC42T17N (GST-Cdc42, T17N) were grown in SC-Leu-Ura medium containing 2% raffinose at 30°C. Galactose was added to a final concentration of 2%, and the cultures were grown for 4 h to induce the expression of GST-fusion proteins. GST or GST-tagged proteins were pulled down by glutathione-Sepharose beads from equal amounts of Triton X-100-solubilized cell lysates. Molecular weight: GST (27 kDa), GST-Cdc42 (46 kDa), HA-Msb1 (130 kDa).
Figure 2
Figure 2. Detection of Msb1 interaction with Bem1, Cdc24, Boi1, and Boi2.
(A) Msb1 interacts with Boi2 and Boi1 by GST pull-down assay. Cells of yeast strain YEF1395 (msb1Δ) carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (Cdc42), pEGKT-BEM1 (Bem1), pEGKT-CDC24 (Cdc24), pEGKT-BOI1 (Boi1), or pEGKT-BOI2 (Boi2), as well as cells of yeast strain JGY2425 (boi1Δ) or JGY2349 (boi2Δ) carrying YEp181-3HA-MSB1/pEGKT or YEp181-3HA-MSB1/pEGKT-BOI1 were used in the assay. (B) Msb1 interacts with the C-terminal region of Boi1 and Boi2 lacking the proline-rich motif. Left panel, the schematic diagram of domain structure in Boi1 and Boi2. P, proline-rich motif. Right panel, GST pull-down assay with GST-tagged Boi2, Boi2-C, and Boi1-C in yeast strains YEF473A (WT), JGY2425 (boi1Δ), and JGY2349 (boi2Δ). (C) Msb1 interacts with Cdc42 in boi1Δ boi2Δ cells. GST pull-down assay was performed in cells of yeast strain YEF473A (WT) and JGY2821 (boi1Δ boi2Δ) carrying YEp181-3HA-MSB1/pEGKT or YEp181-3HA-MSB1/pEGKT-CDC42. Molecular weight of GST-tagged proteins: Cdc42 (46 kDa), Bem1 (86 kDa), Cdc24 (120 kDa), Boi1 (133 kDa), Boi2 (140 kDa), Boi1-C (88 kDa), and Boi2-C (90 kDa).
Figure 3
Figure 3. Functional interaction between Msb1 and Boi1/Boi2.
(A) Morphology of boi1Δ, boi2Δ, and boi1Δ boi2Δ cells overexpressing MSB1. Cells of yeast strains JGY2425 (boi1Δ), JGY2349 (boi2Δ), and JGY2821 (boi1Δ boi2Δ) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing galactose and raffinose for 12 h. (B) Morphology of cells with elevated expression of MSB1 and BOI2. Cells of yeast strain YEF473A carrying plasmids YEp13-MSB1/pUG36 (MSB1↑), YEp13/pUG36-BOI2 (BOI2↑), or YEp13-MSB1/pUG36-BOI2 (MSB1↑ BOI2↑) were grown on SC-Leu-Ura plate containing dextrose at 30°C for 16 h. Bars, 5 µm.
Figure 4
Figure 4. Phenotypes of cells overexpressing MSB1.
(A) Morphology and septin organization (shown by GFP-Cdc3) in cells overexpressing MSB1. Cells of yeast strain JGY881 (GFP-CDC3) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing galactose and raffinose at 30°C for 16 h. DIC and GFP fluorescence images were taken. (B, C) Chitin deposition (B) as well as 1,3-β-glucan and mannan distribution (C) were visualized in cells of strain YEF473A carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) grown in SC-Ura medium containing galactose and raffinose at 30°C. The cells were stained for chitin, 1,3-β-glucan, and mannan. (D) Msb1 localizes to sites of aberrant glucan deposition. Cells of strain JGY139A (GAL1-GFP-MSB1) was grown in SC-Ura medium containing galactose and raffinose at 30°C. Cells were stained for 1,3-β-glucan with aniline blue. Bars, 5 µm.
Figure 5
Figure 5. Msb1 overproduction inhibits the growth and glucan synthesis in rho1-104 cells.
(A) Overexpression of MSB1 inhibits the growth of rho1-104 cells. Cells of yeast strain NY1537 (WT) and NY1538 (rho1-104) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 3 d. (B) Overexpression of MSB1 in rho1-104 cells causes the accumulation of small-budded cells. The percentage of small buds in the population of budding cells overexpressing MSB1 as in panel A was quantitated. More than 200 buds were scored. (C) Glucan distribution in rho1-104 cells overexpressing MSB1. Cells as in panel A were stained for 1,3-β-glucan. Bar, 5 µm.
Figure 6
Figure 6. Msb1 inhibits Rho1 function and interacts with Rho1 in vivo.
(A) Overexpression of MSB1 inhibits the growth of rho1-3 cells. Cells of yeast strain NY2284 (WT), NY2285 (rho1-2), and NY2286 (rho1-3) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 3 d. (B) Overexpression of MSB1 does not inhibit the growth of cdc42-Ts cells. Cells of yeast strain YEF473A (WT), YEF2258 (cdc42-201), and JPC241 (cdc42G60D) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing dextrose (Dex) or galactose and raffinose (Gal) at 30°C. Pictures were taken after 4 d. (C) Msb1 interacts with Rho1 by GST pull-down assay. Cells of yeast strain YEF1395 (msb1Δ) carrying YEp181-3HA-MSB1 along with pEGKT (GST), pEGKT-CDC42 (Cdc42), or pEGKT-RHO1 (Rho1) were used in the assay. Molecular weight: GST (27 kDa), GST-Cdc42 (46 kDa), GST-Rho1 (48 kDa).
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