Msb1 overproduction inhibits the growth and glucan synthesis in <i>rho1-104</i> cells.

<p>(<b>A</b>) Overexpression of <i>MSB1</i> inhibits the growth of <i>rho1-104</i> cells. Cells of yeast strain NY1537 (WT) and NY1538 (<i>rho1-104</i>) 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>B</b>) Overexpression of <i>MSB1</i> in <i>rho1-104</i> cells causes the accumulation of small-budded cells. The percentage of small buds in the population of budding cells overexpressing <i>MSB1</i> as in panel A was quantitated. More than 200 buds were scored. (<b>C</b>) Glucan distribution in <i>rho1-104</i> cells overexpressing <i>MSB1</i>. Cells as in panel A were stained for 1,3-β-glucan. Bar, 5 µm.</p

Msb1 Interacts with Cdc42, Boi1, and Boi2 and May Coordinate Cdc42 and Rho1 Functions during Early Stage of Bud Development in Budding Yeast

<div><p>Msb1 is not essential for growth in the budding yeast <i>Saccharomyces cerevisiae</i> since <i>msb1</i>Δ cells do not display obvious phenotypes. Genetic studies suggest that Msb1 positively regulates Cdc42 function during bud development, since high-copy <i>MSB1</i> suppressed the growth defect of temperature-sensitive <i>cdc24</i> and <i>cdc42</i> mutants at restrictive temperature, while deletion of <i>MSB1</i> showed synthetic lethality with <i>cdc24</i>, <i>bem1</i>, and <i>bem2</i> 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 <i>rho1-104</i> and <i>rho1-3</i> but not <i>rho1-2</i> 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.</p></div

Functional interaction between Msb1 and Boi1/Boi2.

<p>(<b>A</b>) Morphology of <i>boi1</i>Δ, <i>boi2</i>Δ, and <i>boi1</i>Δ <i>boi2</i>Δ cells overexpressing <i>MSB1</i>. Cells of yeast strains JGY2425 (<i>boi1</i>Δ), JGY2349 (<i>boi2</i>Δ), and JGY2821 (<i>boi1</i>Δ <i>boi2</i>Δ) carrying pEGKT316 (Vec) or pEGKT316-MSB1 (GAL-MSB1) were grown in SC-Ura medium containing galactose and raffinose for 12 h. (<b>B</b>) Morphology of cells with elevated expression of <i>MSB1</i> and <i>BOI2</i>. 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.</p

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Msb1 inhibits Rho1 function and interacts with Rho1 <i>in vivo</i>.

<p>(<b>A</b>) Overexpression of <i>MSB1</i> inhibits the growth of <i>rho1-3</i> cells. Cells of yeast strain NY2284 (WT), NY2285 (<i>rho1-2</i>), and NY2286 (<i>rho1-3</i>) 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>B</b>) Overexpression of <i>MSB1</i> does not inhibit the growth of <i>cdc42</i>-Ts cells. Cells of yeast strain YEF473A (WT), YEF2258 (<i>cdc42-201</i>), and JPC241 (<i>cdc42^G60D</i>) 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. (<b>C</b>) Msb1 interacts with Rho1 by GST pull-down assay. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) 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).</p

Msb1 localizes to sites of polarized growth and interacts with Cdc42.

<p>(<b>A</b>) GFP-Msb1 localization during bud development. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) carrying plasmid pRS426-GFP-MSB1 were grown in SC-Ura medium and examined for GFP fluorescence. Bar, 5 µm. (<b>B</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-CDC42^Q61L (GST-Cdc42, Q61L), or pEGKT-CDC42^T17N (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).</p

Detection of Msb1 interaction with Bem1, Cdc24, Boi1, and Boi2.

<p>(<b>A</b>) Msb1 interacts with Boi2 and Boi1 by GST pull-down assay. Cells of yeast strain YEF1395 (<i>msb1</i>Δ) 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 (<i>boi1</i>Δ) or JGY2349 (<i>boi2</i>Δ) carrying YEp181-3HA-MSB1/pEGKT or YEp181-3HA-MSB1/pEGKT-BOI1 were used in the assay. (<b>B</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 (<i>boi1</i>Δ), and JGY2349 (<i>boi2</i>Δ). (<b>C</b>) Msb1 interacts with Cdc42 in <i>boi1</i>Δ <i>boi2</i>Δ cells. GST pull-down assay was performed in cells of yeast strain YEF473A (WT) and JGY2821 (<i>boi1</i>Δ <i>boi2</i>Δ) 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).</p

Phenotypes of cells overexpressing <i>MSB1</i>.

<p>(<b>A</b>) Morphology and septin organization (shown by GFP-Cdc3) in cells overexpressing <i>MSB1</i>. Cells of yeast strain JGY881 (<i>GFP-CDC3</i>) 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>B</b>, <b>C</b>) Chitin deposition (<b>B</b>) as well as 1,3-β-glucan and mannan distribution (<b>C</b>) 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. (<b>D</b>) Msb1 localizes to sites of aberrant glucan deposition. Cells of strain JGY139A (<i>GAL1-GFP-MSB1</i>) 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.</p

Interaction of Rga1 with Rho3 may require additional sequence upstream of Rga1’s RhoGAP domain.

<p><b>(A)</b> Two-hybrid assay of the interaction of Rga1-C segments with Rho3 and Cdc42. Cells were grown on SC-Leu-Ura-Ade plate. Picture was taken after 3 days at 30°C. Prey plasmids pGAD-C1 (AD vector) and pGAD-RGA1-C segments were used. <b>(B)</b> Cells of strain YEF473A carrying plasmids pEGKT306 (Vec), pEGKT306-RGA1-C538, and pEGKT306-RGA1-C783 were grown on SC-Ura (Dex) and SRG-Ura (Gal) plates for 4 days at 30°C. <b>(C)</b> DIC images of cells as in (B) grown on SRG-Ura plate for 16 hr at 30°C.</p

Rga1 interacts with Rho3 <i>in vivo</i>.

<p><b>(A)</b> Two-hybrid assay of the interaction between Rga1 and four Rho GTPases. The assay was performed as described in <i>Materials and Methods</i>. Cells were grown on SC-Leu-Trp-Ade plate. Picture was taken after 4 days at 30°C. Growth indicates interaction between the DBD and AD fusion proteins. <b>(B)</b> Two-hybrid assay of the interaction of Rga1-C211 and Rga1-C598 with Rho3 and Cdc42. Cells were grown on SC-Leu-Ura-Ade plate. Picture was taken after incubation at 30°C for 3 days. <b>(C)</b> GFP-Rho3 and PMT-GFP-Rho3 localization. YEF473A cells carrying pUG36-RHO3 (GFP-Rho3) and pUG36-PMT-RHO3 (PMT-GFP-Rho3) were visualized for GFP fluorescence. <b>(D)</b> BiFC assay between Rga1 and Rho3. Cells of strain JGY2653 (<i>rho3</i>Δ) carrying pVN1-PMT-RHO3^Q74L/pVC1, pVN1-PMT-RHO3^Q74L/pVC1-RGA1, and pVN1/pVC1-RGA1 pairs were grown in SC-Ura-His medium. Green fluorescence was examined by fluorescence microscopy. Bars, 5 μm.</p