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

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

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

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

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

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

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

Kin2’s localization to the polarity site is essential for its multicopy suppression of late secretory mutants.

Cells of strains JGY27B (sec1-1), JGY28B (sec2-41), JGY82A (sec15-1) carrying pRS426 (Vec), pRS426-KIN2, pRS426-KIN2-NT, pRS426-KIN2-N7, and pRS426-KIN2-N5 were spotted on SC-Ura plate at 1:10 serial dilution and incubated at permissive (24°C) and their respective restrictive temperatures. Pictures were taken after 4 days.</p

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases, Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall

<div><p>MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast <i>Saccharomyces cerevisiae</i>. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains—TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 <i>in vivo</i>. Our study provides new insight in the localization, function, and regulation of Kin2.</p></div