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.
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.
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.
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.
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.
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).
Table 1.
S. cerevisiae strains used in this study.