![Figure 4. Bud3 activates Cdc42 in vivo. (A) PBD-RFP polarization in cdc24ts cells carrying each multicopy plasmid after shifting to 37°C for 3 h. Percentages of cells with Cdc42-GTP clusters are shown. (right) The Bud3 and Bud3ΔCR2 protein levels in cdc24ts cells carrying each multicopy plasmid. Relative amount of each protein (numbers at bottom) is estimated using Bem1 as a control. (bottom) Quantification of PBD-RFP clusters in individual polarized cells is shown as a percentage of the whole-cell intensity with means (horizontal lines) ± SEM (error bars). *, P = 0.86; **, P < 10−3. (B) Cdc42-GTP polarization in WT cells overexpressing GFP-Bud3 or GFP-Bud3ΔCR2 (7 h in galactose [Gal]) or in uninduced cells (raffinose [Raff]). About 79% of medium- or large-budded cells (n = 100) overexpressing GFP-Bud3 had elongated buds (a/b [ratio of major and minor axes] > 2; often a/b > 4), whereas <1% of cells overexpressing GFP-Bud3ΔCR2 had elongated buds (n = 100). Cells overexpressing GFP-Bud3ΔCR2 had slightly more elongated buds (a/b = ∼1.5) than uninduced cells (a/b = ∼1.2). Line scans show PBD-RFP intensity of numbered cells, normalized to the peak intensity of the cell #1. Bars, 3 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/206/1/10.1083_jcb.201402040/6/jcb_201402040_fig4.jpeg?Expires=1771638045&Signature=SpCx4dmWe0GLjT11jlov71Y4Eir15bxXQ~lWZGlZz8gx8Hn~fb3ENnFUMCZiurNFr21Y8u6AsvTXb7Ic3hrW1I14j4l13eqCG8LGJqyEC~LYC5K7nxG~ep8zFWtGboVOQFmD9sFIcyqVopHL9OkedyBuD1HUc1LeIoxcT5aoBcTZpmDngpD345mUsT6B8A99aqfj2WLlSpa11S1c1nHFFlhtyHO9P0-~O~tWHmMJhsuxBtLVNYPfpngDWk73fM2Or4yTre6ytrotBCcR5xEwzM6ixPhUH4O~Ynl2OUTJMuT1uOPerEf6i4TH~oNLN6HXAmM5yo22mbiMCHAZSJFBuw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Bud3 activates Cdc42 in vivo. (A) PBD-RFP polarization in cdc24ts cells carrying each multicopy plasmid after shifting to 37°C for 3 h. Percentages of cells with Cdc42-GTP clusters are shown. (right) The Bud3 and Bud3ΔCR2 protein levels in cdc24ts cells carrying each multicopy plasmid. Relative amount of each protein (numbers at bottom) is estimated using Bem1 as a control. (bottom) Quantification of PBD-RFP clusters in individual polarized cells is shown as a percentage of the whole-cell intensity with means (horizontal lines) ± SEM (error bars). *, P = 0.86; **, P < 10−3. (B) Cdc42-GTP polarization in WT cells overexpressing GFP-Bud3 or GFP-Bud3ΔCR2 (7 h in galactose [Gal]) or in uninduced cells (raffinose [Raff]). About 79% of medium- or large-budded cells (n = 100) overexpressing GFP-Bud3 had elongated buds (a/b [ratio of major and minor axes] > 2; often a/b > 4), whereas <1% of cells overexpressing GFP-Bud3ΔCR2 had elongated buds (n = 100). Cells overexpressing GFP-Bud3ΔCR2 had slightly more elongated buds (a/b = ∼1.5) than uninduced cells (a/b = ∼1.2). Line scans show PBD-RFP intensity of numbered cells, normalized to the peak intensity of the cell #1. Bars, 3 µm.
