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. 2012 Oct 1;23(19):3814–3826. doi: 10.1091/mbc.E12-05-0400

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© 2012 Chen et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — The Cdc42p effector Gic2p may regulate Bni1p. (A, B) Gic2p binds to a region of Bni1p distinct from the GBD, SBD, or BBD. (A) Gic2p-Myc was coimmunoprecipitated with overexpressed GFP-Bni1p full-length (FL; DLY14515) and Bni1p^3Δ (DLY14516) but not with FH1–FH2 (DLY14517). The stronger binding to full-length Bni1p is consistent with previous findings that Gic2p can also bind to Bud6p/Spa2p. Negative controls: DLY 14513, 13857. (B) The uncharacterized N-terminal Bni1p regions upstream of the GBD (ND1), between the GBD and SBD (ND2), and between the SBD and FH1 (ND3) were fused to the catalytic fragment and expressed as GFP-tagged proteins from the GAL1 promoter (DLY14885, 14886, 14887). The ND2 construct (but not the others) coimmunoprecipitated Gic2p-Myc. (C) Gic1p and Gic2p become critical in rewired cells and when the Bni1p GBD, SBD, and BBD are deleted. The gic1Δ gic2Δ growth defect (DLY14169) at 37°C is greatly exacerbated when combined with BEM1-GFP-SNC2 (DLY14170) or with BNI1^3Δ (DLY14601). Serial dilutions of the indicated strains were spotted on yeast extract/peptone/dextrose media and grown for 2.5 d at the indicated temperature. (D, E) DIC images of exponentially growing cells of indicated genotypes at 24°C, showing strong synthetic polarity defects even at permissive temperature. Scale bar, 5 μm.