Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast

Abstract

Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells, but the effectors mediating this control remain unknown. Through the use of the complete yeast genomic sequence, we have identified two novel Cdc42p targets, Gic1p and Gic2p, which contain consensus Cdc42/Rac interactive-binding (CRIB) domains and bind specifically to Cdc42p-GTP. Gic1p and Gic2p colocalize with Cdc42p as cell polarity is established during the cell cycle and during mating in response to pheromones. Cells deleted for both GIC genes exhibit defects in actin and microtubule polarization similar to those observed in cdc42 mutants. Finally, the interaction of the Gic proteins and Cdc42p is essential, as mutations in the CRIB domain of Gic2p that eliminate Cdc42p binding disrupt Gic2p localization and function. Thus, Gic1p and Gic2p define a novel class of Cdc42p targets that are specifically required for cytoskeletal polarization in vivo.

Figure 1

Gic1p and Gic2p, two novel Cdc42p binding proteins. (A) Alignment of the CRIB domains of Gic1p and Gic2p to several Ste20/PAK-related kinases. Consensus amino acid residues are shown in bold (Burbelo et al. 1995). Sequences are from the following sources: Ste20p and Cla4p (Leberer et al. 1992; Ramer and Davis 1993; Cvrckova et al. 1995), Shk1p (Markus et al. 1995), and PAK1 (Manser et al. 1994). (B) Alignment of Gic1p (top) and Gic2p. Identical amino acid residues are indicated with black boxes. Similar amino acids are shaded.

Figure 2

Gic1p and Gic2p bind Cdc42p-GTP. (A) Gic2p binds to Cdc42p-GTP. Sepharose beads containing purified yeast Gic2p were assayed for their ability to retain E. coli expressed Cdc42p preloaded with either GTPγS (lanes 1,3,4), or GDP (lane 2). (Lane 1) Control beads. (Lanes 2–4) Gic2-HAp beads. The following Cdc42 proteins were analyzed: (Lanes 1–3) wild-type Cdc42p. (Lane 4) Cdc42p^T35A. Cdc42p was detected by immunoblotting. (B) Overproduction of an amino-terminal fragment of Gic2p inhibits cell growth (overexpressed from the GAL1 promoter). Cells accumulate with a large unbudded morphology. This growth defect could be suppressed by a high-copy-number plasmid carrying CDC42, but not by high-copy-number plasmids carrying no insert (VEC), CDC24, RHO1, CLA4, BEM1, or CDC42^T35A.

Figure 3

Gic2p is expressed in a cell cycle-dependent manner. Cells were synchronized by releasing temperature-sensitive cdc15 cells from their block in late mitosis. Aliquots were collected every 15 min and analyzed by immunoblotting. The time of bud emergence is indicated as BE. (A) Gic2p; (B) Cln2p; (C) actin.

Figure 4

Gic1p and Gic2p colocalize with Cdc42p to regions of polarized growth. Epitope-tagged Gic1p, Gic2p, and Cdc42p, expressed from their native promoters on centromeric plasmids, were detected by indirect immunofluorescence. Gic1p, Gic2p, and Cdc42p proteins localize to the incipient bud site and the surfaces of small buds (A), and to the tips of polarized mating projections (B).

Figure 5

Gic1p and Gic2p encode redundant proteins required for efficient growth and cell polarization in vivo. (A) Strains harboring deletions in GIC1 or GIC2 grow at rates indistinguishable from the wild-type parental strain. Strains deleted for both GIC1 and GIC2 grow slowly at 30°C. (B–I) Wild-type (B,C,F,G) and gic1Δ gic2Δ cells (D,E,H,I) were observed by DIC (B,D) and epifluorescence microscopy (C,E,F–I). Actin (C,E), DNA (F,I), and microtubules (G,H) are shown.

Figure 6

Gic1p and Gic2p are required for polarized morphogenesis but not for MAP kinase signaling in response to pheromones. (A) Activity of the MAP kinase signaling pathway triggered by pheromones was measured by induction of the FUS1–LacZ reporter, and plotted as percent Miller Units relative to wild-type controls. (Lane 1) No α-factor added. (Lanes 2–5) α-factor added for 1 hr. The following strains were analyzed: (1 and 2) wild-type; (3) gic1Δ; (4) gic2Δ; 5: gic1Δ gic2Δ. (B) gic1Δ gic2Δ (top panel) and wild-type cells (bottom panel) are able to arrest their cell cycle in response to α-factor as assessed by halo assay. (C) gic double mutants (a and c), but not cells lacking GIC2 (b and d), are defective for polarized morphogenesis in response to α-factor. (a and b) Phase contrast photographs; (c and d) actin visualized by rhodamine-phalloidin. (D) Mating efficiencies of wild-type and gic mutant strains to a far1-c tester (Valtz and Peter 1997).

Figure 7

Interaction between Gic2p and Cdc42p is essential for Gic2p function and localization. (A) The growth defect of gic1Δ gic2Δ cells could be complemented by plasmids expressing wild-type GIC2 but not gic2^crib−. (1) gic1Δ gic2Δ strain carrying vector. (2) gic1Δ gic2Δ carrying vector with a GIC2 insert. (3) gic1Δ gic2Δ carrying vector with a gic2^crib− insert. (4) A gic1 strain carrying no plasmid for comparison to sector 2. (B) Wild-type and Gic2p^crib− proteins are present at equal levels. Lane numbers correspond to those of panel A. Gic2 protein was detected by immunoblotting with specific Gic2p-antibodies. (C) Asymmetric localization of Gic2p is dependent on a functional CRIB domain. Localization of epitope-tagged wild-type Gic2p (top panels) is contrasted with the localization of epitope-tagged Gic2p^crib− (bottom panels), both expressed from a high-copy-number plasmid.

Figure 8

Genetic interaction between the Gic proteins, Bem1p, and Cdc42p. (A) Overexpression of Gic1p but not Gic2p is able to partially restore growth of cells lacking Bem1p function at 39°C. (B) Overexpression of wild-type Cdc42p but not Cdc42p in its active GTP-bound form (G12V) is able to suppress the growth defect of gic1Δgic2 mutants at 37°C.

Figure 9

A pathway for cytoskeletal polarization mediated by Cdc42p in yeast. Cell cycle progression or extracellular signals lead to the conversion of GDP to GTP on Cdc42p, possibly by activation of the exchange factor Cdc24p. GTP–Cdc42p then binds and thereby localizes the effectors Gic1p and Gic2p to sites of polarized growth, which in turn contribute to polarization of the actin cytoskeleton. Cdc42p might exert some of its effects on the cytoskeleton not only through Gic1p and Gic2p but also through other targets including Bni1p, Ste20p, and Cla4p. Bni1p is known to interact also with other members of the Rho-GTPase family.