Cdc24 binds preferentially to GTP[γS]-bound Bud1 in vitro. (A) Association of GST–Bud1 and six-histidine-tagged Cdc24. Purified GST–Bud1 and GST (final concentration, approximately 750 nM) were preincubated with 1 mM GDP or GTP[γS] and then incubated with six-histidine-tagged Cdc24 (final concentration, 15 nM). Cdc24 was incubated with either GTP[γS]-bound Bud1 (lane 1) or GDP-bound Bud1 (lane 2), or with GST that had been preincubated with GTP[γS] (lane 3) or GDP (lane 4). Proteins bound to glutathione-Sepharose beads were collected and analyzed by SDS/PAGE. Approximately equal amounts of GST–Bud1 and GST proteins were recovered for each reaction as judged by Coomassie blue staining of the gel (data not shown). Association of Cdc24 was determined by immunoblotting with polyclonal antibodies against Cdc24. (B) Association of GST–Bud1 and MBP–Cdc24 in vitro. MBP–Cdc24 (750 nM) was incubated either with GST–Bud1 (75 nM) preloaded with GTP[γS] (lane 1) or GDP (lane 2), or with GST–Bud1 that had not been preincubated with any nucleotides (lane 3). MBP (750 nM) was also incubated with GST–Bud1 (75 nM) preloaded either with GTP[γS] (lane 4) or GDP (lane 5), or with GST–Bud1 that had not been preincubated with any nucleotides (lane 6). Proteins bound to amylose resin were collected and analyzed by SDS/PAGE. Approximately equal amounts of MBP–Cdc24 and MBP were recovered for each reaction as judged by Coomassie blue staining of the gel (data not shown). Association of GST–Bud1 was determined by immunoblotting with polyclonal antibodies against GST.

Bud1 effector domain mediates interaction between Bud1 and Cdc24. (A) Overexpression of Bud1^T35A fails to suppress a temperature-sensitive cdc24 mutation. Strains with the temperature-sensitive cdc24 mutation (Y147) transformed with 2-μm plasmid (pRS425) carrying BUD1, bud1^T35A, or vector alone were streaked on SD-Leu plate containing 1 M sorbitol and incubated at 25°C or 36°C for 3 days. (B) The Bud1^T35A protein fails to interact with Cdc24 in vitro. MBP–Cdc24 (750 nM) was incubated with wild-type GST–Bud1 (15 nM) preloaded with GTP[γS] (lane 1) or GDP (lane 2), or with GST–Bud1^T35A (15 nM) preloaded with GTP[γS] (lane 3) or GDP (lane 4). Proteins were analyzed as in Fig. 1B. GST–Bud1^T35A was expressed by using plasmid HP649, which is the same as pRS4 (15) except for a single amino acid substitution at residue 35.

Bem1 binds preferentially to GDP-bound Bud1 in vitro. Purified GST–Bud1, GST–Bud1^T35A, and GST (approximately 750 nM) were preincubated with 1 mM GDP or GTP[γS] and then incubated with six-histidine-tagged Bem1 (15 nM). Bem1 was incubated with GTP[γS]–Bud1 (lane 1), GDP–Bud1 (lane 2), GTP[γS]–Bud1^T35A (lane 3), GDP–Bud1^T35A (lane 4), GST preincubated with GTP[γS] (lane 5), or GDP (lane 6) or with GST–Bud1 (lane 7) or GST–Bud1^T35A (lane 8) that had not been preloaded with any nucleotides. Proteins bound to glutathione-Sepharose beads were collected and analyzed by SDS/PAGE. Association of Bem1 was determined by immunoblotting with polyclonal antibodies against Bem1 (ref. 30 and Kathy Corrado and J.R.P., unpublished results). A negative control is shown in lane 9, in which no GST–Bud1 or GST was added.

Subcellular fractionation and solubilization of Bud1 from the particulate fraction. (A) Subcellular fractionation and solubilization of wild-type Bud1. Cells of yeast strain expressing HA-epitope-tagged Bud1 were broken open by bead beating and centrifuged at 500 × g to produce whole cell extract (wce); the wce was subsequently centrifuged at 10,000 × g to produce pellect (P) and supernatant (S) fractions (lanes 1 and 2). Aliquots of the pellet fractions (P) were washed with the same lysis buffer (lanes 3 and 4) or treated with 500 mM NaCl (lanes 5 and 6), 2% Triton (lanes 7 and 8), and 1% SDS (lanes 9 and 10) before centrifuging at 10,000 × g again (34). Both pellet and supernatant fractions from equal amounts of cells (OD₆₀₀ unit) were loaded on 10% polyacrylamide gel and subjected to immunoblot analysis using monoclonal antibodies against HA epitope to detect HA-tagged Bud1. As a control for fractionation, identical samples were also subjected to immunoblot analysis using antibodies against the yeast endoplasmic reticulum membrane protein Sec61 (36). (B) Subcellular fractionation of Bud1^G12V and Bud1^K16N. Cells of yeast strain expressing HA-epitope-tagged Bud1^G12V or Bud1^K16N were treated in the same way as described in A.

Scheme for function of a GTPase cascade in determination of yeast cell polarity. In this model, the GTPase Bud1 determines the localization to the bud site of proteins that organize the actin cytoskeleton—Bem1, Cdc24, and the GTPase Cdc42. First, a tetrapartite complex is formed at the bud site, at which the Bud2, the GTPase-activating protein for Bud1, appears to be located (H.-O.P., unpublished results). Bud2 stimulates conversion of Bud1–GTP to Bud1–GDP, which allows a reconfiguration of components at the bud site and activation of Cdc24. This guanine nucleotide exchange factor, Cdc24, then stimulates formation of Cdc42–GTP, which is presumably the active species for organizing the actin cytoskeleton. Bud1–GDP is converted to Bud1–GTP by its guanine-nucleotide exchange factor, Bud5. Multiple rounds of this cycle may result in a critical concentration of Bem1 and Cdc42–GTP at the presumptive bud site, which stimulates polarization of the actin cytoskeleton toward that point (see text for details).