The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecularweight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Gin to and Asp to in the putative GTP-binding and hydrolysis domains and one mutation (Cys to in the putative isoprenylation site. The incapable of complementing the cdc42-l' mutation and was recessive to both wild-type and cdc42-1'. In double-mutant alleles, the cdc42srel188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.The Saccharomyces cerevisiae CDC42 gene product is involved in the control of several morphogenetic events during the cell cycle, including the generation of cell polarity, development of normal cell shape, localization of secretion, and deposition of cell-surface material (1,2,20). Its predicted amino acid sequence (20) is similar to that of members of the ras superfamily of low-molecular-weight GTP-binding proteins from S. cerevisiae and higher eukaryotes (16), especially in those domains that have been implicated in the binding and hydrolysis of GTP and in carboxylterminal modifications. CDC42 shows the greatest degree of sequence similarity to the products of two related human cDNAs, CDC42Hs (39) and G25K (27), which encode isoforms of the low-molecular-weight GTP-binding protein previously referred to as Gp or G25K (11, 33). Both of these human gene products are 80% identical (88% related amino acids) in predicted amino acid sequence to the S. cerevisiae CDC42 gene product, which we have redesignated CDC42Sc, and they are 95% identical to each other. These human genes, when expressed in S. cerevisiae under the control of a yeast promoter on a multicopy plasmid, can functionally complement the cdc42-1's mutation (27, 39). The Gp/G25K protein is an excellent in vitro phosphosubstrate for the epidermal growth factor receptor tyrosine kinase (17), but its in vivo function is unknown.In order to further elucidate the role of the CDC42Sc gene product in the control of cell polarity, we have generated new mutations in CDC42Sc by using site-directed mutagenesis (see Fig. 1). A carboxyl-terminal mutation predicted to * Corresponding author.eliminate isoprenylation results in a nonfunctional product. Mutations in the putative guanine-nucleotide-binding domain lead to a lethal phenotype that can be suppressed by combination with the carboxyl-terminal mutation. This lethality is manifested as several different and unique morphological abnormalities, which have provided clues to the function of the CDC42Sc gene product in controlling cell polarity. MATERIALS AND METHODSReagents. Enzymes, M13 dideoxy sequencing kit, mutagenesis kit, and other reagents were obtained from standard commercial sources and used according to the suppliers' Media, growth conditions, and strains. Conditions for...

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

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Kringle domains are found in several plasma proteins of blood coagulation and fibrinolysis. A murine monoclonal antibody, designated alpha HII-5, was produced against a synthetic peptide representing residues 216-231 of human prothrombin kringle 2. The sequence of the hexadecapeptide (Glu-Asn-Phe-Cys-Arg-Asn-Pro-Asp-Gly-Asp-Glu-Glu-Gly-Val-Gly-Cys) is conserved in several kringle-containing proteins, represents a predicted region of high local hydrophilicity in prothrombin kringle 2, and contains the anionic (Asp-223 and Asp-225) residues that contribute to lysine binding by plasminogen kringle 4. In a solution-phase immunoassay, antibody alpha HII-5 bound prothrombin and the kringle 5 light chain fragment of plasminogen (miniplasminogen), but not plasminogen or plasmin. In contrast, using a solid-phase assay with antigen immobilized onto a surface (polystyrene microtiter plates, glass, or nitrocellulose) antibody alpha HII-5 specifically bound prothrombin, plasminogen, recombinant tissue plasminogen activator (tPA), and the apo(a) subunit of lipoprotein(a). By immunoblotting analysis antibody alpha HII-5 bound determinants on prothrombin fragment 2 and plasminogen kringle 5. These observations suggest that a subset of kringle domains on plasma proteins, including prothrombin kringle 2 and plasminogen kringle 5, contains a homologous antigenic determinant in the region of the kringle lysine-binding site. In contrast to prothrombin kringle 2, the homologous peptide site on plasminogen is not available for antibody binding except when plasminogen is adsorbed to a nonphysiological surface, or when kringles 1-4 are removed.