RESUMO
The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.
Assuntos
Alquil e Aril Transferases , Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Transferases/genética , Proteínas rho de Ligação ao GTP , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Análise Mutacional de DNA , Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Fenótipo , Prenilação de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Especificidade por Substrato , Supressão Genética , Temperatura , Transferases/química , Transferases/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
Based on structure, activity, and expression, the Drosophila drosulfakinin I peptide (DSK I; FDDY(OSO3H)GHMRFamide) is similar to the vertebrate peptide, cholecystokinin. Dromyosuppressin (DMS; TDVDHVFLRFamide) is an abundant peptide isolated from adult Drosophila which shares a high degree of sequence homology with peptides isolated from chicken, cockroach, fleshfly, and locust. DSK I and DMS, encoded by different precursors, have similar expression patterns in larval brain tissue; each localizes to cells in the anterior and medial protocerebrum. Because of the precedence for coexistence of neural messengers, it was of interest to determine the cellular expression patterns relative to one another. The question of whether the two peptides were expressed in the same cells was resolved using an immunofluorescent double-labeling technique developed for sequence-specific antisera raised in separate animals of the same species. Double labeling was done using a combination of indirect and direct immunofluorescence. DSK I and DMS were shown to localize to different cells in close proximity to one another in the larval brain. The non-overlapping expression patterns of these peptides illustrate the complete lack of cross-staining with this technique.