RESUMEN
H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Ácido Palmítico/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Neuritas , Proteína Oncogénica p21(ras)/química , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Covalent attachment of lipids appears to be an important mechanism by which many proteins interact with membranes. As we learn more about how lipids and adjacent amino acids participate in addressing proteins to specific membranes within the cell, it should be possible to design more elegant and precise membrane targeting systems that can be used to guide proteins to functionally relevant destinations.
Asunto(s)
Ácidos Mirísticos/metabolismo , Ácidos Palmíticos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al GTP/metabolismo , Vectores Genéticos , Mamíferos , Datos de Secuencia Molecular , Mutagénesis Insercional , Ácido Mirístico , Oligodesoxirribonucleótidos , Ácido Palmítico , Reacción en Cadena de la Polimerasa/métodos , Prenilación de Proteína , Señales de Clasificación de Proteína/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Mapeo Restrictivo , Proteínas ras/biosíntesisRESUMEN
The Saccharomyces cerevisiae genes ELM1, ELM2, and ELM3 were identified on the basis of the phenotype of constitutive cell elongation. Mutations in any of these genes cause a dimorphic transition to a pseudohyphal growth state characterized by formation of expanded, branched chains of elongated cells. Furthermore, elm1, elm2, and elm3 mutations cause cells to grow invasively under the surface of agar medium. S. cerevisiae is known to be a dimorphic organism that grows either as a unicellular yeast or as filamentous cells termed pseudohyphae; although the yeast-like form usually prevails, pseudohyphal growth may occur during conditions of nitrogen starvation. The morphologic and physiological properties caused by elm1, elm2, and elm3 mutations closely mimic pseudohyphal growth occurring in conditions of nitrogen starvation. Therefore, we propose that absence of ELM1, ELM2, or ELM3 function causes constitutive execution of the pseudohyphal differentiation pathway that occurs normally in conditions of nitrogen starvation. Supporting this hypothesis, heterozygosity at the ELM2 or ELM3 locus significantly stimulated the ability to form pseudohyphae in response to nitrogen starvation. ELM1 was isolated and shown to code for a novel protein kinase homolog. Gene dosage experiments also showed that pseudohyphal differentiation in response to nitrogen starvation is dependent on the product of CDC55, a putative B regulatory subunit of protein phosphatase 2A, and a synthetic phenotype was observed in elm1 cdc55 double mutants. Thus, protein phosphorylation is likely to regulate differentiation into the pseudohyphal state.