RESUMEN
Stp1 and Stp2 are two homologous transcription factors activated in response to extracellular amino acid stimuli. Here we show that both ubiquitin-dependent degradation of Stp1 and Stp2 and their intracellular localization are differentially regulated. We have found that the E2 ubiquitin-conjugating enzyme Cdc34 is required for degradation of both full-length and processed Stp1, but not Stp2. We have also found that Grr1, the F-box component of the SCF(Grr1) E3 ubiquitin ligase, is the primary factor in degradation of full-length Stp1, whereas both Grr1 and Cdc4 are required for degradation of processed Stp1. Our localization studies showed that full-length Stp1 is localized both in the cytoplasm and at the cell periphery, whereas full-length Stp2 is localized only diffusely in the cytoplasm. We identified two nuclear localization signals of Stp1 and found that the N-terminal domain of Stp1 is required for localization of full-length Stp1 to the cell periphery. We also found that Stp2 is the primary factor involved in basal activation of target gene expression. Our results indicate that the functions of two seemingly redundant transcription factors can be separated by differential degradation and distinct cellular localization.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina Proteasas/metabolismo , Factores de Transcripción/metabolismo , Aminoácidos/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina Proteasas/genética , Factores de Transcripción/genética , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context. Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1.