RESUMEN
Evidence and arguments are summarized that suggest that intrinsic (tryptophan) protein fluorescence provides an excellent and convenient signal for monitoring both GEF (guanine nucleotide exchange factor) and GAP (GTPase activating protein) activity of a large number of small GTPases. In addition, post-translational modifications of Rab proteins occurring in a region known to be a hot spot for such modifications also lead to fluorescence changes that can be accurately monitored in a time-dependent manner. It is suggested that intrinsic fluorescence should be the first method chosen for monitoring such reactions of tryptophan-containing small GTPases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Imagen Óptica/métodos , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas Activadoras de GTPasa/genética , Humanos , Factores de Transcripción/genéticaRESUMEN
After the pathogenic bacterium Legionella pneumophila is phagocytosed, it injects more than 250 different proteins into the cytoplasm of host cells to evade lysosomal digestion and to replicate inside the host cell. Among these secreted proteins is the protein DrrA/SidM, which has been shown to modify Rab1b, a main regulator of vesicular trafficking in eukaryotic cells, by transfer of adenosine monophosphate (AMP) to Tyr(77). In addition, Legionella provides the protein SidD that hydrolytically reverses the covalent modification, suggesting a tight spatial and temporal control of Rab1 function by Legionella during infection. Small angle x-ray scattering experiments of DrrA allowed us to validate a tentative complex model built by combining available crystallographic data. We have established the effects of adenylylation on Rab1 interactions and properties in a quantitative way. In addition, we have characterized the kinetics of DrrA-catalyzed adenylylation as well as SidD-catalyzed deadenylylation toward Rab1 and have determined the nucleotide specificities of both enzymes. This study enhances our knowledge of proteins subverting Rab1 function at the Legionella-containing vacuole.
Asunto(s)
Proteínas Bacterianas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Legionella pneumophila/enzimología , Enfermedad de los Legionarios/enzimología , Procesamiento Proteico-Postraduccional , Proteínas de Unión al GTP rab1/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/genéticaRESUMEN
The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.
