Dataset Information

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein.

ABSTRACT: 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.

SUBMITTER: Muller MP

PROVIDER: S-EPMC3471704 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein.

Müller Matthias P MP Shkumatov Alexander V AV Oesterlin Lena K LK Schoebel Stefan S Goody Philip R PR Goody Roger S RS Itzen Aymelt A

The Journal of biological chemistry 20120807 42

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 ...[more]

PMID: 22872634

Similar Datasets

Project description:UnlabelledLegionella pneumophila is an intracellular pathogen that resides within a membrane-bound compartment that is derived from vesicles exiting the endoplasmic reticulum (ER). To create this compartment, these bacteria use a type IV secretion system to deliver effector proteins that subvert host cell functions. Several Legionella effector proteins modulate the function of the host protein Rab1, which is a GTPase that is recruited to the Legionella-containing vacuole (LCV). Here, we examined which of the Rab1-directed enzymatic activities displayed by Legionella effectors are important for localizing the Rab1 protein to the LCV membrane. The guanine nucleotide exchange factor (GEF) domain in the effector protein DrrA (SidM) was essential for Rab1 recruitment to the LCV and Rab1 AMPylation by the nucleotidyltransferase domain in DrrA was important for Rab1 retention. Legionella organisms producing mutant DrrA proteins that were severely attenuated for GEF activity in vitro retained the ability to localize Rab1 to the LCV. Rab1 localization to the LCV mediated by these GEF-defective mutants required AMPylation. Importantly, we found that efficient localization of Rab1 to the LCV occurred when Rab1 GEF activity and Rab1 AMPylation activity were provided by separate proteins. Rab1 phosphocholination (PCylation) by the effector protein AnkX, however, was unable to substitute for Rab1 AMPylation. Lastly, the defect in Rab1 localization to the LCV in AMPylation-deficient strains of Legionella was partially suppressed if the GTPase-activating protein (GAP) LepB was eliminated. Thus, our data indicate that AMPylation of Rab1 is an effective strategy to maintain this GTPase on the LCV membrane.ImportanceActivities that enable the intracellular pathogen Legionella pneumophila to subvert the function of the host protein Rab1 were investigated. Our data show that a posttranslational modification called AMPylation is critical for maintaining a pool of Rab1 on the LCV membrane. AMPylation of Rab1 led to the accumulation of GTP-bound Rab1 on the LCV membrane by protecting the protein from inactivation by GAPs. Importantly, PCylation of Rab1 by the Legionella effector protein AnkX was neither necessary nor sufficient to maintain Rab1 on the LCV, indicating that AMPylation and PCylation represent functionally distinct activities. We conclude that modification of Rab1 by AMPylation is an effective strategy to spatially and temporally regulate the function of this GTPase on a membrane-bound organelle.

Dataset Information

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein.

Publications

Characterization of enzymes from Legionella pneumophila involved in reversible adenylylation of Rab1 protein.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets