Spatial and Temporal Aspects of Rab1 Recruitment by Legionella pneumophila.

摘要

After being internalized by a eukaryotic host cell, Legionella pneumophila remains in a membrane bound compartment that recruits vesicles exiting the endoplasmic reticulum. To mediate vesicle fusion, L. pneumophila delivers several proteins that modulate the activity of Rab1. LepB demonstrates GTPase activating protein (GAP) activity towards Rab1 under in vitro conditions, but an in vivo role for LepB has not been demonstrated. Spinning-disk confocal microscopy and fluorescence recovery after photobleaching (FRAP) was used to track the recruitment of YFP-Rab1 to vacuoles containing L. pneumophila. The proportion of Rab1+ vacuoles containing L. pneumophila (LCVs) and the dynamics of Rab1 dissociation from LCVs were not affected by the LepB protein. Importantly, live cell imaging generated novel phenotypes, including the identification of Rab1+ tubules and enhanced Rab1 recovery at the Golgi compartment compared to the LCV. YFP-Rab1 is recruited before ARF1-CFP to LCVs and is independent of the Legionella- encoded ARF1 GEF RalF. I subsequently performed a systematic analysis of bacterial effector activities required for the recruitment, retention, and dissociation of Rab1 at the LCV. L. pneumophila effector AnkX transfers a phosphocholine moiety onto Rab1. DrrA is a Legionella effector that contains an adenylylation domain that also inserts an AMP moiety onto Rab1. Further, DrrA contains a central guanine nucleotide exchange factor (GEF) domain. Mutations were previously identified in DrrA that severely attenuated GEF activity in vitro. RAW cells were infected with strains overproducing DrrA GEF mutants and Rab1 recruitment at the LCV was assessed by immunofluorescence. All GEF mutants were reduced, but not abolished, for Rab1 LCV localization compared to wild-type DrrA. AMPylation was required for the basal level of Rab1 localization in both wild-type and GEF-attenuated DrrA proteins. AMPylating activity provided in trans to AMPylation-deficient DrrA restored the ability of Rab1 to be localized to LCVs, but was not sufficient for initial recruitment. We found no detectable role for phosphocholination in affecting Rab1 dynamics at LCVs. Finally, LepB dissociated Rab1 from LCVs in the absence, but not in the presence, of AMPylating activity. We present a model where the GEF domain first recruits Rab1 to the LCV, the AMPylation domain next modifies it and protects it from dissociation, and upon deAMPylation, is inactivated by LepB and removed from the LCV.