The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides1,2,3,4,5,6. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation7. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions.
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We thank E. Gulcicek and K. Stone at the Yale Keck Proteomic Facility for advice and providing the high-resolution MS/MS analysis, K. Reinish and X. Wu for providing purified connecdenn, L. Lucast and P. De Camilli for assistance with lipid analysis, X. Pan for assistance in constructing AnkX plasmids, A. Hubber for technical assistance and H. Newton for editorial assistance. This work was supported by an Anna Fuller Fellowship (S.M.), National Institutes of Health (NIH) Grants F32 AI082927 (J.M.), and NIH grants R01-AI064559, R01-AI048770 and Northeast Biodefense Center Grant U54-AI057158-Lipkin (C.R.R.).
The authors declare no competing financial interests.
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Mukherjee, S., Liu, X., Arasaki, K. et al. Modulation of Rab GTPase function by a protein phosphocholine transferase. Nature 477, 103–106 (2011). https://doi.org/10.1038/nature10335
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