Modulation of Rab GTPase function by a protein phosphocholine transferase

Abstract

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 resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. 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.

Figure 1. Legionella infection mediates two different post-translational modifications to Rab1

a, 3X-FLAG-Rab1 was isolated from HEK293 FcγRII cells after infection with the indicated Legionella strains. LC-MS/MS analysis produced the extracted ion chromatograms. The peak in each graph indicates the amount of Rab1 peptide TITSSYYR with no modifications (m/z = 496.0), peptide containing an AMP moiety (m/z = 660.5), and peptide with an unknown modification (m/z = 578.5). b, MS/MS spectra obtained for the AMPylated Rab1 peptide (top) and the Rab1 peptide with the unknown modification (bottom) showing mass to charge ratios of their fragments upon collision-induced dissociation. The AMPylated peptide (top spectrum) has a mass shift of 329 starting at y3 (y3 – y7), indicating a modification site at the first Tyr from the N-terminus of the peptide. The peptide with the unknown modification had a mass increase of 165 starting at y4 (y4 – y6), indicating the second Ser from the N-terminus was modified. c, Autoradiographs reveal AMPylated Rab1 from in vitro reactions containing the recombinant proteins indicated on the top of each panel and the radiolabeled nucleotides indicated below.

Figure 2. The Legionella effector AnkX functions as a Rab phosphocholine transferase

a, LC-MS/MS analysis of 3X-FLAG-Rab1 isolated from HEK293 cells that were either untransfected or transfected with a plasmid encoding GFP-tagged AnkX, AnkX_H229A or DrrA. Extracted ion chromatograms indicate the amount of Rab1 peptide TITSSYYR with no modification (m/z = 496) and peptide with the unknown modification (m/z = 578.5). b, MS³ analysis on the Rab1 peptide TITSSYYR with the unknown modification. The m/z = 184 peak corresponding to the protonated moiety attached to the Rab1 peptide was selected and subjected to further dissociation. Indicated are the fragments identified in the MS³ spectrum that matched the fragments predicted upon dissociation of the protonated phosphocholine molecule. c, The peak in each graph indicates the amount of Rab1 peptide TITSSYYR with no modifications (m/z = 496) and phosphocholinated peptide (m/z = 578.5) after in vitro incubation of Rab1 with either DrrA or AnkX in the presence of CDP-choline. d, Immunoblots from in vitro reactions that contained Rab1 and the indicated amounts of AnkX. Blots were probed to detect phosphocholinated Rab1 (anti-PC) and total Rab1 (anti-Rab1) in each reaction.

Figure 3. AnkX and DrrA have overlapping but non-identical Rab specificities

a, Extracted ion chromatograms of 3X-FLAG-Rab6 isolated from HEK293 cells producing either DrrA or AnkX. Shown are graphs for the unmodified Rab6 peptide SLIPSYIR (m/z = 475) and the AMPylated (m/z = 639.5) and phosphocholinated (m/z = 557.5) forms of this peptide. b, Extracted ion chromatograms of 3X-FLAG-Rab35 isolated from HEK293 cells producing either DrrA or AnkX. Shown are graphs for the unmodified Rab35 peptide TITSTYYR (m/z = 503.0), the AMPylated peptide (m/z = 667.5) and the phosphocholinated peptide (m/z = 585.5). c, 3X-FLAG-Rab35 was isolated from HEK293 FcγRII cells after infection with the indicated Legionella strains. The peak in each graph indicates the amount of Rab1 peptide TITSSYYR with no modifications (m/z = 503), peptide containing an AMP moiety (m/z = 667.5), and phosphocholinated peptide (m/z = 585.5).

Figure 4. AnkX-mediated phosphocholination modulates the function of Rab1 and Rab35

a, Schematic representation of the AnkX protein showing the location of the FIC domain and the four predicted ankyrin repeat homology domains (A1-A4). The amino acid sequence in a conserved region of the FIC domain containing the essential His229 residue is shown. b, Secretion of alkaline phosphatase into the culture supernatant was measured for HEK293 cells producing either GFP, GFP-AnkX or GFP-AnkX_H229A as indicated on the x-axis. Data are the mean ± standard deviation (s.d.) calculated from three independent sample wells. c, The disruption of early endosomes was assessed in COS7 cells producing either GFP-AnkX, GFP-AnkX_H229A or GFP alone after staining for EEA1 (mean ± s.d., n = 200, *P<0.005 compared to the GFP alone control). d, Binding of recombinant Connecdenn to 3X-FLAG-Rab35_S22N isolated from cells producing either GFP-CBU_2078 or GFP-AnkX or GFP-AnkX_H229A was assessed by co-precipitation. The Coomassie-stained SDS-PAGE gel indicates the amount of Connecdenn and 3X-FLAG-Rab35_S22N in each precipitate. The anti-phosphocholine immunoblot indicates that Rab35_S22N isolated from cells producing GFP-AnkX was phosphocholinated. e, Binding of purified His-tagged DrrA_340–533 to 3X-FLAG-Rab1A isolated from cells producing either GFP-CBU_2078 or GFP-AnkX or GFP-AnkX_H229A was assessed by co-precipitation. The immunoblots indicate the amounts of DrrA_340–533 and the levels of phosphocholinated 3X-FLAG-Rab1A present in each precipitate.