Legionella pneumophila regulates the small GTPase Rab1 activity by reversible phosphorylcholination

Abstract

Effectors delivered into host cells by the Legionella pneumophila Dot/Icm type IV transporter are essential for the biogenesis of the specialized vacuole that permits its intracellular growth. The biochemical function of most of these effectors is unknown, making it difficult to assign their roles in the establishment of successful infection. We found that several yeast genes involved in membrane trafficking, including the small GTPase Ypt1, strongly suppress the cytotoxicity of Lpg0695(AnkX), a protein known to interfere severely with host vesicle trafficking when overexpressed. Mass spectrometry analysis of Rab1 purified from a yeast strain inducibly expressing AnkX revealed that this small GTPase is modified posttranslationally at Ser 76 by a phosphorylcholine moiety. Using cytidine diphosphate-choline as the donor for phosphorylcholine, AnkX catalyzes the transfer of phosphorylcholine to Rab1 in a filamentation-induced by cAMP(Fic) domain-dependent manner. Further, we found that the activity of AnkX is regulated by the Dot/Icm substrate Lpg0696(Lem3), which functions as a dephosphorylcholinase to reverse AnkX-mediated modification on Rab1. Phosphorylcholination interfered with Rab1 activity by making it less accessible to the bacterial GTPase activation protein LepB; this interference can be alleviated fully by Lem3. Our results reveal reversible phosphorylcholination as a mechanism for balanced modulation of host cellular processes by a bacterial pathogen.

Innate immunity kinase TAK1 phosphorylates Rab1 on a hotspot for posttranslational modifications by host and pathogen

“…Interestingly, the intracellular pathogen L. pneumophila is well documented to hijack the function of Rab1 in infected cells by posttranslational modification of nearby switch II residues, including adenylylation, also known as AMPylation, of Y80 and phosphocholination of S79 (Fig. 3D) (15,16,28).…”

“…Multiple studies have shown that phosphocholination and AMPylation of Rab1 switch II manipulate Rab1 function by blocking the ability of Rab1 to interact with GEFs and GAPs (16,28,30), because switch II serves as the primary interface for these binding events. To investigate whether phosphorylation of Rab1T75 may have a similar effect, we assayed the ability of the Legionella Rab1 GEF DrrA (GEF domain only, residues 340-533) to catalyze the displacement of mant-GDP from WT and mutant Rab1 in vitro (Fig.…”

“…As discussed earlier, these modifications serve as locks on the Rab1 nucleotide state and block interactions with the host enzymes normally responsible for regulating Rab1. Two additional Legionella enzymes, SidD and Lem3, have cognate Rab1-demodifying activities (28,39). Legionella maintains exquisite and tightly regulated control of Rab1 to mature its replication vacuole by carefully timing the sequential secretion of these effectors and subsequent recruitment of Rab1 to the Legionella-containing vacuole.…”

Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport

“…As in the case of adenylylation, there was a major effect on the interaction with GDI, which was essentially abolished and would therefore lead to stabilization of membrane attachment, and in this case preferentially in the inactive form due to the preference of the phosphocholination reaction for Rab : GDP. Similarly to the situation with adenylylation, L. pneumophila also secretes a protein that can reverse the modification, in this case removing the phosphocholine group hydrolytically (Tan et al, 2011;Goody et al, 2012). The similarity with adenylylation also extends to the present lack of understanding of the biological purpose of the modification, apart from the general feature that Rabs are stabilized in their membrane-bound form.…”

Posttranslational modifications of Rab GTPases help their insertion into membranes

“…Phosphocholination could however affect the interaction with GEFs, as shown for phosphocholinated Rab35 and its GEF connecdenn (15). However, interestingly, Legionella also expresses a dephosphorylcholinase, Lgp0696, that can reverse AnkX-mediated modification of Rab1 (17), and thus possibly permits binding of Rab1 to its GEF(s). Posttranslational modifications of Rab GTPases represent a tentative mechanism to explain, at least in part, how Rab GTPases can be targeted to membranes.…”