Abstract
Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Previous work has posited that Sde proteins solely target serine (Ser) residues within acceptor protein substrates. We show here that SdeC-mediated pR-Ub modification results from a stepwise reaction that also modifies tyrosine (Tyr) residues. Unexpectedly, the presence of an HA tag on Ub resulted in poly-pR-ubiquitination, consistent with the HA tag acting as an acceptor target. Interrogation of phosphoribosyl-linked HA-Ub revealed that Tyr4 was the preferred targeted residue, based on LC-MS/MS analysis of the crosslinked product. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. Although previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr → Ser replacement mutants. This work demonstrates that pR-ubiquitination by the Sde family is not limited to Ser-modification as previously proposed, and broadens the potential sites targeted by this family.
Highlights
Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS)
Phosphoribosylubiquitination is promoted by SdeC in two independent steps The crystal structures of the Legionella SdeA demonstrate that the ART and NP active sites face in opposite directions and are located 50 Å apart.[37,38,39,41]
To develop an assay that allows rapid analysis of modified forms of Ub and facile interrogation of the independent activities, agarose bead-immobilized purified recombinant SdeC variants[15] were used to allow easy separation of enzyme from substrates. This strategy centered around sequential incubation of Ub with a resin-bound SdeC variant followed by removal of the immobilized SdeC, allowing additional immediate treatment of the modified Ub with a different SdeC variant in the presence of an acceptor protein (Fig. 1C)
Summary
Legionella pneumophila establishes a replication vacuole by translocating hundreds of protein effectors through a type IV secretion system (T4SS). Among these translocated effectors are members of the Sde family, which catalyze phosphoribosyl-linked ubiquitination (pR-Ub) of host targets. Further analysis using synthetic HA variants revealed promiscuous modification of Tyr, as crosslinking was prevented only by constructing a triple mutant in which all three Tyr within the HA sequence were substituted with Phe. previous work has indicated that Ser is the sole acceptor residue, we found no evidence of Ser preference over Tyr using Tyr - Ser replacement mutants. These findings support a critical role for pR-Ub in promoting L. pneumophila infection in amoebal species
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