Dataset Information

Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

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.

SUBMITTER: Zhang M

PROVIDER: S-EPMC8496037 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Members of the <i>Legionella pneumophila</i> Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Zhang Mengyun M McEwen Joseph M JM Sjoblom Nicole M NM Kotewicz Kristin M KM Isberg Ralph R RR Scheck Rebecca A RA

RSC chemical biology 20210729 5

<i>Legionella pneumophila</i> 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 tha ...[more]

PMID: 34704056

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Project description:Legionella pneumophila grows intracellularly within a host membrane-bound vacuole that is formed in response to a bacterial type IV secretion system (T4SS). T4SS translocated Sde proteins promote phosphoribosyl-linked ubiquitination of endoplasmic reticulum protein Rtn4, but the role played by this modification is obscure due to lack of clear growth defects of mutants. To identify the steps in vacuole biogenesis promoted by these proteins, mutations were identified that unmasked growth defects in Δ sde strains. Mutations in the sdhA , ridL and legA3 genes aggravated the Δ sde fitness defect, resulting in disruption of the Legionella -containing vacuole (LCV) membrane within 2 hrs of bacterial contact with host cells. Depletion of Rab5B and sorting nexin 1 partially bypassed loss of Sde proteins, consistent with Sde blocking early endosome and retrograde trafficking, similar to roles previously demonstrated for SdhA and RidL proteins. Sde protein protection of LCV lysis was only observed shortly after infection, presumably because Sde proteins are inactivated by the metaeffector SidJ during the course of infection. Deletion of SidJ extended the time that Sde proteins could prevent vacuole disruption, indicating that Sde proteins are negatively regulated at the posttranslational level and are limited to protecting membrane integrity at the earliest stages of replication. Transcriptional analysis was consistent with this timing model for an early point of execution of Sde protein. Therefore, Sde proteins act as temporally-regulated vacuole guards during establishment of the replication niche, possibly by constructing a physical barrier that blocks access of disruptive host compartments early during biogenesis of the LCV.Significance statementMaintaining replication compartment integrity is critical for growth of intravacuolar pathogens within host cells. By identifying genetically redundant pathways, Legionella pneumophila Sde proteins that promote phosphoribosyl-linked ubiquitination of target eukaryotic proteins are shown to be temporally-regulated vacuole guards, preventing replication vacuole dissolution during early stages of infection. As targeting of reticulon 4 by these proteins leads to tubular endoplasmic reticulum aggregation, Sde proteins are likely to construct a barrier that blocks access of disruptive early endosomal compartments to the replication vacuole. Our study provides a new framework for how vacuole guards function to support biogenesis of the L. pneumophila replicative niche.

Dataset Information

Members of the Legionella pneumophila Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Publications

Members of the <i>Legionella pneumophila</i> Sde family target tyrosine residues for phosphoribosyl-linked ubiquitination.

Similar Datasets

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