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Protein interaction partners of drosophila Unr identified by pull down of wild type and an interdomain mutant protein

ABSTRACT: To identify an RNP interactome of Unr, V5 tagged wildtype protein and a mutant containing several interdomain contacts, that shows reduced protein expression were tansfected in SL2 cells after a siRNA knoch-down of the endogenous protein levels. After pulling down the transfected protein with an antibody against the V5-tag differences interacting proteins were identified using mass spectrometry. One set was done with an RNase treatment and one without, to identify interaction partners that rely on RNA binding. The lower expression level of the mutant protein allowed us to take this as a specific background control.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Cell Suspension Culture, Epithelial Cell

SUBMITTER: Frank Stein

LAB HEAD: Janosch Hennig

PROVIDER: PXD018115 | Pride | 2020-07-14

REPOSITORIES: Pride

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Project description:Intracellular bacterial pathogens inject a cocktail of effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. Salmonella enterica serovar Typhimurium (STm) uses two Type 3 secretion system (T3SS) to deliver a suite of effector proteins into host cells, however, knowledge of their host-targets remains sparse. To systematically map STm effector-host protein-protein interactions (PPIs) during active infection, we generated a library of 32 STm strains expressing affinity-tagged effector proteins from their endogenous chromosomal loci. Tagged-effector strains were then used to infect macrophages or epithelial cells, and PPIs were detected by Affinity-Purification Quantitative Mass-Spectrometry (AP-QMS) under both native and cross-linking conditions. We recovered 25 previously described effector-host PPIs, along with 421 novel interactions across the two cell lines tested, as well as several effector-effector PPIs. While effectors converged on specific host cellular processes, which varied across cell types, most had multiple host targets. Using reciprocal co-immunoprecipitations, we could validated 13 out of 22 new PPIs, demonstrating an accuracy of at least 59% for the AP-QMS approach. To illustrate the utility of this resource for discovering novel infection biology, we first showed that SseJ and SseL cooperate to control cholesterol transport at the Salmonella Containing Vacuole (SCV) via the Niemann-Pick C1 protein (NPC1). Second, we uncovered that PipB directly binds and recruits the organelle contact site protein PDZD8 to the SCV. Third, we elucidated a novel mechanism whereby the effector kinase SteC promotes host actin bundling, by directly interacting and phosphorylating formin-like proteins. Overall, we provide a systems-wide host-bacterial pathogen physical interactome resource, to our knowledge the first in an infection context, with broad implications for effector function and cooperation.

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Protein interaction partners of drosophila Unr identified by pull down of wild type and an interdomain mutant protein

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