Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome ofStreptococcus mutans

Author:

Qin Hua,Anderson David,Zou Zhengzhong,Higashi Dustin,Borland Christina,Kreth Jens,Merritt Justin

Abstract

AbstractMecA is a highly conserved adaptor protein encoded by prokaryotes from theBacillotaphylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiontStreptococcus mutanslikely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactionsin vivo. In addition, we further develop a new application of SLCA to supportin vivomeasurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of MecA regulatory function still has yet to be discovered.

Publisher

Cold Spring Harbor Laboratory

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