Characterizing the Role of YeaG in Cytoplasmic Protein Degradation under Nitrogen-Limitation in Escherichia coli

Abstract

In Escherichia coli, cytoplasmic protein degradation is thought to be primarily mediated by four ATP-dependent proteases: ClpP, Lon, HslV, and FtsH. Recent work from our lab demonstrated that substantial cytoplasmic protein degradation occurs under nitrogen limitation. However, deletion of the non-essential ATP-dependent proteases (ΔclpP, ΔhslV, Δlon) does not prevent this protein turnover, and similar observations have been made with an ftsH knockout mutant. These findings suggest the existence of an uncharacterized proteolytic pathway responsible for protein degradation under nitrogen stress. YeaG, a poorly characterized protein containing AAA+ ATPase and serine/threonine kinase domains, is a compelling candidate for a protein involved in this pathway because its expression is strongly upregulated upon nitrogen depletion. To enable rapid interrogation of candidate genes involved in this response, we developed and validated a β-galactosidase-based assay as a proxy for nitrogen starvation-induced protein turnover. This assay identified YeaG as a key factor in the response and further implicated two additional uncharacterized proteins, YeaH and YcgB, whose deletion phenocopied the yeaG knockout. We then confirmed by chemostat-based heavy isotope labeling and quantitative proteomics that loss of YeaG largely abolishes nitrogen starvation-induced cytoplasmic protein degradation, and that mutations predicted to disrupt either the AAA+ ATPase domain or the kinase domain produce a similar loss-of-function phenotype. AlphaFold-Multimer structural modeling further predicts that YeaG, YeaH, and YcgB form a multiprotein complex, suggesting that these proteins may constitute a previously uncharacterized proteolytic regulatory module. Collectively, these findings identify YeaG as a key regulator of cytoplasmic protein degradation under nitrogen starvation and implicate a YeaG-YeaH-YcgB module in proteome remodeling under nitrogen stress.