The Escherichia coli S2P intramembrane protease RseP regulates ferric citrate uptake by cleaving the sigma factor regulator FecR

Abstract

Escherichia coli RseP, a member of the site-2 protease family of intramembrane proteases, is involved in the activation of the σ E extracytoplasmic stress response and elimination of signal peptides from the cytoplasmic membrane. However, whether RseP has additional cellular functions is unclear. In this study, we used mass spectrometry–based quantitative proteomic analysis to search for new substrates that might reveal unknown physiological roles for RseP. Our data showed that the levels of several Fec system proteins encoded by the fecABCDE operon ( fec operon) were significantly decreased in an RseP-deficient strain. The Fec system is responsible for the uptake of ferric citrate, and the transcription of the fec operon is controlled by FecI, an alternative sigma factor, and its regulator FecR, a single-pass transmembrane protein. Assays with a fec operon expression reporter demonstrated that the proteolytic activity of RseP is essential for the ferric citrate–dependent upregulation of the fec operon. Analysis using the FecR protein and FecR-derived model proteins showed that FecR undergoes sequential processing at the membrane and that RseP participates in the last step of this sequential processing to generate the N-terminal cytoplasmic fragment of FecR that participates in the transcription of the fec operon with FecI. A shortened FecR construct was not dependent on RseP for activation, confirming this cleavage step is the essential and sufficient role of RseP. Our study unveiled that E. coli RseP performs the intramembrane proteolysis of FecR, a novel physiological role that is essential for regulating iron uptake by the ferric citrate transport system.

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

“…Samples were then mixed with an equal volume of 2× SDS sample buffer plus 2ME and boiled for 5 min. The proteins were separated by SDS-PAGE using a 15% bis-tris gel and MES SDS running buffer ( 61 ) and visualized using a PhosphorImager BAS5000 (Cytiva).…”

Cryo-EM structure of the entire FtsH-HflKC AAA protease complex

“…Cells were grown, and the total cellular proteins were precipitated with 5% trichloroacetic acid, washed with acetone, and dissolved in SDS sample buffer. Immunoblotting was carried out essentially as described previously (Yokoyama et al, 2021). Proteins were separated by SDS-PAGE and electroblotted onto an Immobilon-P membrane filter (Millipore Sigma).…”

Increased Amino Acid Absorption Mediated by Lacticaseibacillus rhamnosus IDCC 3201 in High-Protein Diet-Fed Mice

“…PepO has distinct cleavage site for a s1 -casein fragment 1-23, and the protease IspA, which is frequently found mainly in the Bacillus species, is crucial role in stationary phase, where cell growth being to stop [ 24 , 25 ]. Lastly, RseP stimulates transcriptional factor of σ E extra-cytoplasmic stress response and in turn, eliminates signal peptides of extracelluar proteins in the secondary processing in cytoplasmic membrane [ 26 ]. On the other hands, The PepR and PepX which are proline-specific peptidases are found in only other L. rhamnosus strains, while PepXP (prolyl dipeptidyl aminopeptidase) are only found in in L. rhamnosus IDCC3201 and these genes are frequently found in Lactococcus lactis strains [ 27 , 28 ].…”