Role of the signal sequence in proteorhodopsin biogenesis in E. coli

• Published in: Biotechnology and bioengineering Vol. 116; no. 4; pp. 912 - 918
• Main Authors: Soto‐Rodríguez, Jessica, Baneyx, François
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.04.2019
• Subjects: Biosynthesis; Cell Membrane - chemistry; Cell Membrane - genetics; Cell Membrane - metabolism; E coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Hydrogen-Ion Concentration; Industrial Microbiology; Light; membrane protein; Membrane proteins; Mutagenesis; Protein Sorting Signals; Proteins; Protons; Retina; rhodopsin; Rhodopsins, Microbial - chemistry; Rhodopsins, Microbial - genetics; Rhodopsins, Microbial - metabolism; secretion; Signal peptidase; Signal peptidase I; Signal recognition particle; rhodopsin; membrane protein; secretion
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.26878

Blue‐absorbing proteorhodopsin (BPR) from marine bacteria is a retinal‐bound, light‐activated, outwards proton transporter containing seven α‐helical transmembrane segments (TMS). It is synthesized as a precursor species (pre‐BPR) with a predicted N‐terminal signal sequence that is cleaved to yield the mature protein. While optimizing the production of BPR in Escherichia coli to facilitate the construction of bioprotonic devices, we observed significant pre‐BPR accumulation in the inner membrane and explored signal sequence requirements and export pathway. We report here that BPR does not rely on the Sec pathway for inner membrane integration, and that although it greatly enhances yields, its signal sequence is not necessary to obtain a functional product. We further show that an unprocessable version of pre‐BPR obtained by mutagenesis of the signal peptidase I site exhibits all functional attributes of the wild‐type protein and has the advantage of being produced at higher levels. Our results are consistent with the BPR signal sequence being recognized by the signal recognition particle (SRP; a protein that orchestrates the cotranslational biogenesis of inner membrane proteins) and serving as a beneficial “pro” domain rather than a traditional secretory peptide. Blue‐absorbing proteorhodopsins (BPR) are light‐activated proton transporters that reside in the inner membrane of marine bacteria but are synthesized with a cleavable signal sequence typical of secretory proteins. Here we show that although it improves yields, the signal sequence is not required for BPR to traffic to the plasma membrane, and that a variant with an uncleavable signal sequence is fully functional and accumulates at higher levels than the wild type protein in the membranes of E. coli.