Molecular communication of the membrane insertase YidC with translocase SecYEG affects client proteins

Anja Steudle,Dirk Spann,Eva Pross,Andreas Kuhn,Ross E Dalbey,Sri Karthika Shanmugam

doi:10.1038/s41598-021-83224-x

Anja Steudle, Dirk Spann + Show 4 more

Open Access

https://doi.org/10.1038/s41598-021-83224-x

Copy DOI

Abstract

The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.

Highlights

Most bacterial membrane proteins are membrane-inserted during their biosynthesis by the Sec translocase and the YidC insertase[1]
M13 procoat was pulse-labelled for 1 min with 35S-methionine and analysed by polyacrylamide gel electrophoresis (PAGE) after immunoprecipitation with an antiserum to coat
A low-resolution structure was presented that suggests that the lateral gate of SecY and the TM3 of YidC are in contact[23]

Summary

Introduction

Most bacterial membrane proteins are membrane-inserted during their biosynthesis by the Sec translocase and the YidC insertase[1]. YidC is capable of catalyzing membrane insertion of small proteins on its own, e.g. the coat proteins of the filamentous phage M13 and Pf38–10. In this YidC-only pathway, the client protein enters by the hydrophobic slide generated by TM3 and TM511. To determine the specific interaction between SecYEG and YidC the proteins were purified and their binding was measured in detergent solution and in reconstituted proteoliposomes using fluorescence resonance energy transfer (FRET). A mutant YidC which is impaired in Sec-dependent insertion was deficient for SecYEG interaction and no FRET occurred in the co-reconstituted proteoliposomes

Methods

Results

Conclusion