Abstract
The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of the C-terminal tails of these proteins to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in both E. coli and human, leading to protein defects, loss of function, and genetic diseases. In E. coli, we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.