The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop‐transfer signals in the mitochondrial inner membrane

The TIM23 complex mediates membrane insertion of presequence‐containing mitochondrial proteins via a stop‐transfer mechanism. Stop‐transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop‐transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23‐mediated stop‐transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2‐deletion or ‐overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix‐facing positive charges, is reduced. These results imply that the Mgr2‐mediated gatekeeper function is important for controlling membrane sorting of marginal stop‐transfer signals.