Analysis of inner membrane lateral sorting at the presequence translocase.

Abstract

The translocase of the mitochondrial inner membrane (TIM23) complex mediates the import and membrane insertion of presequence-carrying mitochondrial proteins. It is experimentally challenging to determine whether the segment of the polypeptide is imported to the matrix or inserted into the inner membrane. Utilizing the unique topogenesis of Mgm1p, a versatile experimental approach to study the TIM23-mediated membrane insertion is developed and described in this chapter. This method combines a simple SDS-gel based assay with the quantification of the relative fractions of membrane inserted and non-inserted products, enabling the quantitative measurement of the membrane insertion efficiencies of a transmembrane segment into the mitochondrial inner membrane.