Multispanning inner membrane proteins with internal targeting signals are transported into mitochondria by the carrier import pathway. TOM70 is the receptor for this class of precursor proteins and directs them to the TOM complex. The TIM22 complex mediates inner membrane insertion utilizing the Δψ as driving force. Only a hand full of substrates has been experimentally defined and the mechanism of transport is only poorly understood. Here we will carry out analyses on the function of the human and yeast carrier translocases. We aim to define the mitochondrial sublocalization of the carrier pathway to understand transfer of precursors from TOM to the TIM22 complex. The identification of links between the TIM22 complex and human diseases has led us to address the dysfunctions of the carrier pathway in metabolically active human cells. A central goal is to define the molecular pathology of AGK and TIM22 dysfunction.