Chantal Priesnitz

Abstract

The respiratory chain complexes, localised in the mitochondrial inner membrane, shuttle electrons from NADH or FADH₂ through their redox systems. The released energy is used to generate a proton gradient across the inner membrane, which drives the ATP production by the ATP synthase. The electrons are finally transferred to molecular oxygen by the cytochrome c oxidase (complex IV, COX). Complex IV contains subunits of dual genetic origin with eight nuclear- and three mitochondrially encoded components. COX proteins produced in the cytosol must be imported into mitochondria. This involves the translocase of the outer membrane (TOM complex) and the presequence translocase (TIM23 complex), that mediates transport of preproteins into the inner membrane or mitochondrial matrix. The translocation of preproteins depends on the membrane potential (Δψ) and the ATP powered presequence translocase-associated motor (PAM complex). Core component of this import motor is the chaperone mtHsp70. Further elements are the nucleotide exchange factor Mge1, Tim44 and the PAM proteins Pam16, Pam17 and Pam18. Mitochondrially encoded COX subunits on the other hand are inserted into the membrane in a co-translational manner by the oxidase assembly (OXA) machinery. Subsequently, assembly intermediates composed of nuclear and mitochondrial COX proteins are built-up and finally the mature complex IV is formed. This highly complicated process involves more than 20 proteinaceous assembly factors. Recent findings indicate that components of the PAM machinery also associate to respiratory chain complexes and assembly intermediates. The aim of this project is to analyse the role of these factors in the coordination of protein import and COX assembly.

Methods

• Yeast cell culture
• Isolation of mitochondria
• Cell free translation of membrane proteins
• In vitro protein import into yeast mitochondria
• Blue native PAGE, SDS PAGE, Western blot
• Affinity purification