Our main interest is the intracellular protein trafficking in eukaryotic cells. About 30-50% of the human proteome consists of membrane proteins or proteins that have to be targeted to a site of action distant to where they are synthesized. This highlights the paramount importance of correct intracellular protein targeting, which is crucial for the architecture, organization and function of all cells. It is hence not surprising that more than 50% of the current Pharma Industry targets are against membrane proteins. Our focus is on the protein import mechanisms in mitochondria, the organelles that control several key cellular processes in normal physiology (ATP production, metal ion homeostasis) but are also involved in several medical conditions (neurological disorders, cancer) and cell death.
As nearly all mitochondrial proteins (on average 10-15% of the proteome) are imported from the cytosol, these organelles have an elaborate and versatile protein machinery (translocases) to cope with these import processes. To study these, we use yeast mitochondria as
(i) we can combine the power of yeast genetics (genome completely sequenced,
all gene knockouts, tagged proteins, interactome databases etc are readily available) with biochemical approaches,
(ii) the yeast mitochondrial proteome is currently the most complete (95% of proteins identified)
(iii) isolated yeast mitochondria can be readily isolated to high homogeneity and in a
functional form that can be kept for months.
We combine three main methodologies ranging from in vivo studies (intact cells, about 6000 proteins), through in organello studies (isolated mitochondria and import assays, about 900 proteins)down to in vitro studies using isolated proteins and their complexes. In this context we use an interdisciplinary approach combining molecular biology (cloning, yeast genetics, mutagenesis), cell biology (cellular fractionation, confocal fluorescence microscopy), biochemistry (protein purification, chemical crosslinking, immunoprecipitations, several liquid chromatography methods, mass spectrometry), biophysics (circular dichroism, isothermal titration calorimetry, light scattering, surface plasmon resonance, fluorescence).
The particular issues that we want to resolve include
(i) how membrane proteins are targeted to the mitochondrial inner membrane
(ii) how the TIM10 chaperone system for membrane proteins assembles and functions
(iii) what is the molecular basis of a novel oxidative folding pathway that we have
discovered in mitochondria and is essential for the assembly of a number of intermembrane space proteins.