Biogenesis of multispan proteins of the mitochondrial outer membrane

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The mitochondrial outer membrane (MOM) facilitates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Biogenesis of this membrane requires targeting of newly synthesized proteins to the organelle and their integration into the lipid bilayer. An important group of such MOM proteins are those that span the membrane with multiple α-helical segments. Despite ever-increasing evidence for the biological significance of these proteins, our understanding of their biogenesis process is scarce. The general objective of this study is to define the molecular mechanisms that underlie the biogenesis of α-helical multispan MOM proteins. We plan to address the following questions: 1) How do the membrane insertion factors Mim1 and Mim2 facilitate the membrane integration of MOM multispan proteins? 2) How does the import receptor Tom70 discriminate between MOM multispan proteins and carrier proteins destined to the inner membrane? and 3) What is the mammalian functional homologue of the fungal MIM complex?To that end, we will analyze in vitro, in organello and in vivo the import of precursor proteins into wild type mitochondria and into organelles from manipulated strains. To address aim 1, the membrane integration of multispan proteins will be studied by purifying Mim1 and Mim2 from a cell-free system. The ability of the detergent-solubilized Mim proteins to recognize MOM substrates will be investigated. Furthermore, the two proteins will be combined for the formation of the MIM complex and then the complex will be reconstituted into artificial lipid vesicles. To test whether the MIM complex builds the minimal machinery for membrane integration and functions as integrase, its functionality in such proteoliposomes will be monitored by testing their ability to mediate membrane integration of substrate proteins. Aim 2 will be addressed by mapping with photo-induced site-specific cross-linking the regions in either outer or inner membrane substrates, and within Tom70 itself, that are involved in receptor-substrate interactions. The identity of the cross-linking adducts will be investigated by Western blotting and mass spectrometry. Furthermore, the influence of the binding of various substrate proteins to Tom70 on the association of this receptor with either the rest of the TOM complex or the MIM complex will be studied by cross-linking and pull-down assays. Finally, to address Aim 3, mammalian functional homologues of Mim1/2 will be searched for by using yeast expression libraries comprising mouse or rat cDNAs. We will search for those cDNAs whose protein product is able to rescue the growth defect of yeast cells deleted for both MIM1 and MIM2 (mim1Δmim2Δ). The function of potential candidates will be verified in both yeast and mammalian cells. Taken together, this proposed study will shed new light on crucial processes in the biogenesis of mitochondrial outer membrane proteins.