Insights into mitochondrial metabolism through structure and function analysis of outer and inner membrane transporters.

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Membrane proteins, as determinants of mitochondrial structure and function, are the focus of this NSERC-funded program. In previous phases, we developed an understanding of the broad range of activities linked to mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC), through investigation of strains of Neurospora crassa harbouring porin variants or lacking the protein. The first aim of the proposed research builds on this information and preliminary work to determine the role porin plays in the homeostasis of the mitochondrial outer membrane (MOM), in terms of lipid content, biophysical properties and protein complex formation. Information derived from this work will reveal the physical interconnections between lipid and protein in membranes that are critical to their function. The second aim is to determine the folded state of native porin and the sequence determinants thereof, building on the methods for isolation of highly purified porin from the N. crassa MOM that were developed in the last granting period. Detailed structural information is limited to vertebrate porins folded in detergent following expression in E. coli and the proposed structural studies will allow assessment of heterologously expressed porin as a model for the native form. In addition, structural analysis of a fungal porin and comparison with vertebrate porins will reveal how similar functions can be achieved by proteins of very low primary sequence similarity. The final aim of the proposed research is to identify and characterize protein(s) potentially involved in mitochondrial drug efflux and the regulatory systems that control them. Porin acts as a general pore, allowing small molecules access to the inner membrane and the transporters therein. This flow has the potential to allow toxins and antibiotics to enter mitochondria. Given the soil habitat of fungi, it is expected that antibiotics with mitochondrial targets will cross the inner membrane of the organelle. We hypothesized that processes similar to bacterial efflux exist for the removal of antibiotics from mitochondria, and recently we demonstrated that isolated N. crassa mitochondria can, in an energy-dependent manner, efflux ethidium bromide, a model compound used to assess bacterial drug efflux. The transport mechanisms used by eukaryotes to protect mitochondria in complex soil environments are important aspects of fungal biology and have implications for biocontrol in agriculture. Taken together, the information gleaned from the proposed studies will advance our understanding of mitochondrial membrane proteins as structural and transport molecules required for eukaryotic cell function. Through this work, graduate and undergraduate students will receive excellent training in membrane biology, important preparation for future careers in discovery and applied research in the life sciences.**