Mitochondrial Biogenesis - Protein Translocation into Mitochondria Our research encompasses two major areas: Understanding the mechanism of protein import into mitochondria and determining the process by which defects in mitochondrial protein translocation lead to disease.

A basic question in cell biology is the mechanism by which a protein reaches its correct location within the cell. Of all the organelles in a mammalian cell, the mitochondrion is the most complex because two membranes must be crossed. In addition to the metabolic role, the mitochondria is a key player in many cellular processes including apoptosis, metal ion homeostasis and aging. My specific interests lie in mitochondrial biogenesis, particularly the mechanism by which proteins are imported into the mitochondrial inner membrane.

approaches Our laboratory uses a combined genetic and biochemical approach to understand the fundamental process of protein import in the model organism Saccharomyces cerevisiae. Protein import is highly conserved between yeast, plants, and animals. We are particularly interested in the process by which inner membrane proteins are imported.

The Link Between Defects In Mitochondrial Protein Import And Disease: An X-linked disease deafness/dystonia syndrome (Mohr-Tranebjaerg syndrome) is caused by a defect in mitochondrial protein import. Our studies in mammalian systems are aimed at understanding the molecular basis for this disease.

Mechanistic Studies The mitochondrion contains several protein import and export pathways (Fig 1). The general protein import pathway for precursors carrying a cleavable, N-terminal targeting sequence has been well-characterized. The coordinated action of the translocase of the outer membrane (TOM) and the Tim23p/Tim17p translocase of the inner membrane (TIM23) mediates passage of the precursor from the cytoplasm to the matrix (Fig. 1, 2). Components of the TOM and TIM complexes are integral membrane proteins, and many are essential for cell viability. In the TOM complex of yeast mitochondria, Tom20, 22, 37, and 70 are receptors, mediating transfer of the precursor from cytosolic chaperones to the TOM channel consisting of Tom5, 6, 7, and 40. Tim23 of the inner membrane contains a hydrophilic domain that functions as a receptor for the precursor as it emerges from the TOM channel. The precursor passes through the TIM23 channel (Tim23 and Tim17) and ATP hydrolysis by the translocation motor of Tim44, mHsp70, and mGrpE completes translocation.

Many precursors lack a cleavable targeting sequence, carrying instead their targeting and sorting information within the "mature" part of the polypeptide chain. This category of proteins includes at least 34 members of the mitochondrial carrier family, which span the membrane six times, as well as numerous other inner membrane proteins, including the import components themselves. The TIM22 pathway mediates import of these proteins (Fig. 3) (12). Components include the tiny Tim proteins of the intermembrane space (Tim8, Tim9, Tim10, Tim12, and Tim13) (8, 9, 10) and the inner membrane proteins Tim18, Tim22, and Tim54 (9, 14). The tiny Tim proteins act as putative chaperones to guide unfolded inner membrane proteins across the aqueous intermembrane space (11), while the inner membrane complex mediates insertion of the precursor into the inner membrane (14).

We are interested in understanding the mechanistic process by which proteins are imported into the mitochondrial inner membrane. Our studies focus on the model system Saccharomyces cerevisiae because the protein import machines are conserved between plants, animals, and fungi. By using a combined genetic and biochemical approach, we have constructed temperature-sensitive mutants and exploited them to dissect the mechanism of protein import (8). The tiny Tim proteins of the mitochondrial intermembrane space act as putative chaperones to guide precursor across the intermembrane space. The Tim8/Tim13 complex and the Tim9/Tim10 complex have different substrate specificity however both complexes bind to the transmembrane spanning domains of inner membrane precursors. This specific protein-protein interactions shields the hydrophobic residues from the aqueous intermembrane space thus enabling the tiny Tim complexes to escort the precursor to the inner membrane translocons (19, 21). The precursor then engages the TIM22 complex of the inner membrane. Membrane proteins Tim18, Tim22, and Tim54 may form a translocation pore to mediate insertion of the carriers into the inner membrane, while peripheral proteins Tim9, Tim10, and Tim12 assist in the process.

The Link Between Defects In Mitochondrial Protein Import And Disease A mutation in DDP1, the mammalian homolog of Tim8, leads to an X-linked disease (Mohr-Tranebjaerg syndrome) in which males have progressive deafness, blindness, and dystonia (10). This is the first disease caused by a defect in protein import, presumably because some key inner membrane proteins are present in decreased amounts. Because the spectrum of mitochondrial diseases varies, an interesting question is why this disease primarily affects the nervous system in contrast to other mitochondrial diseases affecting both nervous and muscular systems. Through a collaboration with Dr. Lisbeth Tranebjaerg at the University of Tromso, Norway, we are trying to determine the molecular basis of this disease.

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