Abstract 157 Cardiology: Mouse Models and Basic Mechanisms of Cardiac Disease Platform, Tuesday, 5/4

Mitochondrial biogenesis and function depends upon transport of nuclearly encoded proteins across the mitochondrial membranes. This is presumed to occur via post-translational import in vivo. However, we have shown that ribosomes target and bind to mitochondria in a reaction controlled by the transit peptide of the nascent chain and GTP-binding proteins, thus suggesting import could occur by a co-translational process similar to movement of proteins across the endoplasmic reticulum (ER). To identify and characterize G-binding proteins that might participate in ribosome binding, we used ultraviolet crosslinking and biochemical techniques to identify a family of GTP-binding proteins in the mitochondrial membranes. One of these proteins, termed G54, demonstrated significant binding activity for GTP in intact mitochondria. Biochemical analysis localized G54 to a protected site in the membranes that has access to GTP in intact mitochondria, and GTP is hydrolyzed to GDP while bound to G54. Pulse-chase experiments show that GDP is stably bound by G54 but is rapidly released upon ribosome binding to intact mitochondria. Pyrimidine nucleotides will not compete with GTP for G54. Adenine nucleotides do compete with GTP but only if present in great excess indicating specificity of G54 for GTP. ADP and ATP cause a concentration-dependent shift in GTP binding between two similar sized G-binding proteins at 54 and 52 kDa size. G54 is also present in beef heart mitochondria but does not bind ATP. Induction of the permeability transition by Mastoparan does not affect GTP binding by G54 indicating that a membrane potential is not necessary for G54 activity. These results identify a new group of G-binding proteins in the mitochondrial membranes one of which, termed G54, interacts with ribosomes and is present across species. They strongly suggest that mitochondrial protein import may involve a novel GTP-binding cycle, and that ribosomes binding to mitochondria initiate this cycle in a manner similar to co-translational import into the ER. Thus, import of proteins into mammalian mitochondria may be more complex than the post-translational model suggests. This pathway may be important in mitochondrial biogenesis, and could be susceptible to disruption, such as during ischemia/reperfusion injury in heart or brain, leading to delayed injury or cell death.