All eucaryotic organisms contain mitochondria, complex organelles that generate metabolic energy in the form of ATP. Mitochondria are also involved in several key cellular processes, including the metabolism of amino acids and lipids, and the assembly of iron-sulfur clusters. The majority of mitochondria! proteins are synthesized on cytosolic ribosomes and imported into the matrix by the translocase of the inner membrane (TIM) complex, which is composed of a transmembrane protein-conducting channel and an import motor. Translocation is driven by two factors: the electric membrane potential of the inner membrane, and ATP hydrolysis catalyzed by the mitochondrial heat-shock protein mtHsp70, termed Ssc1 in yeast. Other components of the import motor which stimulate the ATPase activity of Ssc1 include Tim44, Pam18 and Pam16. Tim44 coordinates the localization of the import motor to the channel of the translocon, facilitating the binding of polypeptide substrates as they are translocated across the inner membrane. Pam18 is a specific J protein that stimulates the ATPase activity of Ssc1. Interestingly, Pam16 has sequence similarity to the J domain of Pam18 but does not stimulate the ATPase activity of Ssc1. Instead, Pam16 interacts with Pam18 to form a stable complex which is required for efficient peptide import. This research is aimed at better understanding the role of this Pam18:Pam16 heterodimer, which is essential for cell survival. The structure and function of the active conformation of Pam18:Pam16 will be determined, and its regulation of Ssc1 activity will be examined to determine the physiological significance of such a regulatory mechanism. These studies will employ a number of different approaches, including yeast genetics, cell biology, mutagenesis, X-ray crystallography, and in vitro biochemical studies. The association of the Pam18:Pam16 heterodimer with the core translocon will then be studied by reconstitution of the TIM complex, which will enable a number of studies made difficult by the insolubility of the membrane-bound components of the complex. The development of these techniques will initiate a broad undertaking to better understand the overall mechanism of protein translocation in the context of the inner membrane environment. The proper function of mitochondria is essential to a number of cellular processes, including cell development, calcium signaling, apoptosis, and aging. Malfunctions in mitochondria are thus responsible for a number of severe human diseases including heart disease, cancer, diabetes and neurological disorders. A better understanding of mitochondrial protein import, which is critical for mitochondrial biogenesis, is therefore of great importance to public health.