The terminal stage of ATP synthesis in the mitochondria of animal and human cells takes place within the inner membrane and requires 3 separate participants, the ATP synthase (F0F1). a double motor enzyme, the phosphate carrier (PIC), and the adenine nucleotide carrier (ANQ. During the past progress period that led to the discovery of several novel mechanistic and structural features of the mitochondrial ATP synthase (PNAS, 1998;JBC, 1999;JBC, 2000;Proteins, 2003), we discovered also that the above proteins exist as a single ATP synthase/PIC/ANC complex (>0.7 million Da) that we named the """"""""ATP synthasome"""""""" (JBC, 2003). In addition to purifying this 17 subunit type complex, and identifying detergents that maintain it in a soluble, stable, dispersed form, we obtained a 3-D structure at 2.3 nm resolution by reconstructing negatively stained images of single molecules (JBC, 2004). Most recently, we have shown that the ATP svnthasome forms 2-dimensional (2D) crystalline sheets and is a functionally relevant complex as oligomycin. the classical inhibitor of mitochondrial oxidative phosphorylation. only inhibits the ATP svnthase when PIC and ANC are bound. Finally, in a project related to the primary mission of the NCI who funded this work, we found that the antimetabolite 3-bromopyruvic acid (3-BrPA) targets the ATP synthasome, rapidly and completely depletes cell ATP, and eradicates advanced stage cancers in all 19 treated cases in a rodent model (BBRC, 2004). Future work will entail 3 Specific Aims as follows: 1. Determine to what extent the ATP synthasome alone catalyzes partial reactions characteristic of the terminal steps of oxidative phosphorylation in mitochondria, assess the functional significance of placing PIC and ANC near the ATP synthase's motor components, and determine whether PIC and ANC form a heterodimer. 2. Elucidate at molecular resolution a topological map in 3-D in which those 8 subunit types (PIC, ANC, d, e, f, g, F6 &A6L), unique to the mitochondrial ATP synthasome, can be visualized relative to each other and to the F1 and F0 motor components common to all ATP synthases. 3. Vigorously pursue efforts in 2 different laboratories, that of the P.I. and that of J. Deisenhofer, to obtain 3-D crystals of the ATP synthasome that diffract to atomic resolution. Collectively, these novel studies are fundamental to understanding mitochondrial function in mammalian cells and have direct relevance both to identifying new effective cancer therapies and to understanding mitochondrial associated diseases. These studies also have direct relevance to using nanotechnology in medicine as the ATP synthasome is comprised of 2 reversible nanomotors.
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