Abstract

The F₁F₀ ATP synthase is an ancient and complex enzyme, which has been studied extensively with respect to its structure and function. More recently the role that the enzyme plays in human disease has been the focus of much attention, in part from observations that this archetypical mitochondrial protein has other locations in the cell. This new area of investigation raises important questions not only about exactly where the ATP synthase resides among cellular compartments but also its interactions in different locations.

This dissertation describes the identification of F₁F ₀, as well as the rest of the mitochondrial respiratory chain on the cell surface (i.e. plasma membrane) in an osteosarcoma cell line, and also adds to the knowledge of the interactions of the complex in the mitochondrial membrane, which is very different from that in the plasma membrane.

Specifically, it had been shown that F₁F₀ is present on the surface of some cell types, where it plays a role in multiple biological processes including angiogenesis and vasoregulation. Currently the mechanism by which this large complex arrives at the cell surface is not understood. We have performed a detailed immunocytochemical study and determined that subunits of both F₁ and F₀ subcomplexes are present on the surface of several human cell types. Moreover, we present evidence that the entire respiratory chain is present on the surface, in a distribution pattern similar to that of the F₁F₀. These data help decide between possible mechanisms by which the F₁F₀ arrives at the cell surface.

Recent studies have established that the F₁F₀ is not an isolated enzyme within the mitochondrial inner membrane, but is physically associated with its substrate carrier proteins, adenine nucleotide translocase (ANT) and phosphate carrier protein (PCP), into an 'ATP synthasome' protein supercomplex. Here we describe the development and characterization of a monoclonal antibody against ANT that can be used to immunocapture the synthasome. We show that this protein supercomplex is expressed in both heart and liver, and that it has a differential phosphorylation pattern in these two tissue types, perhaps implying tissue specific regulation via post-translational modification.