The field of study is hepatic heme metabolism. The long term objectives of this project are: (A) to understand the mechanism(s) governing intracellular heme distribution in the hepatocyte and the role of protein mediation in these processes; and (B) to study how these processes respond to - or may themselves affect - the heme/iron status of the organism both in physiological and pathological states. Knowledge in this area of research may provide a firm basis for the development of better therapeutic intervention in the porphyrias and greater insight on the mechanisms of drug metabolism. The primary hypothesis we advance is that intracellular heme distribution is protein mediated in all of its phases. Specific cytosolic proteins that bind heme and may participate in heme transport have been identified. One of these proteins, heme binding protein (HBP) isolated in this laboratory from rat liver cytosol, promotes the efflux of heme from mitochondria, the site of heme synthesis. A protein with immunologic similarity to HBP but of slightly greater molecular weight (15.5 kDa vs 14 kDa) has also been identified, named p15.5, and found to localize in euchromatin-containing regions of the nucleus and nucleolus. We are proposing: I. To characterize nuclear p15.5, establish its relation to HBP and define its possible role in heme transport into the nucleus. For this aim, p15.5 will be purified and its interaction with heme and DNA studied. Furthermore, we will establish the relation between p15.5 in the nucleus and cytosolic HBP. In labeling experiments, we will explore whether p15.5 is in fact a post-translational modification of HBP, and by electronspray ionization mass spectrometry if these two proteins differ in their amino acid sequence. We will also investigate the possibility that HBP/p15.5 play a role in the uptake of heme into the nucleus by microinjecting fluorescently-labeled protein + heme into single cultured hepatocytes and studying the heme-dependent nuclear import of HBP/p15.5 in a cell-free nuclear system. II. To analyse the role of HBP in heme transport from mitochondria to other cellular organelles, isolated mitochondria will be used to determine the mechanism of HPB-promoted heme efflux. We will test for the presence of HBP-binding mitochondrial membrane protein(s) by cross-linking and adsorption experiments, and investigate the energy dependence of the process. In addition, we will examine the ability of HBP to reconstitute holo-cytochrome P-450 in apo- cytochrome P-450-rich microsomes separated from mitochondria by a membrane permeable to proteins in an effort to elucidate the role of HBP in heme transport from mitochondria to microsomes. III. We propose to characterize and purify two ca. 40 and 45 kDa heme-binding cytosolic proteins and test their possible function in heme transport in the described nuclear and mitochondrial systems.
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