Rats bearing the Zajdela hepatoma tumor and T3-treated hypothyroid rats were used to study the role of protein degradation in the process of mitochondrial biogenesis. It was shown that the activity, protein and mRNA levels of the ATPdependent Lon protease increased in rapidly growing Zajdela hepatoma cells. The increase in the rate of mitochondrial biogenesis by thyroid hormone was similarly accompanied by enhanced expression of the Lon protease. The results imply that mitochondrial biogenesis in mammalian cells is, at least partially, regulated by the matrix Lon protease.z 1999 Federation of European Biochemical Societies.

Point and deletion mutations and a general depletion of mammalian mitochondrial DNA (mtDNA) give rise to a wide variety of medical syndromes that are refractory to treatment, possibly including aging itself. While gene therapy directed at correcting such deficits in the mitochondrial genome may offer some therapeutic benefits, there are inherent problems associated with a direct approach. These problems are primarily due to the high mitochondrial genome copy number in each cell and the mitochondrial genome being "protected" inside the double-membrane mitochondrial organelle. In an alternative approach there is evidence that genes normally present in the mitochondrial genome can be incorporated into the nuclear genome. To extend such studies, we modified the Chinese Hamster Ovary (CHO) mtDNA-located ATPase6 gene (possessing a mutation which confers oligomycin resistance- oli(r)) by altering the mtDNA code to the universal code (U-code) to permit the correct translation of its mRNA in the cytoplasm. The U-code construct was inserted into the nuclear genome (nucDNA) of a wild type CHO cell. The expressed transgene products enabled the transformed CHO cell lines to grow in up to 1000 ng mL(-1) oligomycin, while untransformed sensitive CHO cells were eliminated in 1 ng mL(-1) oligomycin. This approach, termed allotopic expression, provides a model that may make possible the transfer of all 13 mtDNA mammalian protein-encoding genes to the nucDNA, for treatments of mtDNA disorders. The CHO mtATPase6 protein is 85% identical to both the mouse and human mtATPase6 protein; these proteins are highly conserved in the region of the oligomycin resistance mutation. They are also well conserved in the regions of the oligomycin resistance mutation of the mouse, and in the region of a mutation found in Leigh's syndrome (T8993G), also called NARP (neurogenic weakness, ataxia, retinitis pigmentosum). It is likely that the CHO oli(r) mtATPase6 Ucode construct could impart oligomycin-resistance in human and mouse cells, as well as function in place of the mutant ATPase subunit in a NARP cell line. Preliminary experiments on human cybrids homoplasmic for the NARP mutation (kindly supplied by D.C. Wallace), transformed with our construct, display an increased oligomycin resistance that supports these suppositions.

Hereditary hemochromatosis is characterized by iron overload in parenchymal tissues, such as the liver. Mutations in the hereditary hemochromatosis protein, HFE, result in the most common type of this disease. Two binding partners of HFE, which also participate in regulation of iron homeostasis, have been identified: transferrin receptor 1 (TfR1) and 2 (TfR2). Recent efforts focused on understanding how HFE, TfR1, and TfR2 communicate to modulate hepcidin levels in the body, but basic questions such as the relative levels of these players have been overlooked. Thus, the aim of this study was to determine in vivo concentrations of HFE, TfR1, and TfR2 in human liver tissues. RT‐PCR analysis showed that the level of TfR2 mRNA was 100‐ and 200‐ fold higher than that of TfR1 and HFE, respectively. The molar concentration of TfR2 protein (1.95 nmoles/g protein) was about 4.5 times higher than that of TfR1 (0.43 nmoles/g protein) as judged by quantitative immunoblotting. The level of HFE protein was below 0.20 nmoles/g protein, indicating that HFE is present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This observation suggests that HFE might be the limiting factor during reorganization of HFE/TfR1 and HFE/TfR2 complexes in vivo in response to holo‐transferrin concentrations. This work was supported by NIH Grant RO1‐DK072166 (C.A.E.) and in part by MRF of Oregon ACEBD0082 (M.C.).