Rapid increases in atractyloside-insensitive binding of purine nucleotides (ADP or GDP) and in a polypeptide of 32 000 occur in brown adipose tissue mitochondria of the rat during acclimation to cold. The increased binding is apparent within 1 h and reaches a maximum after 3--7 days of exposure to 4 degrees C. The increase in the 32 000 peptide occurs more slowly and reaches a maximum after 2--3 weeks. There is a simultaneous decrease in a polypeptide of 96 000, apparent after 1 day and reaching a maximum after 1--2 weeks. Results are interpreted in terms of the appearance of an increased amount of the purine nucleotide-sensitive proton conductance pathway in association with the development of an enhanced thermogenic capacity of brown adipose tissue mitochondria during acclimation of the rat to cold.

The proposed function of Cdc4p, an essential contractile ring protein in Schizosaccharomyces pombe, is that of a myosin essential light chain. However, five conditionally lethal cdc4 alleles exhibit complementation in diploids. Such interallelic complementation is not readily explained if the sole function of Cdc4p is that of a myosin essential light chain. Complementation of cdc4 alleles could occur only if different mutant forms can assemble into an active oligomeric complex or if Cdc4p has more than one essential function. To search for other proteins that may interact with Cdc4p, we performed a two-hybrid screen and identified two such candidates: one similar to Saccharomyces cerevisiae Vps27p and the other a putative phosphatidylinositol (PI) 4-kinase. Binding of Cdc4p to the latter and to myosin heavy chain (Myo2p) was confirmed by immunosorbent assays. Deletion studies demonstrated interaction between the Cdc4p C-terminal domain and the PI 4-kinase C-terminal domain. Furthermore, interaction was abolished by the Cdc4p C-terminal domain point mutation, Gly 107 to Ser. This allele also causes failure of cytokinesis. Ectopic expression of the PI 4-kinase C-terminal domain caused cytokinesis defects that were most extreme in cells carrying the G107S allele. We suggest that Cdc4p plays multiple roles in cytokinesis and that interaction with a PI 4-kinase may be important for contractile ring assembly and/or function.

A large fraction (30-50%) of the various proteins synthesized within isolated rat liver mitochondria were degraded to amino acids within 60 min after synthesis. Incomplete mitochondrial polypeptides resulting from the incorporation of puromycin were degraded even more extensively (80% per hr). Protein breakdown was measured by the appearance of acid-soluble radioactivity and by the disappearance of labeled polypeptides detected on NaDodSO4/polyacrylamide gel electrophoresis. The amino acids generated by proteolysis were transported rapidly out of the mitochondria and no peptide intermediates accumulated in the organelle. This degradative process did not involve lysosomes or lysosomal enzymes and was markedly stimulated by ATP either generated within the mitochondria or supplied exogenously. An inhibitor of respiration (cyanide) or uncouplers of oxidative phosphorylation (oligomycin, dinitrophenol) reduced proteolysis when mitochondria were provided substrates for ATP generation. When exogenous ATP was provided, these agents did not affect proteolysis, but degradation was then sensitive to atractyloside, an inhibitor of adenine nucleotide transport. Vanadate, an inhibitor of various ATPases, blocked proteolysis even in the presence of ATP and caused a marked stabilization of nearly all polypeptide bands. Thus, mitochondria-like bacteria or the cytosol of animal cells-contain a pathway for complete degradation of proteins which seems to selectively remove polypeptides with abnormal structures. Within this organelle, ATP hydrolysis appears necessary for an initial step in this degradative process.The proteins comprising the mitochondria, like other proteins in mammalian cells, are subject to continual turnover (1). It has generally been assumed that mitochondria are degraded within the lysosome (2-5), and under certain conditions whole mitochondria or mitochondrial enzymes have been demonstrated within autophagic vacuoles (2-4). However, different mitochondrial proteins turn over at distinct rates. Outer membrane proteins tend to be degraded at a faster rate than those of the inner membrane (6). Furthermore, individual proteins within the same mitochondrial compartment can also have different turnover rates. In the matrix, several enzymes have tl/2 ranging from 70 min to 1-2 days, whereas the bulk of mitochondrial proteins turn over with a tl/2 of 3-5 days (6-8). Such observations cannot be explained by indiscriminate degradation of this organelle within the lysosome.One possible explanation for this heterogeneity in degradative rates is that proteins may be excreted by the mitochondria for degradation in the cytoplasm or lysosome. Another possibility is that there exists within mitochondria a proteolytic system that selectively hydrolyzes the short-lived enzymes. Several groups have reported proteases associated with the mitochondrial fraction of mammalian cells (9-14). However, an intramitochondrial localization has not been demonstrated definitively for any of these enzymes. The presence in the mitochon...

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Exposure of a rat to cold (4 °C) is known to induce a biphasic change in brown adipose tissue mitochondria, believed to reflect alterations in the thermogenic, purine nucleotide sensitive proton conductance pathway; an initial rapid and large increase in purine nucleotide binding, unaccompanied by any marked change in the 32 000 polypeptide which is the binding site for these nucleotides, is followed by a slower increase in concentration of the 32 000 polypeptide accompanied by a further increase in purine nucleotide binding. The initial rapid effect of cold stress was mimicked by intravenous infusion of noradrenaline; neither the effect of cold exposure for 24 h nor the effect of intravenous infusion of noradrenaline was prevented by cycloheximide. In contrast, the slow adaptive changes in the mitochondria did not occur in response to prolonged (2 weeks) treatment with noradrenaline, although such treatment did induce the expected tissue hypertrophy accompanied by mitochondrial proliferation. Cold-induced (1 week) increases in purine nucleotide binding and 32 000 polypeptide were not prevented by oxytetracycline. The increase in purine nucleotide binding during the 2nd day of cold exposure was prevented by cycloheximide. The effect of cycloheximide on the increase in the 32 000 polypeptide could not be assessed because sufficiently long-term experiments could not be done with this compound. Thus, the initial response to cold stress appears to involve unmasking of mitochondrial proton conductance pathway sites, most probably mediated by noradrenaline. The slower adaptive response occurs in parallel with tissue hypertrophy, which itself may be mediated by noradrenaline, and appears to require cytosolic but not mitochondrial protein synthesis. However, the changes in mitochondrial composition which result in an increased concentration of proton conductance pathway sites are not mediated by noradrenaline.