Degradation of mitochondrial translation products in Saccharomyces cerevisiae mitochondria was studied by selectively labelling these entities in vivo in the presence of cycloheximide and following their fate in isolated mitochondria. One-third to one-half of the mitochondrial translation products are shown to be degraded, depending on the culture growth phase, with an approximate half-life of 35 min. This process is shown to be ATP-dependent, enhanced in the presence of puromycin and inhibited by chloramphenicol. Further, the proteolysis is suppressed by detergents and is insensitive to antisera against yeast proteinases A and B when measured in mitochondria or 'inside-out' submitochondrial particles. It is concluded that the breakdown of mitochondrial translation products is most probably due to the action of endogenous proteinase(s) associated with the mitochondrial inner membrane. This proteinase is inhibited by phenylmethanesulphonyl fluoride, leupeptin, antipain and chymostatin.
To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at nonpermissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.Molecular chaperone proteins bind non-native protein structures and stabilize them against aggregation (1). By this means, they ensure proper folding of newly synthesized proteins, provide protection against heat denaturation, and mediate the vectorial translocation of polypeptides across biological membranes (2-7). Furthermore, evidence is accumulating that chaperone proteins play a pivotal role in ATP-dependent proteolytic processes (8 -10). Chaperone and proteolytic activities thereby constitute a quality control system which prevents the possibly deleterious accumulation of misfolded polypeptides in the cell. For instance, the degradation of misfolded polypeptides by Lon-like proteases in Escherichia coli or mitochondria depends on Hsp70 proteins which prevent the aggregation of substrate polypeptides (11)(12)(13)(14)(15). In addition to classical chaperone proteins that cooperate with ATP-dependent proteases during proteolysis, intrinsic chaperone-like properties have been assigned to some ATP-dependent proteases themselves which may be crucial for the degradation of non-native polypeptides (9, 10). The best studied cases are the hetero-oligomeric Clp proteases of prokaryotes whose regulatory subunits exert ATP-dependent chaperone activity (16,17).Several ATP-dependent proteases have been identified in mitochondria, which mediate the selective degradation of proteins in this organelle (10, 18,19). These proteases are required for maintenance of the respiratory competence of yeast cells suggesting important regulatory functions during the biogenesis of mitochondria. An ATP-dependent protease, highly homologous to Escherichia coli Lon protease, has been identified in the mitochondrial matrix space (20, 21). The corresponding genes from humans (22, 23) and yeast (24, 25) were cloned and termed PIM1 (for proteolysis in mitochondria) or LON. PIM1-mediated proteolysis is required for the maintenance of mitochondrial genome integ...
(3/4) This review summarizes data about structural and functional organization of steroidogenic P450-dependent enzymatic systems. Problems of catalysis of steroid substrate transformation, special features of mitochondrial type P450scc topogenesis, and abilities of some microbial electron transport proteins to support P450 activity in vitro and in vivo are considered. Principal steps in the creation and catalytic properties of transgenic strains of Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica expressing both mammalian steroidogenic P450s and the corresponding electron transport proteins are also described. Achievements and prospects of using such transgenic strains for biotechnological synthesis and pharmacological screening are considered.
There is a vast body of literature on the quality control of protein folding and assembly into multisubunit complexes. Such control takes place everywhere in the cell. The correcting mechanisms involve cytosolic and organellar proteases; the result of such control is individual molecules with proper structure and individual complexes both with proper stoichiometry and proper structure. Obviously, the formation of organelles as such requires some additional criteria of correctness and some new mechanisms of their implementation. It is proposed in this article that the ability to carry out an integral (key) function may serve as a criterion of correct organelle assembly and that autophagy can be accepted as a mechanism eliminating the assembly mistakes.z 1999 Federation of European Biochemical Societies.
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