Mitochondrial ATP-dependent Protease Research Articles

Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe–S proteins

Friedreich ataxia (FRDA) is a rare hereditary neurodegenerative disease characterized by progressive ataxia and cardiomyopathy. The cause of the disease is a defect in mitochondrial frataxin, an iron chaperone involved in the maturation of Fe-S cluster proteins. Several human diseases, including cardiomyopathies, have been found to result from deficiencies in the activity of specific proteases, which have important roles in protein turnover and in the removal of damaged or unneeded protein. In this study, using the muscle creatine kinase mouse heart model for FRDA, we show a clear progressive increase in protein levels of two important mitochondrial ATP-dependent proteases, Lon and ClpP, in the hearts of muscle creatine kinase mutants. These proteases have been shown to degrade unfolded and damaged proteins in the matrix of mitochondria. Their upregulation, which was triggered at a mid-stage of the disease through separate pathways, was accompanied by an increase in proteolytic activity. We also demonstrate a simultaneous and significant progressive loss of mitochondrial Fe-S proteins with no substantial change in their mRNA level. The correlative effect of Lon and ClpP upregulation on loss of mitochondrial Fe-S proteins during the progression of the disease may suggest that Fe-S proteins are potential targets of Lon and ClpP proteases in FRDA.

A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae

Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis–Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.

Characterization of Peptides Released from Mitochondria: EVIDENCE FOR CONSTANT PROTEOLYSIS AND PEPTIDE EFFLUX

Conserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis. Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids that are released from mitochondria. Here, we have characterized peptides extruded from mitochondria by mass spectrometry and identified 270 peptides that are exported in an ATP- and temperature-dependent manner. The peptides originate from 51 mitochondrially and nuclearly encoded proteins localized mainly in the matrix and inner membrane, indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle. Pulse-labeling experiments in logarithmically growing yeast cells revealed that approximately 6-12% of preexisting and newly imported proteins is degraded and contribute to this peptide pool. Under respiring conditions, we observed an increased proteolysis of newly imported proteins that suggests a higher turnover rate of respiratory chain components and thereby rationalizes the predominant appearance of representatives of this functional class in the detected peptide pool. These results demonstrated a constant efflux of peptides from mitochondria and provided new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis.

Nucleotide sequence for cDNA of bovine mitochondrial ATP-dependent protease and determination of N-terminus of the mature enzyme from the adrenal cortex

We have determined the cDNA sequence encoding bovine mitochondrial ATP-dependent Lon protease. Since the 5'-end region of the cDNA was highly GC-rich and thus could not be amplified by the 5'-RACE method, a genomic DNA fragment containing an in-frame ATG was isolated and sequenced. The translated amino acid sequence contained 961 amino acids with a calculated molecular weight 106,665. Sequence similarities of the bovine enzyme to human and E. coli orthologs were 92 and 27%, respectively. The N-terminal amino acid sequence seemed to be a mitochondrial targeting signal. To determine the cleavage site of the signal sequence we analyzed the mature enzyme purified from bovine adrenocortical mitochondria. Analysis of CNBr-digested peptides revealed that the N-terminus was heterogeneous. We suggest that nonspecific aminopeptidase might remove several amino acids from the N-terminus after mitochondrial processing peptidase has cleaved Gly(67)-Leu(68) or Leu(68)-Trp(69).

Activation of mitochondrial ATP-dependent protease by peptides and proteins.

We examined the effect of peptides or protein on the proteolytic and ATPase activities of mitochondrial ATP-dependent LON protease purified from bovine adrenal cortex. Peptides/proteins including angiotensin I which stimulated ATPase activity without hydrolysis of any peptide bonds stimulated proteolysis of 125I-labeled substrates at low concentrations; whereas at high concentrations they competitively inhibited proteolysis, thus displaying a biphasic activity profile. All peptides and proteins thus examined stimulated degradation of 125I-labeled substrates. When an ATP analog was substituted for ATP, only inhibition; i.e., no stimulation, of proteolysis by unlabeled peptides was observed. Without activator peptides, degradation of [125I] peptides was higher in the presence of an ATP analog than that in the presence of ATP. ADP, a product of the ATPase reaction, inhibited the proteolytic activity in the absence of an activator peptide but not in its presence. From analogy to E. coli ATP-dependent protease La (LON), we suggest that the activator peptides stimulated the proteolysis by releasing enzyme-bound ADP.

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae.

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.

Enhanced mitochondrial biogenesis is associated with increased expression of the mitochondrial ATP-dependent Lon protease

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 ATP-dependent 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.

Possible Function of SP-22, a Substrate of Mitochondrial ATP-Dependent Protease, as a Radical Scavenger

SP-22 was found to be a substrate protein of a mitochondrial ATP-dependent protease in bovine adrenal cortex. Its amino acid sequence was homologous to that of some prokaryotic and eukaryotic proteins such as thioredoxin peroxidase (formerly called thiol-specific antioxidant) in yeast and mammalian brains and the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium. In the present study, we found SP-22 to have the ability to scavenge reactive oxygen species, thus protecting radical-sensitive proteins such as tryptophan hydroxylase, glutamine synthetase and hemoglobin from oxidation. The protecting activity was enhanced by the addition of horse serum. The "serum factor(s)" seemed to be protein(s), since the stimulating activity was macromolecular and precipitated with 35% ammonium sulfate. Possible physiological roles of SP-22 in adrenocortical mitochondria are discussed.

Purification and characterization of a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex.

We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.

PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae.

The PIM1 nuclear gene in the yeast Saccharomyces cerevisiae encodes a mitochondrial ATP-dependent protease that exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and one from Myxococcus xanthus. In addition, Pim1 is 1133 amino acids long and has a putative mitochondrial import signal in the N-terminal region. Enzymatic comparisons of normal PIM1+ and deficient pim1-delta strains revealed that the ATP-dependent protease is located within the mitochondrial matrix. The pim1-delta strains are unable to utilize nonfermentable substrates as the sole carbon source and are unable to maintain functional mitochondrial DNA, indicating that the Pim1 protease is required for mitochondrial function. PIM1 mRNA is constitutively expressed but is increased after thermal stress, suggesting that Pim1 may play a role in the heat shock response.

Yeast mitochondrial ATP-dependent protease: purification and comparison with the homologous rat enzyme and the bacterial ATP-dependent protease La

Homogenous ATP-dependent protease has been isolated for the first time from mitochondria of the yeast Saccharomyces cerevisiae. The enzyme molecule consists of six 120 kDa subunits. It is a serine protease with an absolute ATP requirement for its activity. Basic enzymatic characteristics of the yeast protease are similar to those of the corresponding rat mitochondrial enzyme and of the E. coli protease La. The yeast enzyme immunochemically cross-reacts with the bacterial protease La.