cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol.
ABSTRACT We identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria. It was termed mitochondrial import stimulation factor or MSF [Hachiya, N. et al. (1993) EMBO J. 12, 1579-1586]. It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE. MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner. We report here the cDNA cloning and characterization of MSF. Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins. We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library. The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity. The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system. Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state. Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF. Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events.
Article: Overproduction of PDR3 suppresses mitochondrial import defects associated with a TOM70 null mutation by increasing the expression of TOM72 in Saccharomyces cerevisiae.[show abstract] [hide abstract]
ABSTRACT: Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol. We previously reported that the presequence of the F(1)-ATPase beta subunit precursor (pre-F(1)beta) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P. Hajek, J. Y. Koh, L. Jones, and D. M. Bedwell, Mol. Cell. Biol. 17:7169-7177, 1997). We also found that a mutant form of pre-F(1)beta with a minimal targeting signal (Delta 1,2 pre-F(1)beta) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation. We have now analyzed the consequences of reducing the pre-F(1)beta targeting signal to a minimal unit in more detail. We found that Delta 1,2 pre-F(1)beta is more dependent upon the Tom70p receptor for import than WT pre-F(1)beta is, resulting in a growth defect on a nonfermentable carbon source at 15 degrees C. Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Delta 1,2 pre-F(1)beta import signal in an import-competent conformation. We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Delta 1,2 pre-F(1)beta in a tom70 Delta strain. The overproduction of PDR3 mediated this effect by increasing the import of Delta 1,2 pre-F(1)beta into mitochondria. This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions. Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue. These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis.Molecular and Cellular Biology 12/2001; 21(22):7576-86. · 5.53 Impact Factor
Article: Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis.[show abstract] [hide abstract]
ABSTRACT: Age-related impairment of functionality of the central nervous system (CNS) is associated with increased susceptibility to develop many neurodegenerative diseases. Increased oxidative stress in the CNS of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deficits. Glutamate dysregulation, mitochondrial dysfunction and impaired protein synthesis are observed in aged brains, along with increased protein oxidation. Interestingly, all of these age-related cellular alterations can be improved by caloric restriction (CR), which can also improve the plasticity and recovery of the CNS. Although the beneficial effects of CR on brains are well established, the mechanism(s) of its action remains unclear. In order to gain insight into the mechanism of CR in the brain, we located the brain regions that are benefited the most from reduced oxidative stress by CR. Along with other brain regions, striatum (ST) showed significantly decreased bulk protein carbonyl levels and hippocampus (HP) showed decreased bulk protein 3-nitrotyrosine (3-NT) levels in CR aged rats when compared to those of age matched controls. To determine which proteins were oxidatively modified in these brain regions, we used parallel proteomics approach to identify the proteins that are altered in oxidation and expression. The specific carbonyl levels of pyruvate kinase M2 (PKM2), alpha-enolase (ENO1), inositol monophosphatase (INSP1), and F1-ATPase Chain B (ATP-F1B) were significantly decreased in ST of aged CR rats. In contrast, the expression levels of phosphoglycerate kinase 1 (PKG1), inosine monophosphate cyclohydrolase (IMPCH) and F1-ATPase Chain A (ATP-F1A) were significantly increased in the ST of CR rats. In the hippocampus of CR rats, the specific 3-NT levels of malate dehydrogenase (MDH), phosphoglycerate kinase 1 (PKG1) and 14-3-3 zeta protein were significantly decreased and expression levels of DLP1 splice variant 1 (DLP1), mitochondrial aconitase (ACO2), dihydrolipoamide dehydrogenase (DLDH), neuroprotective peptide H3 (NPH3), and eukaryotic translation initiation factor 5A (eIF-5A) are increased. Moreover, an unnamed protein product (UNP1) with similar sequence to initiation factor 2 (IF-2) was decreased in the HP of CR rats. Our data support the hypothesis that CR induces a mild metabolic stress response by increasing the production of neurotrophic proteins, therefore, priming neurons against apoptosis. Moreover, our study shows that the improvement of glutamate dysregulation, mitochondrial dysfunction and protein synthesis by CR is, at least partially, due to the CR-mediated alteration of the oxidation or the expression of PKM2, ENO1, INSP1, ATP-F1B, PKG1, IMPCH, ATP-F1A MDH, PKG1 and 14-3-3 zeta protein, DLP1, ACO2, DLDH, NPH3, eIF-5A and UNP1. This study provides valuable insights into the mechanisms of the beneficial factors on brain aging by CR.Neurobiology of Aging 08/2006; 27(7):1020-34. · 6.19 Impact Factor
Article: The 14-3-3 protein epsilon isoform expressed in reactive astrocytes in demyelinating lesions of multiple sclerosis binds to vimentin and glial fibrillary acidic protein in cultured human astrocytes.[show abstract] [hide abstract]
ABSTRACT: The 14-3-3 protein family consists of acidic 30-kd proteins expressed at high levels in neurons of the central nervous system. Seven isoforms form a dimeric complex that acts as a molecular chaperone that interacts with key signaling components. Recent studies indicated that the 14-3-3 protein identified in the cerebrospinal fluid of various neurological diseases including multiple sclerosis (MS) is a marker for extensive brain destruction. However, it remains unknown whether the 14-3-3 protein plays an active role in the pathological process of MS. To investigate the differential expression of seven 14-3-3 isoforms in MS lesions, brain tissues of four progressive cases were immunolabeled with a panel of isoform-specific antibodies. Reactive astrocytes in chronic demyelinating lesions intensely expressed beta, epsilon, zeta, eta, and sigma isoforms, among which the epsilon isoform is a highly specific marker for reactive astrocytes. Furthermore, protein overlay, mass spectrometry, immunoprecipitation, and double-immunolabeling analysis showed that the 14-3-3 protein interacts with both vimentin and glial fibrillary acidic protein in cultured human astrocytes. These results suggest that the 14-3-3 protein plays an organizing role in the intermediate filament network in reactive astrocytes at the site of demyelinating lesions in MS.American Journal Of Pathology 09/2004; 165(2):577-92. · 4.89 Impact Factor