Tom20-mediated mitochondrial protein import in muscle cells during differentiation

2000 ◽  
Vol 279 (5) ◽  
pp. C1393-C1400 ◽  
Author(s):  
Janice Y. Grey ◽  
Michael K. Connor ◽  
Joseph W. Gordon ◽  
Masato Yano ◽  
Masataka Mori ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Matrix Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Muscle Cells ◽  
In Vitro Assays ◽  
Organelle Biogenesis ◽  
Outer Membrane Receptor ◽  
The Matrix

Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40–60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.

Thyroid hormone modifies mitochondrial phenotype by increasing protein import without altering degradation

1998 ◽  
Vol 275 (6) ◽  
pp. C1508-C1515 ◽  
Author(s):  
Elaine E. Craig ◽  
Alan Chesley ◽  
David A. Hood
Keyword(s):  
Thyroid Hormone ◽  
Outer Membrane ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Chimeric Protein ◽  
Hormone Treatment ◽  
Outer Membrane Receptor ◽  
Cardiac Muscle Cells ◽  
Precursor Proteins ◽  
The Matrix

The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.

scholarly journals Characterization of Mmp37p, aSaccharomyces cerevisiaeMitochondrial Matrix Protein with a Role in Mitochondrial Protein Import

2006 ◽  
Vol 17 (9) ◽  
pp. 4051-4062 ◽  
Author(s):  
Michelle R. Gallas ◽  
Mary K. Dienhart ◽  
Rosemary A. Stuart ◽  
Roy M. Long
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Temperature Sensitive ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Close Proximity ◽  
The Matrix ◽  
Targeting Signal

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

Influence of aging on protein import into cardiac mitochondria

1997 ◽  
Vol 272 (6) ◽  
pp. H2983-H2988 ◽  
Author(s):  
E. E. Craig ◽  
D. A. Hood
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
In Vitro Transcription ◽  
Heat Shock Protein 60 ◽  
Cardiac Mitochondria ◽  
Age Related ◽  
The Matrix ◽  
Matrix Compartment

This study was undertaken to determine whether age-related changes in the content and composition of cardiac mitochondria could be due, in part, to alterations in mitochondrial protein import. Precursor proteins malate dehydrogenase and ornithine carbamoyltransferase were synthesized by in vitro transcription and translation and were incubated with mitochondria isolated from the hearts of young (4-mo), old (22-mo), and senescent (28-mo) rats. Mitochondria from senescent animals exhibited a twofold higher import rate of both precursors into the matrix compartment compared with mitochondria from young and old animals. The expression of glucose regulated protein 75 and heat shock protein 60, two matrix chaperonins that are essential for import, was elevated in the mitochondria of both old and senescent animals before the observed changes in import. Import was equally affected in senescent and young heart mitochondria by inhibition of cardiolipin, a mitochondrial phospholipid involved in protein translocation. The results indicate that the altered mitochondrial phenotype evident in the aging myocardium cannot be accounted for by reduced rates of protein import. Furthermore, levels of cardiolipin and matrix chaperonins do not appear to be rate-limiting steps in the import process. These data suggest that the protein import step of mitochondrial assembly is subject to adaptations under pathophysiological conditions.

Contractile activity-induced adaptations in the mitochondrial protein import system

1998 ◽  
Vol 274 (5) ◽  
pp. C1380-C1387 ◽  
Author(s):  
Mark Takahashi ◽  
Alan Chesley ◽  
Damien Freyssenet ◽  
David A. Hood
Keyword(s):  
Outer Membrane ◽  
Malate Dehydrogenase ◽  
Mitochondrial Biogenesis ◽  
Protein Import ◽  
Contractile Activity ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
The Matrix ◽  
Stimulating Factor ◽  
Import Receptor

We previously demonstrated that subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondrial subfractions import proteins at different rates. This study was undertaken to investigate 1) whether protein import is altered by chronic contractile activity, which induces mitochondrial biogenesis, and 2) whether these two subfractions adapt similarly. Using electrical stimulation (10 Hz, 3 h/day for 7 and 14 days) to induce contractile activity, we observed that malate dehydrogenase import into the matrix of the SS and IMF mitochondia isolated from stimulated muscle was significantly increased by 1.4- to 1.7-fold, although the pattern of increase differed for each subfraction. This acceleration of import may be mitochondrial compartment specific, since the import of Bcl-2 into the outer membrane was not affected. Contractile activity also modified the mitochondrial content of proteins comprising the import machinery, as evident from increases in the levels of the intramitochondrial chaperone mtHSP70 as well as the outer membrane import receptor Tom20 in SS and IMF mitochondria. Addition of cytosol isolated from stimulated or control muscles to the import reaction resulted in similar twofold increases in the ability of mitochondria to import malate dehydrogenase, despite elevations in the concentration of mitochondrial import-stimulating factor within the cytosol of chronically stimulated muscle. These results suggest that chronic contractile activity modifies the extra- and intramitochondrial environments in a fashion that favors the acceleration of precursor protein import into the matrix of the organelle. This increase in protein import is likely an important adaptation in the overall process of mitochondrial biogenesis.

scholarly journals Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes

2008 ◽  
Vol 8 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Ross F. Waller ◽  
Carole Jabbour ◽  
Nickie C. Chan ◽  
Nermin Celik ◽  
Vladimir A. Likić ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Markov Models ◽  
Mitochondrial Protein ◽  
Atp Synthesis ◽  
Cellular Level ◽  
Outer Membrane Receptor ◽  
Mitochondrial Protein Import ◽  
Severe Reduction ◽  
Close Relatives

ABSTRACT Microsporidia are a group of highly adapted obligate intracellular parasites that are now recognized as close relatives of fungi. Their adaptation to parasitism has resulted in broad and severe reduction at (i) a genomic level by extensive gene loss, gene compaction, and gene shortening; (ii) a biochemical level with the loss of much basic metabolism; and (iii) a cellular level, resulting in lost or cryptic organelles. Consistent with this trend, the mitochondrion is severely reduced, lacking ATP synthesis and other typical functions and apparently containing only a fraction of the proteins of canonical mitochondria. We have investigated the mitochondrial protein import apparatus of this reduced organelle in the microsporidian Encephalitozoon cuniculi and find evidence of reduced and modified machinery. Notably, a putative outer membrane receptor, Tom70, is reduced in length but maintains a conserved structure chiefly consisting of tetratricopeptide repeats. When expressed in Saccharomyces cerevisiae, EcTom70 inserts with the correct topology into the outer membrane of mitochondria but is unable to complement the growth defects of Tom70-deficient yeast. We have scanned genomic data using hidden Markov models for other homologues of import machinery proteins and find evidence of severe reduction of this system.

scholarly journals Residues of Tim44 Involved in both Association with the Translocon of the Inner Mitochondrial Membrane and Regulation of Mitochondrial Hsp70 Tethering

2008 ◽  
Vol 28 (13) ◽  
pp. 4424-4433 ◽  
Author(s):  
Dirk Schiller ◽  
Yu Chin Cheng ◽  
Qinglian Liu ◽  
William Walter ◽  
Elizabeth A. Craig
Keyword(s):  
Amino Acid ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Inner Mitochondrial Membrane ◽  
The Matrix ◽  
Mitochondrial Hsp70 ◽  
Amino Acid Segment

ABSTRACT Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.

scholarly journals Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

2016 ◽  
Vol 113 (31) ◽  
pp. E4467-E4475 ◽  
Author(s):  
Sandro Käser ◽  
Silke Oeljeklaus ◽  
Jiří Týč ◽  
Sue Vaughan ◽  
Bettina Warscheid ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
In Vitro Assays ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Genomes ◽  
Dual Function ◽  
Complex Assembly

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

scholarly journals Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria

2006 ◽  
Vol 174 (5) ◽  
pp. 631-637 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
Daigo Ishikawa ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Protein Import ◽  
The Matrix

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix

1996 ◽  
Vol 109 (12) ◽  
pp. 2895-2904 ◽  
Author(s):  
T. Sasaki ◽  
H. Wiedemann ◽  
M. Matzner ◽  
M.L. Chu ◽  
R. Timpl
Keyword(s):  
Matrix Protein ◽  
Human Fibroblasts ◽  
Extracellular Matrix Protein ◽  
In Vitro Assays ◽  
Collagen Vi ◽  
Immunogold Staining ◽  
The Matrix ◽  
Immunological Assays

The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with cross-striated fibrils of collagen I and collagen VI microfibrils.

scholarly journals Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

1994 ◽  
Vol 5 (4) ◽  
pp. 465-474 ◽  
Author(s):  
C Wachter ◽  
G Schatz ◽  
B S Glick
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Inner Membrane ◽  
In Vitro System ◽  
Mitochondrial Inner Membrane ◽  
Precursor Proteins ◽  
The Matrix

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.

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