scholarly journals Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting

1991 ◽  
Vol 273 (3) ◽  
pp. 719-724 ◽  
Author(s):  
G H D Clarkson ◽  
J Neagle ◽  
J G Lindsay
Keyword(s):  
Succinate Dehydrogenase ◽  
Large Subunit ◽  
Limited Proteolysis ◽  
Proteinase K ◽  
Similar Data ◽  
Submitochondrial Particles ◽  
Hydrophobic Membrane ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex.

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.

Removal of a hydrophobic domain within the mature portion of a mitochondrial inner membrane protein causes its mislocalization to the matrix

1990 ◽  
Vol 10 (5) ◽  
pp. 1873-1881
Author(s):  
S M Glaser ◽  
B R Miller ◽  
M G Cumsky
Keyword(s):  
Amino Acids ◽  
Mitochondrial Matrix ◽  
Mature Protein ◽  
Wild Type ◽  
Inner Membrane ◽  
Hydrophobic Domain ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Inner Membrane Protein ◽  
Cognate Protein

We have examined the import and intramitochondrial localization of the precursor to yeast cytochrome c oxidase subunit Va, a protein of the mitochondrial inner membrane. The results of studies on the import of subunit Va derivatives carrying altered presequences suggest that the uptake of this protein is highly efficient. We found that a presequence of only 5 amino acids (Met-Leu-Ser-Leu-Arg) could direct the import and localization of subunit Va with wild-type efficiency, as judged by several different assays. We also found that subunit Va could be effectively targeted to the mitochondrial inner membrane with a heterologous presequence that failed to direct import of its cognate protein. The results presented here confirmed those of an earlier study and showed clearly that the information required to "sort" subunit Va to the inner membrane resides in the mature protein sequence, not within the presequence per se. We present additional evidence that the aforementioned sorting information is contained, at least in part, in a hydrophobic stretch of 22 amino acids residing within the C-terminal third of the protein. Removal of this domain caused subunit Va to be mislocalized to the mitochondrial matrix.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

2001 ◽  
Vol 114 (21) ◽  
pp. 3915-3921 ◽  
Author(s):  
Stefan J. Kerscher ◽  
Andrea Eschemann ◽  
Pamela M. Okun ◽  
Ulrich Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Functional Expression ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Cytosolic Side ◽  
Respiratory Membrane

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

scholarly journals Calcium-dependent opening of a non-specific pore in the mitochondrial inner membrane is inhibited at pH values below 7. Implications for the protective effect of low pH against chemical and hypoxic cell damage

1991 ◽  
Vol 278 (3) ◽  
pp. 715-719 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Protective Effect ◽  
Adenine Nucleotide ◽  
Cell Damage ◽  
Liver Mitochondria ◽  
Low Ph ◽  
Hypoxic Cell ◽  
Inner Membrane ◽  
Calcium Dependent ◽  
Mitochondrial Inner Membrane ◽  
The Matrix

1. The rate of opening of the Ca(2+)-induced non-specific, cyclosporin A-inhibited, pore of the mitochondrial inner membrane of rat heart and liver mitochondria at pH 6.0 was less than 10% of that at pH 7.4. 2. The effect could not be explained by inhibition of Ca2+ uptake into the mitochondria, or of the matrix peptidyl-prolyl cis-trans isomerase (PPIase), or of the Ca(2+)-induced conformational change of the adenine nucleotide translocase. 3. It is suggested that the proposed interaction of matrix PPIase with the ‘c’ conformation of the adenine nucleotide carrier in the presence of Ca2+ [Griffiths & Halestrap (1991) Biochem. J. 274, 611-614] is inhibited by low pH. 4. The relevance of this to the protective effect of low pH on hypoxic and chemical-induced cell damage is discussed.

AAA proteases of mitochondria: quality control of membrane proteins and regulatory functions during mitochondrial biogenesis

2001 ◽  
Vol 29 (4) ◽  
pp. 431-436 ◽  
Author(s):  
T. Langer ◽  
M. Käser ◽  
C. Klanner ◽  
K. Leonhard
Keyword(s):  
Quality Control ◽  
Membrane Proteins ◽  
Matrix Space ◽  
Inner Membrane ◽  
Membrane Surfaces ◽  
Mitochondrial Inner Membrane ◽  
Catalytic Sites ◽  
The Matrix ◽  
The Stability ◽  
Regulatory Functions

An ubiquitous and conserved proteolytic system regulates the stability of mitochondrial inner membrane proteins. Two AAA proteases with catalytic sites at opposite membrane surfaces form a membrane-integrated quality control system and exert crucial functions during the biogenesis of mitochondria. Their activity is modulated by another membrane-protein complex that is composed of prohibitins. Peptides generated upon proteolysis in the matrix space are transported across the inner membrane by an ATP-binding cassette transporter. The function of these conserved components is discussed in the present review.

scholarly journals Cholesterol increase in mitochondria: its effect on inner-membrane functions, submitochondrial localization and ultrastructural morphology

1993 ◽  
Vol 289 (3) ◽  
pp. 703-708 ◽  
Author(s):  
S Echegoyen ◽  
E B Oliva ◽  
J Sepulveda ◽  
J C Díaz-Zagoya ◽  
M T Espinosa-García ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Atpase Activity ◽  
Succinate Dehydrogenase ◽  
Outer Membrane ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Inner Membrane ◽  
Ultrastructural Morphology ◽  
Mitochondrial Inner Membrane

The effect of cholesterol incorporation on some functions of the mitochondrial inner membrane and on the morphology of rat liver mitochondria was studied. Basal ATPase and succinate dehydrogenase activities remained unchanged after cholesterol was incorporated into the mitochondria; however, uncoupled ATPase activity was partially inhibited. The presence of several substrates and inhibitors did not change the amount of cholesterol incorporated, which was localized mostly in the outer membrane. Electron-microscope observations revealed the presence of vesicles between the outer and inner membranes; these vesicles increased in number with the amount of cholesterol incorporated. The data suggest that cholesterol induces the formation of vesicles from the outer membrane, and modifies the activity of stimulated ATPase.

scholarly journals d-Lactate transport and metabolism in rat liver mitochondria

2002 ◽  
Vol 365 (2) ◽  
pp. 391-403 ◽  
Author(s):  
Lidia de BARI ◽  
Anna ATLANTE ◽  
Nicoletta GUARAGNELLA ◽  
Giovanni PRINCIPATO ◽  
Salvatore PASSARELLA
Keyword(s):  
Lactate Dehydrogenase ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Lactate Metabolism ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Inside Out

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize d-lactate. We found the following: (1) externally added d-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation) = 2] and membrane potential (Δψ) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by α-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (d-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate d-lactate traffic across the mitochondrial inner membrane: the d-lactate/H+ symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the d-lactate/oxoacid antiporter (which mediates both the d-lactate/pyruvate and d-lactate/oxaloacetate exchanges) and d-lactate/malate antiporter studied by monitoring photometrically the appearance of the d-lactate counteranions outside mitochondria. The d-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the Vmax values and in the inhibition and pH profiles and were shown to regulate mitochondrial d-lactate metabolism in vitro. The d-lactate translocators and the d-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for d-lactate-dependent gluconeogenesis.

scholarly journals The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Heike Rampelt ◽  
Iva Sucec ◽  
Beate Bersch ◽  
Patrick Horten ◽  
Inge Perschil ◽  
...  
Keyword(s):  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Transmembrane Segments ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
The Matrix ◽  
Mitochondrial Carrier Family ◽  
Mitochondrial Intermembrane Space

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

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