scholarly journals Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

1988 ◽  
Vol 256 (2) ◽  
pp. 529-535 ◽  
Author(s):  
S D Patel ◽  
M W J Cleeter ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Submitochondrial Particles ◽  
Mitochondrial Inner Membrane ◽  
Cytoplasmic Face ◽  
The Matrix ◽  
Dimethyl Suberimidate ◽  
Membrane Reaction ◽  
Cytoplasmic Side ◽  
Chemical Cross Linking

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.

scholarly journals The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane

1980 ◽  
Vol 185 (2) ◽  
pp. 315-326 ◽  
Author(s):  
S Smith ◽  
C I Ragan
Keyword(s):  
Complex I ◽  
Mitochondrial Membrane ◽  
Nadh Dehydrogenase ◽  
Inner Mitochondrial Membrane ◽  
Submitochondrial Particles ◽  
Cytoplasmic Face ◽  
The Matrix

The organization of the constituent polypeptides of mitochondrial NADH dehydrogenase was studied by using two membrane-impermeable probes, diazobenzene[35S]sulphonate and lactoperoxidase-catalysed radioiodination. The incorporation of label into the subunits of the isolated enzyme was compared with that obtained with enzyme immunoprecipitated from labelled mitochondria or inverted submitochondrial particles. On the basis of accessibility to these two labels, we divide the polypeptides of Complex I into five groups: those that are apparently buried in the enzyme, those that are accessible to labelling in the isolated enzyme but not in the membrane, those that are exposed on the cytoplasmic face of the membrane, those that are exposed on the matrix face and finally those that are exposed on both faces and are therefore transmembranous. We conclude that NADH dehydrogenase is asymmetrically organized across the inner mitochondrial membrane.

scholarly journals Structural studies on mitochondrial NADH dehydrogenase using chemical cross-linking

1988 ◽  
Vol 256 (2) ◽  
pp. 521-528 ◽  
Author(s):  
S D Patel ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Membrane Lipid ◽  
Protein Domain ◽  
Cross Linking ◽  
Hydrophobic Domain ◽  
Iron Protein ◽  
Dimethyl Suberimidate ◽  
Cross Links ◽  
Monospecific Antisera ◽  
Chemical Cross Linking

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by cross-linking constituent subunits with disuccinimidyl tartrate, (ethylene glycol)yl bis(succinimidyl succinate) and dimethyl suberimidate. Cross-linked products were identified by Western blotting with monospecific antisera to nine subunits of the enzyme. Cross-links between subunits within the flavoprotein, iron-protein and hydrophobic domains of the enzyme were identified. Cross-linking between the 75 kDa iron-protein-domain subunit and the 51 kDa flavoprotein-domain subunit was modulated by the substrate NADH. Cross-linking of subunits of the iron-protein and flavoprotein domains to constituents of the hydrophobic domain was also found. This was further substantiated by photolabelling subunits of the latter region, which were in contact with the membrane lipid, with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. One such subunit of Mr 19,000 could be cross-linked to components of the iron-protein domain.

Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

1986 ◽  
Vol 64 (3) ◽  
pp. 229-237
Author(s):  
Nobuhito Sone ◽  
Cynthia Hou ◽  
Philip D. Bragg
Keyword(s):  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Thermophilic Bacterium ◽  
Cross Linking ◽  
Dimethyl Suberimidate ◽  
The Cross ◽  
Third Dimension ◽  
Chemical Cross Linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

Calcium Ion Binding Within Fibrinogen Fragment D

1981 ◽  
Author(s):  
David W Britton ◽  
Jan S Lawrie ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Cross Linking ◽  
C Terminus ◽  
Dimethyl Suberimidate ◽  
High Concentrations ◽  
The Stability ◽  
Considerable Body ◽  
Chemical Cross Linking ◽  
Compact Conformation

Fibrinogen contains a number of strongly bound calcium. ions and a considerable body of evidence new exists to show that the plasmin degradation product fragment D contains one strongly bound calciumion. It is also established that this calcium ion has a notable effect on the plasmin resistance of the molecule. Previous work from this laboratory strongly suggests that the binding site is located towards the C-terminus of the γ chain. We have also investigated the influence of calcium. ions on the conformation of fragment D by ultracentrifugation and chemical cross-linking. In the presence of calcium ions there is a preponderance of intra-molecular cross-linking even at high concentrations of fragment D using bisimidates such as dimethyl suberimidate and dimethyl adipimidate. In the absence of calcium. ions there is an increase in the extent of inter-molecular cross-linking. From such evidence we would propose that calcium ions stabilise a compact conformation within fragment D. The presence of calcium ions also affects the stability of the D:E complex.

scholarly journals Structural organization of mitochondrial human complex I: role of the ND4 and ND5 mitochondria-encoded subunits and interaction with prohibitin

2004 ◽  
Vol 383 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Ingrid BOURGES ◽  
Claire RAMUS ◽  
Bénédicte MOUSSON de CAMARET ◽  
Réjane BEUGNOT ◽  
Claire REMACLE ◽  
...  
Keyword(s):  
Complex I ◽  
Nadh Dehydrogenase ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Membrane Association ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Complex I Assembly ◽  
Human Complex

Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho° cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

scholarly journals A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

1984 ◽  
Vol 99 (1) ◽  
pp. 188-198 ◽  
Author(s):  
N G Kravit ◽  
C S Regula ◽  
R D Berlin
Keyword(s):  
Molecular Weight ◽  
Room Temperature ◽  
Molecular Form ◽  
Cross Linking ◽  
Electrophoretic Patterns ◽  
Minority Species ◽  
Associated Proteins ◽  
Dimethyl Suberimidate ◽  
Quantitative Examination ◽  
Chemical Cross Linking

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Aggregation of submitochondrial particles by heparin and its application to the study of carnitine transport

1986 ◽  
Vol 235 (1) ◽  
pp. 297-299 ◽  
Author(s):  
R R Ramsay ◽  
P K Tubbs
Keyword(s):  
External Medium ◽  
Competitive Inhibitor ◽  
Submitochondrial Particles ◽  
Mitochondrial Inner Membrane ◽  
Transport Studies ◽  
Low Concentrations ◽  
The Matrix ◽  
Carnitine Transport ◽  
Km Value ◽  
Acylcarnitine Translocase

A novel technique for the separation of submitochondrial particles from the external medium, an essential procedure in transport studies, was devised. Very low concentrations of heparin (5-10 micrograms/ml) aggregate the particles and permit their rapid sedimentation in a micro-centrifuge. The transfer of activated fatty acids into mitochondria for oxidation depends on the exchange of matrix carnitine for external fatty-acylcarnitine. To study the matrix face of the carnitine/acylcarnitine translocase, inverted submitochondrial particles were prepared and loaded with L-[14C]carnitine. As found in intact mitochondria, the Km value for L-carnitine was 8 mM, that for palmitoyl-L-carnitine was two orders of magnitude lower, and 11-trimethylaminoundecanoyl-DL-carnitine was a competitive inhibitor. The properties of the carrier exposed to the outer and to the matrix sides of the mitochondrial inner membrane are thus similar.

scholarly journals Chemical cross-linking of mitochondrial NADH dehydrogenase from bovine heart

1985 ◽  
Vol 227 (2) ◽  
pp. 467-474 ◽  
Author(s):  
M W J Cleeter ◽  
S H Banister ◽  
C I Ragan
Keyword(s):  
Ethylene Glycol ◽  
Active Site ◽  
Protein Fraction ◽  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Iron Protein ◽  
Bovine Heart ◽  
Cross Links ◽  
Hydrophobic Shell ◽  
Chemical Cross Linking

The structure of bovine heart mitochondrial NADH dehydrogenase was investigated by using two cleavable cross-linking agents, disuccinimidyl tartrate and (ethylene glycol)yl bis-(succinimidyl succinate). Cross-linking was analysed primarily by immunoblotting to detect products containing subunits of the iron-protein fraction from chaotropic resolution of the enzyme, namely those of 75, 49, 30 and 13 kDa. By using both the isolated iron-protein fraction and the intact dehydrogenase, cross-links were identified between these four subunits, from these subunits to the largest subunit of the flavoprotein fraction, which contains the active site for NADH, and from these subunits to polypeptides in the hydrophobic shell, which surrounds the hydrophilic iron-protein and flavoprotein fractions.

scholarly journals Mapping protein interactions in the active TOM-TIM23 supercomplex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ridhima Gomkale ◽  
Andreas Linden ◽  
Piotr Neumann ◽  
Alexander Benjamin Schendzielorz ◽  
Stefan Stoldt ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Matrix Protein ◽  
Protein Transport ◽  
Mitochondrial Proteins ◽  
Cross Linking ◽  
Intermembrane Space ◽  
Precursor Proteins ◽  
The Matrix ◽  
Targeting Signals ◽  
Chemical Cross Linking

AbstractNuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

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.

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