Functional dissection of the proton pumping modules of mitochondrial complex I.

Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Two-dimensional native electrophoretic analysis of respiratory supercomplexes from Yarrowia lipolytica.

Mitochondria of the strictly aerobic yeast Yarrowia lipolytica contain respiratory complex I with close functional and structural similarity to the mammalian enzyme. Unlike mammalian mitochondria, however, Yarrowia mitochondria have been thought not to contain supercomplexes. Here, we identify respiratory supercomplexes composed of complexes I, III and IV also in Y. lipolytica. Evidence for dimeric complex I suggests further association of respiratory supercomplexes into respiratory strings or patches. Similar supercomplex organization in Yarrowia and mammalian mitochondria further makes this aerobic yeast a useful model for the human oxidative phosphorylation system. The analysis of supercomplexes and their constituent complexes was made possible by 2-D native electrophoresis, i.e. by using native electrophoresis for both dimensions. Digitonin and blue-native electrophoresis were generally applied for the initial separation of supercomplexes followed by less mild native electrophoresis variants in the second dimension to release the individual complexes from the supercomplexes. Such 2-D native systems are useful means to identify the constituent proteins and their copy numbers in detergent-labile physiological assemblies, since they can reduce the complexity of supramolecular systems to the level of individual complexes.

Small single transmembrane domain (STMD) proteins organize the hydrophobic subunits of large membrane protein complexes.

The large membrane protein complexes of mitochondrial oxidative phosphorylation are composed of central subunits that are essential for their bioenergetic core function and accessory subunits that may assist in regulation, assembly or stabilization. Although sequence conservation is low, a significant proportion of the accessory subunits is characterized by a common single transmembrane (STMD) topology. The STMD signature is also found in subunits of other membrane protein complexes. We hypothesize that the general function of STMD subunits is to organize the hydrophobic subunits of large membrane protein complexes in specialized environments like the inner mitochondrial membrane.

Chapter 8 Two-dimensional native electrophoresis for fluorescent and functional assays of mitochondrial complexes.

Supramolecular assemblies of native membrane protein complexes were solubilized from biological membranes by very mild detergents and isolated by native electrophoresis. The complexity of these higher order structures can be reduced for proteomic investigations by applying less mild native electrophoresis variants in the second dimension. Supercomplexes thereby dissociate into the individual complexes. Clear-native and blue-native electrophoresis variants are useful alternatives for the second native dimension, but clear-native electrophoresis is advantageous for the identification of fluorescence-labeled proteins and for in-gel activity assays that are hampered by Coomassie dye. The 2-D native gels comprising two orthogonal native dimensions are useful to determine the stoichiometry of complexes in supercomplexes. Strips from 2-D native gels can also be used for 3-D SDS-PAGE to identify loosely bound accessory subunits of supercomplexes. The subunit composition of supercomplexes and individual complexes is investigated by 4-D gels. The 4-D protocol starts with isolation of highly pure membrane protein complexes by 2-D native electrophoresis, followed by doubled SDS-PAGE to resolve the subunits.