Extra Subunits Research Articles

Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria.

3D Gel Map of Arabidopsis Complex I

Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana.

L-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Proteomic approach to characterize mitochondrial complex I from plants

Mitochondrial NADH dehydrogenase complex (complex I) is by far the largest protein complex of the respiratory chain. It is best characterized for bovine mitochondria and known to consist of 45 different subunits in this species. Proteomic analyses recently allowed for the first time to systematically explore complex I from plants. The enzyme is especially large and includes numerous extra subunits. Upon subunit separation by various gel electrophoresis procedures and protein identifications by mass spectrometry, overall 47 distinct types of proteins were found to form part of Arabidopsis complex I. An additional subunit, ND4L, is present but could not be detected by the procedures employed due to its extreme biochemical properties. Seven of the 48 subunits occur in pairs of isoforms, six of which were experimentally proven. Fifteen subunits of complex I from Arabidopsis are specific for plants. Some of these resemble enzymes of known functions, e.g. carbonic anhydrases and l-galactono-1,4-lactone dehydrogenase (GLDH), which catalyzes the last step of ascorbate biosynthesis. This article aims to review proteomic data on the protein composition of complex I in plants. Furthermore, a proteomic re-evaluation on its protein constituents is presented.

Reversibility of exercise-induced translocation of Na+-K+ pump subunits to the plasma membrane in rat skeletal muscle.

Exercise-induced translocation of Na+-K+ pump subunits to the sarcolemmal membrane was studied using sarcolemmal giant vesicles as a membrane purification procedure. The subunit content was quantified by Western blotting or by ouabain labeling. Low-intensity treadmill running increased (P<0.01) the alpha1, alpha2, beta1, and beta2 subunit contents by 19-32% in membranes from oxidative muscle fibers and the alpha1, alpha2, and beta2 contents increased by 13-25% in membranes from glycolytic muscle fibers. Ouabain labeling of membranes from mixed fibers was increased by 29% after exercise. A similar increase in subunit content could be induced by 5 min of fatiguing, high-intensity electrical stimulation of isolated soleus muscles. An increased subunit content was just detectable in vesicles produced 30 min after exercise, and the content was completely back to control levels 3 h after exercise. It is concluded that both low-intensity long-lasting running and short-lasting high-intensity contractions are able to induce a translocation of pump subunits to the sarcolemmal membrane. The post-exercise disappearance of the extra subunits (half-time approximately 20 min) from the membrane demonstrates the reversible nature of the translocation process.

A Reductase/Isomerase Subunit of Mitochondrial NADH:Ubiquinone Oxidoreductase (complex I) Carries an NADPH and is Involved in the Biogenesis of the Complex

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.

Direction selectivity of synaptic potentials in simple cells of the cat visual cortex.

Direction selectivity of synaptic potentials in simple cells of the cat visual cortex. J. Neurophysiol. 78: 2772-2789, 1997. The direction selectivity of simple cells in the visual cortex is generated at least in part by nonlinear mechanisms. If a neuron were spatially linear, its responses to moving stimuli could be predicted accurately from linear combinations of its responses to stationary stimuli presented at different positions within the receptive field. In extracellular recordings, this has not been found to be the case. Although the extracellular experiments demonstrate the presence of a nonlinearity, the cellular process underlying the nonlinearity, whether an early synaptic mechanism such as a shunting inhibition or simply the spike threshold at the output, is not known. To differentiate between these possibilities, we have recorded intracellularly from simple cells of the intact cat with the whole cell patch technique. A linear model of direction selectivity was used to analyze the synaptic potentials evoked by stationary sine-wave gratings. The model predicted the responses of cells to moving gratings with considerable accuracy. The degree of direction selectivity and the time course of the responses to moving gratings were both well matched by the model. The direction selectivity of the synaptic potentials was considerably smaller than that of the intracellularly recorded action potential, indicating that a nonlinear mechanism such as threshold enhances the direction selectivity of the cell's output over that of its synaptic inputs. At the input stage, however, the cells apparently sum their synaptic inputs in a highly linear fashion. A more constrained test of linearity of synaptic summation based on principal component analysis was applied to the responses of direction-selective cells to stationary gratings. The analysis confirms that the summation in these cells is highly linear. The principal component analysis is consistent with a model in which direction selectivity in cortical simple cells is generated by only two subunits, each with a different receptive-field position and response time course. The response time course for each of the two subunits is derived for four analyzed cells. Each derived subunit is linear in spatial summation, suggesting that the neurons that comprise each subunit are either geniculate X-cells or receive their primary synaptic input from X-cells. The amplitude of the response of each subunit is linearly related to the contrast of the stimulus. The subunits are nonlinear in the time domain, however: the response to a stationary stimulus whose contrast is modulated sinusoidally in time is nonsinusoidal. The principal component analysis does not exclude models of direction selectivity based on more than two subunits, but such higher-order models would have to include the constraint that the extra subunits form a smooth continuum of interpolation between the properties derived from the two subunit solution.

Characterization of the respiratory chain from cultured Crithidia fasciculata

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplstids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F 0F 1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F 0F 1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F 0 subunits (b and c) and all of the F 1 subunits. The F 0 b subunit has a low degree of similarity to subunit b from other organisms. The F 1 α subunit is extremely small making the β subunit the largest F 1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

A simple, rapid method for purification of epsilon-subunit, coupling factor 6, subunit d, and subunit e from rat liver H(+)-ATP synthase and determination of the complete amino acid sequence of epsilon-subunit.

The rat liver mitochondrial epsilon-subunit, coupling factor 6, subunit d, and subunit e of H(+)-ATP synthase, which are all extra subunits with no counterparts in Escherichia coli, were purified by reverse-phase high performance liquid chromatography. The complete amino acid sequence of the rat epsilon-subunit was determined by automated Edman degradation of the whole protein and derived peptides. The protein contains 50 amino acids and has a molecular mass of 5635 kDa. It is a basic hydrophilic protein with an isoelectric point of 10.5. The sequence of the rat epsilon-subunit is highly homologous with that of the epsilon-subunit of bovine heart and slightly similar to those of the epsilon-subunit of the yeast and sweet potato mitochondria. However, it has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase.

Interchain Disulfide Bonds in Crotamine Self-Association

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.

The influence of mutations upon the synthesis of RNA polymerase subunits in Escherichia coli cells.

The influence of mutations in structural genes of beta and beta subunits of RNA polymerase upon the synthesis of these subunits in E. coli cells have been investigated. An amber-mutation ts22 in the beta subunit gene decreases the intracellular concentration of this subunit and the rate of its synthesis. At the same time the concentration and the rate of beta subunit synthesis is increased. These suggest the compensatory activation of the RNA polymerase operon that takes place under the conditions of shortage of one of the subunits. Reversions as well as more effective suppression of ts22 amber mutation, achieved by streptomycin addition, substitution of su2 by sul, or by specific mutations, result in a rise of beta and drop of beta subunit concentration and synthesis in ts22 mutant. TsX missense-mutation in the beta subunit gene alters the properties of the enzyme increasing, at the same time, the concentration and the rate of synthesis of both beta and beta subunits, particularly at a nonpermissive temperature. This points to an inversely proportional relationship between the rate of synthesis of RNA polymerase subunits and the total intracellular activity of the enzyme. Extra subunits are rapidly degraded in ts22 and tsX mutants. The whole complex of our data and those of others suggest that the regulation of the synthesis of RNA polymerase subunits is accomplished by interaction of a negative and a positive mechanisms of regulation which include not only activators and repressors but the enzyme itself as well.