V.A. Grinkevich

Amino acid sequence of the oligomycin sensitivity-conferring protein (OSCP) of beef-heart mitochondria and its homology with the δ-subunit of the F1-ATPase of Escherichia coli

The complete amino acid sequence of the oligomycin sensitivity‐conferring protein (OSCP) of beef‐heart mitochondria is reported. The protein contains 190 amino acids and has a molecular mass of 20 967. Its structure is characterized by a concentration of charged amino acids in the two terminal segments (N 1–77 and C 128–190) of the protein, whereas its central region is more hydrophobic. The earlier reported homology of the protein with the δ‐subunit of E. coli F1, based on the terminal amino acid sequences of OSCP, is further substantiated.

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1β subunit of the ATP synthase (pF1β) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1β with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1β into intact mitochondria. Import of pF1β through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.

Electron microscopy of two-dimensional crystals of mitochondrial ATP synthase

Two‐dimensional crystals of the mitochondrial ATP synthase up to 0.4 μm in size were obtained from the detergent‐lipid‐protein micelles by detergent dialysis. A projected map of the negatively stained crystal was calculated from electron microscopical images by the Fourier‐filtering procedure at about 2.8 nm resolution. The unit cell (with not more than two ATP synthase molecules) has the following parameters: a = 13.0 nm, b = 25.6 nm and γ = 86°. Two alternative models for the crystal structural organization were suggested, viz. with one or two protein molecules per unit cell.

Proteomic analysis of heart mitochondria from Bos taurus: I. Application of proteomic methods to identification of transmembrane domains of proteins of the internal mitochondrial membrane

This study is part of a large-scale investigation of the proteome of mitochondria from the heart muscle of Bos taurus. We developed a special approach to simplification of the protein mixture by separation of mitochondrial fractions with stable protein compositions. At the first stage of this approach, we isolated and purified internal mitochondrial membranes. The protein composition of this fraction was analyzed by the following proteomic methods: enzymatic or/and chemical cleavage of the proteins, chromatographic fractionation of the complex mixture of the resulting peptides, mass-spectrometric identification of these peptides, and a search for proteins in databases of amino acid sequences. We reliably identified 147 unique proteins with the use of the SwissProt database. The subcellular location and functions of these proteins were analyzed. Approaches to studies of transmembrane domains of integral membrane proteins of the internal mitochondrial membrane were proposed on the basis of proteomic methods of analysis. Considerable coincidence of the experimental data with the results of determination of the 3D structures of the proteins by X-ray analysis was shown.

Proteomic analysis of mitochondria from the Bos taurus heart. II. Identification of mitochondrial soluble proteins

A fraction of the so-called mitochondrial soluble proteins was obtained after the destruction of purified mitochondria by sonication according to the previously found approach to the identification of protein subsets of the Bos taurus heart proteome. A tryptic destruction of these proteins was achieved. Approximately half of the tryptic hydrolysate was separated into two fractions of cysteine-containing and cysteine-free peptides by covalent chromatography on Thiopropyl Sepharose 4B. The cysteine-containing peptides were modified by iodoacetamide. The peptides were mass-spectrometrically identified in all the three fractions of tryptic hydrolysate, and the proteins were searched for in the amino acid sequence databases. There were 213 unique proteins reliably identified.

Mitochondrial H+-ATPase: Identification of Subunits of the F0 Subcomplex that Contact Membrane Lipids

The subunits of the F0 membrane sector of bovine heart mitochondrial H+-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H+-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-14C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([ 125I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F0 membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.

Proteomic analysis of mitochondria from the heart of Bos taurus . III. Identification of proteins of the inner mitochondrial membrane

Proteins of the inner mitochondrial membrane packed into submitochondrial particles (SMP) have been investigated. SMPs were treated with trypsin, and the peptides were separated from the so-called “shaved vesicles”. The “shaved vesicles” were disrupted, and the proteins and peptides obtained were subjected to cleavage by cyanogen bromide and trypsin. The two groups of tryptic peptides obtained were analyzed separately using proteomic methods, namely, chromatographic fractionation of peptides, mass spectrometric identification and a search in amino acid sequence databases. The possibility of non-specific fragmentation was also taken into account when identification of proteins of the inner mitochondrial membrane was performed. Reliable identification of 298 proteins allowed for a more precise estimation of their localization in the cell and analysis of their function.