Subunit 4 of ATP synthase (F₀F₁) from yeast mitochondria

Abstract

One subunit of the membrane portion of yeast ATP synthase was purified. Structural data are reported. This subunit (subunit 4) is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its apparent relative molecular mass is about 25 000. The polypeptide was extracted from the complex with a mixture of chloroform/methanol (l/l) and 0.5 M pyridinium acetate pH 6.0. Purification was performed with a combination of gel permeation chromatography on Sephadex G-75 and high-performance gel permeation chromatography with aqueous solvents containing 5% sodium dodecyl sulfate. The amino acid composition is reported here. The following sequence of the NH,-terminal ten residues was determined: Met-Ser--.The ATP synthase of the inner mitochondria1 membrane can be divided into two portions: the F1 portion which contains the catalytic site for the synthesis and hydrolysis of ATP, and the Fo portion which acts as a proton channel. In yeast, the water-soluble F1 consists of five non-identical subunits (a$,y,6,~) [l, 21 which are imported from the cytoplasm. The membrane-associated Fo is less well defined but it has been shown to contain three mitochondrially synthesized proteins (subunits 6, 8 and 9) [3-81. In addition, ATP synthase also contains three other cytoplasmically synthesized proteins with apparent relative molecular masses of 25 000, 21 000 and 19000. Whatever the method of preparation used, these subunits copurify with the enzyme complex [l, 9, 101 and therefore, it is highly probable that they are genuine subunits of the enzyme complex. In view of the following points, the 25-kDa subunits 4 may be considered as a real subunit of the ATP synthase. a) Two copies of subunit 4 were estimated per enzyme complex [9]. b) Subunit 4 was chemically cross-linked to subunits CI, p and 9 [Ill. c) Subunit 4 was coprecipitated with the complex by monoclonal antibodies raised against whole ATP synthase or subunit p [12, 131. d) A mutational defect in the Oli 2 gene product (subunit 6) results in a deficiency in a 25-kDa polypeptide of the membrane factor [14].e) Torok and Joshi [15] have reported that Copper-ophenanthroline treatment of ATP synthase of beef heart mitochondria results in cross-linking of a 24-25-kDa protein to oligomycin-sensitivity-conferring protein (21 kDa). These

The role of the stalk in the coupling mechanism of F1F0‐ATPases

“…The remainder of its sequence is highly charged and is capable of making important interactions either with other subunits in the stalk or, as in the analogous subunit b in the bacterial F,F,-ATPases, with F, subunits. Homologues of bovine b are present in the rat [29] and yeast [30] mitochondrial enzymes, and a human homologue has also been sequenced [31]. Subunit d has no extensive hydrophobic regions in its sequence [28], and, like F,, it is probably attached to the matrix surface of the inner mitochondrial membrane.…”

ATP4, the structural gene for yeast F0F1 ATPase subunit 4

“…The start of the mature subunit 4 was identified by matching the amino acid sequence predicted from the DNA sequence with the sequence of the NH2-terminal ten residues. Thus the mature subunit 4 consists of 209 amino acid residues with a mass of 23250 Da, which is slightly smaller than the 25 kDa determined by SDS gel electrophoresis [14]. Some discrepancies between the previous reported amino acid composition [14] and the predicted composition from the gene product occurred in the amount of acidic residues.…”

“…The sequence of the NH2-terminal ten residues was reported previously [14]. Using this sequence, we built a probe of mixed oligonuleotides (Fig.…”

“…In this respect, it is of interest to perform detailed structural analysis of these polypeptides. To this end, we have previously isolated the subunit 4 (molecular mass 25 kDa) [14] which is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass as proposed by Todd et al [12]. Subunit 4 could be located at the Fo-FI interface since cross-linkings were performed between this subunit and F1 M and fi subunits, Fo subunit 9 [I 51 and the oligomycin-sensitivity-confering protein (OSCP) [16].…”

“…In this respect, it is of interest to perform detailed structural analysis of these polypeptides. To this end, we have previously isolated the subunit 4 (molecular mass 25 kDa) [14] which is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass as proposed by Todd et al [12]. Subunit …”