Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues

Abstract

We have used site-directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C-terminus of Y8 by site-directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol-specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N-terminus of Y8 is located in the intermembrane space of mitochondria whereas the C-terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross-linking reagents, each with one thiol-specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C-terminal domain of Y8 to subunits d and f, and that of the N-terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross-links, that Y8 may also be part of the stator stalk.

The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification

“…Construction of Y8 Cysteine Replacements-The gene cassettes N9L-D/Y8-1 and N9L-D/Y8-1-FLAG, yeast expression vector pPD72, and yeast strains YM2 and FTC2 were as described previously (37). Briefly, each gene cassette encodes a full-length wild-type Y8 protein bearing a 7-amino acid extension at the N terminus (YSSEISS, numbered from Ϫ7 to Ϫ1), which is retained following import of Y8 into mitochondria and processing of the N9L mitochondrial import sequence by matrix protease.…”

“…Site-directed mutagenesis was carried out on the N9L-D/Y8-1 gene cassette as described (37) to generate 46 single cysteine replacements in the expressed Y8 proteins. The Y8(M1C) FLAG variant was constructed by ligation of a BamHI/KpnI fragment containing the N9L-D sequence and the first 15 nucleotides of the synthetic Y8 gene bearing the M1C mutation into a similarly digested pUC9 vector containing the FTC2 variant.…”

“…The Y8(M1C) FLAG variant was constructed by ligation of a BamHI/KpnI fragment containing the N9L-D sequence and the first 15 nucleotides of the synthetic Y8 gene bearing the M1C mutation into a similarly digested pUC9 vector containing the FTC2 variant. The (M1C) FLAG cassette was then excised by BamHI/NotI restriction digestion and ligated into the pPD72 vector as described (37).…”

“…In a previous study, analysis of allotopically expressed Y8 variants having a cysteine substitution at the N-or C-terminal residue of Y8 demonstrated that the CHD spans the inner mitochondrial membrane with N out -C in orientation (37). Here we have combined cysteine-scanning mutagenesis with allotopic expression to construct a series of single cysteine replacements encompassing the entire length of the Y8 protein (excluding three residues within the C-terminal positively charged region whose replacement would abrogate function (28)).…”

Lack of Mitochondrial Anionic Phospholipids Causes an Inhibition of Translation of Protein Components of the Electron Transport Chain

“…Proteolipid isolation of ATPase subunits was accomplished as described (49) by organic extraction of sonicated highly purified mitochondria. ATP6p, ATP8p, and ATP9p were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining.…”