The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane

Abstract

A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

“…The e-and g-subunits are found in the curved region of the micelle, indicating a role in producing the curvature. This role is consistent with experiments showing that onion-like mitochondria without sharp bends in their inner membranes form in yeast on deletion of subunit e (22) or g (25) or mutation of subunits e, g, or b to cause loss of subunit g from the complex (24,(39)(40)(41). The observed curvature suggests that the monomeric enzyme in isolation would produce an unusual membrane topology, which would be under strain in a planar lipid bilayer.…”

The Modification of the Conserved GXXXG Motif of the Membrane-spanning Segment of Subunit g Destabilizes the Supramolecular Species of Yeast ATP Synthase

“…In addition, a spontaneous conversion into rho Ϫ cells was observed. As reported previously (15,17), there is a correlation between the increase in the spontaneous rho Ϫ cell conversion (rho Ϫ cells that are devoid of oxidative phosphorylation are unable to grow with lactate as carbon source) and the increase in the generation time of mutant strains. It is also possible to correlate the increase in the spontaneous rho Ϫ cell conversion with the increase in the insensitivity of the mitochondrial ATPase activity of the five mutants toward oligomycin (an inhibitor of membranous domain of the mitochondrial ATP synthase) under the experimental conditions used (pH 8.4 and Triton X-100) ( Table I).…”

“…Two complementary oligonucleotides, 5Ј-TATAAACATCACCACCACCACCACCACCACTA-AGCTTTT-3Ј and 5Ј-GAATTAAAAAGCTTAGTGGTGGTGGTGGTGG-TGGTGATG-3Ј, were used to introduce the (His) 6 sequence into the C terminus of subunit gC75S/L109C by the PCR mutagenesis procedure. The point mutant eC28S was constructed by integration of the mutated version of the TIM11 gene at the chromosomic locus in the deleteddisrupted yeast strain (17). All strains containing the subunit eC28S and different mutations in subunit g were constructed by integration of the mutated versions of the ATP20 gene at the chromosomic locus in the ⌬g/eC28S yeast strain.…”

“…The presence of a cysteine residue (Cys 28 ) placed after the membrane domain of subunit e made it possible to establish, by cross-linking experiments, that two subunits e are close to each other in the membrane. This disulfide bond was shown to significantly stabilize ATP synthase dimerization/oligomerization in intact mitochondria (17). The subunit e also contains a putative coiled-coil region in its C-terminal part, which is involved in the stabilization of the dimeric forms of the detergentsolubilized ATP synthase complexes (18).…”

Conserved transmembrane glycine residues in the Shigella flexneri polysaccharide co-polymerase protein WzzB influence protein–protein interactions

“…This motif also exists in the TM2 region of WzzB SF (at aa positions 305-309) and might also facilitate TM interactions among different monomers of Wzz. The importance of tightly packed TM region GXXXG motifs in oligomer formation has also been previously suggested (Arselin et al, 2003;Jenei et al, 2009;Overton et al, 2003), and if this motif is similarly responsible for establishing or stabilizing oligomeric Wzz structures, then it is likely that the G-to-A substitutions in the TM2 region have undermined this process. It remains plausible that the changes to G305 and G311 have resulted in Wzz G305A/G311A not being able to interact with wild-type, or preferentially interacting with its own monomers, and thus resulting in a separate Oag-processingassembly mechanism.…”