The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F₁F_o‐ATP synthase

Abstract

Mitochondrial F1Fo-ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear- and mitochondria-encoded subunits. Whereas chaperones for formation of the matrix-exposed hexameric F1-ATPase core domain have been identified, insight into how the nuclear-encoded F1-domain assembles with the membrane-embedded Fo-region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F1-module and peripheral stalk, but not with the assembled F1Fo-ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F1-module to the membrane embedded Fo-domain. We conclude that INAC represents a matrix-exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F1Fo-ATP synthase.

Assembly of F1F0-ATP synthases

“…In addition, several lineage-specific assembly factors have evolved. For instance, the Ina17/Ina22 complex and Fmc1p are only found in fungi [141,170]. Moreover, ALB4 seems to have functionally diversified from a preexisting factor (ALB3) after a gene duplication event, to be recruited in the assembly of the chloroplast ATP synthase [142].…”

“…These results together imply that INAC mediates stator assembly and promotes the linkage of F 1 and F 0 . Indeed, the discovery of INAC suggests the existence of an alternative stator subunit incorporation pathway: in contrast to the recently discovered a8-Atp10p-stator intermediate [117], Lytovchenko et al (2014) [141] postulate the existence of a precursor comprising the complete F 1 module and the stator subunits.…”

“…INAC associates with several F 1 and peripheral stator subunits, but is not a component of the fully assembled ATP synthase. In the absence of either Ina22 or Ina17, a larger fraction of the F 1 portion remains unconnected to the F 0 portion and the peripheral stalk subunits OSCP (related to bacterial δ) and b fail to assemble properly [141]. Nevertheless, ina22 and ina17 strains were still capable of forming fully assembled ATP synthases.…”

“…Late steps in the assembly of the mitochondrial ATP synthase are mediated by the inner membrane assembly complex INAC [141]. INAC is composed of the two transmembrane proteins Ina22 and Ina17, which are found exclusively in the fungal kingdom.…”

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria

“…Gel lanes were cut into 12 equal slices, and proteins were processed for LC-MS analysis including reduction of disulfide bonds, alkylation of free thiol groups, and tryptic digestion, as described previously (44). Proteins coimmunoprecipitated with HA-tagged pATOM36 and Myc-tagged TAC65 were acetone precipitated, reduced, alkylated, and tryptically digested in solution as described before (45).…”