The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

SummaryQuality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein complexes of the mitochondrial outer membrane. To study if outer membrane interactions and maintenance of cristae morphology are directly coupled, we generated mutant forms of mitofilin/Fcj1 (formation of crista junction protein 1), a core component of MINOS. Mitofilin consists of a transmembrane anchor in the inner membrane and intermembrane space domains, including a coiled-coil domain and a conserved C-terminal domain. Deletion of the C-terminal domain disrupted the MINOS complex and led to release of cristae membranes from the inner boundary membrane, whereas the interaction of mitofilin with the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM) were enhanced. Deletion of the coiled-coil domain also disturbed the MINOS complex and cristae morphology; however, the interactions of mitofilin with TOM and SAM were differentially affected. Finally, deletion of both intermembrane space domains disturbed MINOS integrity as well as interactions with TOM and SAM. Thus, the intermembrane space domains of mitofilin play distinct roles in interactions with outer membrane complexes and maintenance of MINOS and cristae morphology, demonstrating that MINOS contacts to TOM and SAM are not sufficient for the maintenance of inner membrane architecture.

The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. We report that MINOS independently interacts with both preprotein translocases of the outer mitochondrial membrane and plays a role in the biogenesis of β-barrel proteins of the outer membrane.

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.