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Summary: The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

Summary: Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric F1Fo-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10ATPsynthase, not on Mic10MICOS. We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F1Fo-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth.

Oxidative phosphorylation takes place at specialized compartments of the inner mitochondrial membrane, the cristae. The elaborate ultrastructure of cristae membranes enables efficient chemi-osmotic coupling of respiratory chain and F1Fo-ATP synthase. Dynamic membrane remodeling allows mitochondria to adapt to changing physiological requirements. The mito-chondrial contact site and cristae organizing system (MICOS) and the oligomeric ATP synthase have been known to govern distinct features of cristae architec-ture. A new study [1] on the crosstalk between these two machineries now sheds light on the mechanisms of cristae formation and maintenance.

The MICOS complex has an essential role in crista junction formation and mitochondrial inner membrane morphology. Here, the authors show that one of its components, Mic60, known to form contact sites between inner and outer membranes, also displays membrane-shaping activity.

Metabolite carriers of the mitochondrial inner membrane are crucial for cellular physiology since mitochondria contribute essential metabolic reactions and synthesize the majority of the cellular ATP. Like almost all mitochondrial proteins, carriers have to be imported into mitochondria from the cytosol. Carrier precursors utilize a specialized translocation pathway dedicated to the biogenesis of carriers and related proteins, the carrier translocase of the inner membrane (TIM22) pathway. After recognition and import through the mitochondrial outer membrane via the translocase of the outer membrane (TOM) complex, carrier precursors are ushered through the intermembrane space by hexameric TIM chaperones and ultimately integrated into the inner membrane by the TIM22 carrier translocase. Recent advances have shed light on the mechanisms of TOM translocase and TIM chaperone function, uncovered an unexpected versatility of the machineries, and revealed novel components and functional crosstalk of the human TIM22 translocase.

The cytosolic chaperone network of Saccharomyces cerevisiae is intimately associated with the emergence and maintenance of prion traits. Recently, the Hsp110 protein, Sse1, has been identified as a nucleotide exchange factor (NEF) for both cytosolic Hsp70 chaperone family members, Ssa1 and Ssb1. We have investigated the role of Sse1 in the de novo formation and propagation of [PSI(+)], the prion form of the translation termination factor, Sup35. As observed by others, we find that Sse1 is essential for efficient prion propagation. Our results suggest that the NEF activity is required for maintaining sufficient levels of substrate-free Ssa1. However, Sse1 exhibits an additional NEF-independent activity; it stimulates in vitro nucleation of Sup35NM, the prion domain of Sup35. We also observe that high levels of Sse1, but not of an unrelated NEF, very potently inhibit Hsp104-mediated curing of [PSI(+)]. Taken together, these results suggest a chaperone-like activity of Sse1 that assists in stabilization of early folding intermediates of the Sup35 prion conformation. This activity is not essential for prion formation under conditions of Sup35 overproduction, however, it may be relevant for spontaneous [PSI(+)] formation as well as for protection of the prion trait upon physiological Hsp104 induction.