MICOS is a conserved multisubunit complex that localizes to mitochondrial cristae junctions and organizes cristae positioning within the organelle. MICOS is organized into two independent subcomplexes; however, the mechanisms that dictate the assembly and spatial positioning of each MICOS subcomplex are poorly understood. Here, we determine that MICOS subcomplexes target independently of one another to sites on the inner mitochondrial membrane that are in proximity to contact sites between mitochondria and the ER. One subcomplex, composed of Mic27/Mic26/Mic10/Mic12, requires ERMES complex function for its assembly. In contrast, the principal MICOS component, Mic60, self-assembles and localizes in close proximity to the ER through an independent mechanism. We also find that Mic60 can uniquely redistribute adjacent to forced mitochondria–vacuole contact sites. Our data suggest that nonoverlapping properties of interorganelle contact sites provide spatial cues that enable MICOS assembly and ultimately lead to proper physical and functional organization of mitochondria.

Mitochondria, so much more than just being energy factories, also have the capacity to synthesize macromolecules including phospholipids, particularly cardiolipin (CL) and phosphatidylethanolamine (PE). Phospholipids are vital constituents of mitochondrial membranes, impacting the plethora of functions performed by this organelle. Hence, the orchestrated movement of phospholipids to and from the mitochondrion is essential for cellular integrity. In this review, we capture recent advances in the field of mitochondrial phospholipid biosynthesis and trafficking, highlighting the significance of interorganellar communication, intramitochondrial contact sites, and lipid transfer proteins in maintaining membrane homeostasis. We then discuss the physiological functions of CL and PE, specifically how they associate with protein complexes in mitochondrial membranes to support bioenergetics and maintain mitochondrial architecture.

The fusion of mammalian inner mitochondrial membranes (IMMs) is mediated by dynamin-like GTPase OPA1. Mutations in human OPA1 cause optic atrophy, but the molecular basis for membrane fusion and pathogenesis is not clear. Here, we determined the crystal structure of the minimal GTPase domain (MGD) of human OPA1. A three-helix bundle (HB) domain including two helices extending from the GTPase (G) domain and the last helix of OPA1 tightly associates with the G domain. In the presence of GDP and BeF₃⁻, OPA1-MGD forms a dimer, the interface of which is critical for the maintenance of mitochondrial morphology. The catalytic core of OPA1 possesses unique features that are not present in other dynamin-like proteins. Biochemical experiments revealed that OPA1-MGD forms nucleotide-dependent dimers, which is important for membrane-stimulated GTP hydrolysis, and an N-terminal extension mediates nucleotide-independent dimerization that facilitates efficient membrane association. Our results suggest a multifaceted assembly of OPA1 and explain the effect of most OPA1 mutations on optic atrophy.

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER–LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein–protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P₂. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.