Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.

Sterol transporters at membrane contact sites regulate TORC1 and TORC2 signaling.

Membrane contact sites (MCSs) function to facilitate the formation of membrane domains composed of specialized lipids, proteins, and nucleic acids. In cells, membrane domains regulate membrane dynamics and biochemical and signaling pathways. We and others identified a highly conserved family of sterol transport proteins (Ltc/Lam) localized at diverse MCSs. In this study, we describe data indicating that the yeast family members Ltc1 and Ltc3/4 function at the vacuole and plasma membrane, respectively, to create membrane domains that partition upstream regulators of the TORC1 and TORC2 signaling pathways to coordinate cellular stress responses with sterol homeostasis.

Ltc1 is an ER-localized sterol transporter and a component of ER-mitochondria and ER-vacuole contacts.

Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER-mitochondria and ER-vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER-mitochondria and ER-vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER-mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER-mitochondria and ER-vacuole contact sites.

MICOS coordinates with respiratory complexes and lipids to establish mitochondrial inner membrane architecture.

The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.

A bimodal distribution of two distinct categories of intrinsically disordered structures with separate functions in FG nucleoporins.

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.

Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin.

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.