The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino-terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.

Coenzyme Q (CoQ) lipids are ancient electron carriers that, in eukaryotes, function in the mitochondrial respiratory chain. In mitochondria, CoQ lipids are built by an inner membrane–associated, multicomponent, biosynthetic pathway via successive steps of isoprenyl tail polymerization, 4-hydroxybenzoate head-to-tail attachment, and head modification, resulting in the production of CoQ. In yeast, we discovered that head-modifying CoQ pathway components selectively colocalize to multiple resolvable domains in vivo, representing supramolecular assemblies. In cells engineered with conditional ON or OFF CoQ pathways, domains were strictly correlated with CoQ production and substrate flux, respectively, indicating that CoQ lipid intermediates are required for domain formation. Mitochondrial CoQ domains were also observed in human cells, underscoring their conserved functional importance. CoQ domains within cells were highly enriched adjacent to ER–mitochondria contact sites. Together, our data suggest that CoQ domains function to facilitate substrate accessibility for processive and efficient CoQ production and distribution in cells.

The fusion of inner mitochondrial membranes requires dynamin-like GTPases, Mgm1 in yeast and OPA1 in mammals, but how they mediate membrane fusion is poorly understood. Here, we determined the crystal structure of Saccharomyces cerevisiae short Mgm1 (s-Mgm1) in complex with GDP. It revealed an N-terminal GTPase (G) domain followed by two helix bundles (HB1 and HB2) and a unique C-terminal lipid-interacting stalk (LIS). Dimers can form through antiparallel HB interactions. Head-to-tail trimers are built by intermolecular interactions between the G domain and HB2-LIS. Biochemical and in vivo analyses support the idea that the assembly interfaces observed here are native and critical for Mgm1 function. We also found that s-Mgm1 interacts with negatively charged lipids via both the G domain and LIS. Based on these observations, we propose that membrane targeting via the G domain and LIS facilitates the in cis assembly of Mgm1, potentially generating a highly curved membrane tip to allow inner membrane fusion.

Widespread repression of protein synthesis rates is a key feature of Endoplasmic Reticulum (ER) stress, mediated by the ER sensor kinase PERK. While select transcripts escape this repression, global translational down-regulation impacts crucial protein levels in all cellular compartments, beyond the ER. How the cell manages this paradox is unclear. PERK has a unique cytoplasmic loop within its kinase domain that binds PERKs target, eIF2α. We identified the mitochondrial protein, ATAD3A, as a new interactor of the loop, binding to a highly conserved region within it. During ER stress, increased interaction between ATAD3A and PERK attenuates PERK signalling to eIF2α, removing the translational block on several mitochondrial proteins. This occurs at novel context-dependent, mitochondria-ER contact sites. The interaction provides a previously unknown mechanism for fine-tuning translational repression at a local level, mitigating the impact of ER stress on mitochondria. Further, it represents a new target for selective modulation of PERK-eIF2α signalling in diseases from cancer to neurodegeneration.

No abstract