Structural analysis of a trimeric assembly of the mitochondrial dynamin-like GTPase Mgm1.

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.

Coenzyme Q biosynthetic proteins assemble in a substrate-dependent manner into domains at ER-mitochondria contacts.

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.

Defining the physiological role of SRP in protein-targeting efficiency and specificity.

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.

Multiple exportins influence thyroid hormone receptor localization.

The thyroid hormone receptor (TR) undergoes nucleocytoplasmic shuttling and regulates target genes involved in metabolism and development. Previously, we showed that TR follows a CRM1/calreticulin-mediated nuclear export pathway. However, two lines of evidence suggest TR also follows another pathway: export is only partially blocked by leptomycin B (LMB), a CRM1-specific inhibitor; and we identified nuclear export signals in TR that are LMB-resistant. To determine whether other exportins are involved in TR shuttling, we used RNA interference and fluorescence recovery after photobleaching shuttling assays in transfected cells. Knockdown of exportins 4, 5, and 7 altered TR shuttling dynamics, and when exportins 5 and 7 were overexpressed, TR distribution shifted toward the cytosol. To further assess the effects of exportin overexpression, we examined transactivation of a TR-responsive reporter gene. Our data indicate that multiple exportins influence TR localization, highlighting a fine balance of nuclear import, retention, and export that modulates TR function.

Multiple novel signals mediate thyroid hormone receptor nuclear import and export.

Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor ß1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor ß1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general.