Cristae, the organized invaginations of the mitochondrial inner membrane, respond structurally to the energetic demands of the cell. The mechanism by which these dynamic changes are regulated and the consequences thereof are largely unknown. Optic atrophy 1 (OPA1) is the mitochondrial GTPase responsible for inner membrane fusion and maintenance of cristae structure. Here, we report that OPA1 responds dynamically to changes in energetic conditions to regulate cristae structure. This cristae regulation is independent of OPA1's role in mitochondrial fusion, since an OPA1 mutant that can still oligomerize but has no fusion activity was able to maintain cristae structure. Importantly, OPA1 was required for resistance to starvation-induced cell death, for mitochondrial respiration, for growth in galactose media and for maintenance of ATP synthase assembly, independently of its fusion activity. We identified mitochondrial solute carriers (SLC25A) as OPA1 interactors and show that their pharmacological and genetic blockade inhibited OPA1 oligomerization and function. Thus, we propose a novel way in which OPA1 senses energy substrate availability, which modulates its function in the regulation of mitochondrial architecture in a SLC25A protein-dependent manner.

Mitochondrial dynamics have been extensively studied in the context of classical cell death models involving Bax-mediated cytochrome c release. Excitotoxic neuronal loss is a nonclassical death signaling pathway that occurs following overactivation of glutamate receptors independent of Bax activation. Presently, the role of mitochondrial dynamics in the regulation of excitotoxicity remains largely unknown. Here, we report that NMDA-induced excitotoxicity results in defects in mitochondrial morphology as evident by the presence of excessive fragmented mitochondria, cessation of mitochondrial fusion, and cristae dilation. Up-regulation of the mitochondrial inner membrane GTPase, Opa1, is able to restore mitochondrial morphology and protect neurons against excitotoxic injury. Opa1 functions downstream of the calcium-dependent protease, calpain. Inhibition of calpain activity by calpastatin, an endogenous calpain inhibitor, significantly rescued mitochondrial defects and maintained neuronal survival. Opa1 was required for calpastatin-mediated neuroprotection because the enhanced survival found following NMDA-induced toxicity was significantly reduced upon loss of Opa1. Our results define a mechanism whereby breakdown of the mitochondrial network mediated through loss of Opa1 function contributes to neuronal death following excitotoxic neuronal injury. These studies suggest Opa1 as a potential therapeutic target to promote neuronal survival following acute brain damage and neurodegenerative diseases.

The cytoplasmic translation factor eEF1A has been implicated in the nuclear export of tRNA species in lower eukaryotes. Here we demonstrate that eEF1A plays a central role in nuclear export of proteins in mammalian cells. TD-NEM (transcription-dependent nuclear export motif), a newly characterized nuclear export signal, mediates efficient nuclear export of several proteins including the von Hippel-Lindau (VHL) tumor suppressor and the poly(A)-binding protein (PABP1) in a manner that is dependent on ongoing RNA polymerase II (RNA PolII)-dependent transcription. eEF1A interacts specifically with TD-NEM of VHL and PABP1 and disrupting this interaction, by point mutations of key TD-NEM residues or treatment with actinomycin D, an inhibitor of RNA PolII-dependent transcription, prevents assembly and nuclear export. siRNA-induced knockdown or antibody-mediated depletion of eEF1A prevents in vivo and in vitro nuclear export of TD-NEM-containing proteins. Nuclear retention experiments and inhibition of the Exportin-5 pathway suggest that eEF1A stimulates nuclear export of proteins from the cytoplasmic side of the nuclear envelope, without entering the nucleus. Together, these data identify a role for eEF1A, a cytoplasmic mediator of tRNA export in yeast, in the nuclear export of proteins in mammalian cells. These results also provide a link between the translational apparatus and subcellular trafficking machinery demonstrating that these two central pathways in basic metabolism can act cooperatively.

These results identify a novel nuclear export motif, further highlight the role of nuclear-cytoplasmic shuttling of E3 ligases in degradation of nuclear substrates, and provide evidence that disease-causing mutations can target subcellular trafficking.Ubiquitylation is a multiprotein pathway that destines marked proteins for degradation by the 26S proteasome (22,59). The conjugation of ubiquitin to proteins requires the action of three different enzymes: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. The process of ubiquitylation begins with the loading of a ubiquitin molecule onto the E1 ubiquitin-activating enzyme. This is followed by the transfer of ubiquitin from the E1 to the E2 ubiquitin-conjugating enzyme. Finally, transfer of ubiquitin from the E2 to the lysine residue of a target substrate is catalyzed by the E3 ubiquitin ligase. Selectivity of this pathway relies heavily on E3 ubiquitin ligases, which ultimately dictate substrate specificity. E3 ubiquitin ligases can act individually or form a multisubunit complex that may include a member of the Cullin family of proteins to covalently modify a vast array of cellular proteins. In view of the essential role of E3 ubiquitin ligases in regulation of many aspects of cellular functions and biological processes, there is mounting evidence that loss of function or deregulation of E3 ligases contributes to the development of disease.Degradation of nuclear substrates by the ubiquitylation system often requires nuclear-cytoplasmic trafficking of both the E3 ubiquitin ligase and the substrate protein (2, 54). One example is the ubiquitin-mediated degradation of the p53 tumor suppressor protein by the Mdm2 (murine double minute 2) E3 ubiquitin ligase (45, 47). Mdm2 shuttles continuously between the nucleus and the cytoplasm in order to efficiently degrade nuclear p53 (12, 53). Cancer-causing point mutations that disrupt nuclear export of Mdm2 are impaired in mediating proteasomal degradation of p53 (37). Nuclear export of the ROC1-SCF Fbw1a E3 ubiquitin ligase is also required for the proteasomal degradation of the Smad3 transcription factor (13). Another example is the cyclin-dependent kinase inhibitor p27 Kip1 , which requires nuclear export by Jab1 for proteasomemediated degradation. A mutant form of p27Kip1 that fails to assemble with Jab1 cannot be exported from the nucleus and is not degraded by the proteasome (54, 58).The von Hippel-Lindau tumor suppressor protein (VHL) is a vital component of the VBC-Cul2 E3 ubiquitin ligase complex, as it acts as the substrate recognition protein to provide specificity to the degradation process (25,27,30,38,39,50). VHL promotes the recruitment, ubiquitylation, and subsequent proteasomal degradation of the alpha subunit of hypoxia-inducible factor (HIF) in an oxygen-dependent manner (26,41). Under conditions of normal oxygen tension (normoxia), HIF␣ is hydroxylated at key prolyl residues within the oxygen-dependent degradation domain by prolyl hydroxylases (5,8,24,26). This p...

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