OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand.

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.

The mitochondrial inner membrane GTPase, optic atrophy 1 (Opa1), restores mitochondrial morphology and promotes neuronal survival following excitotoxicity.

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 non-classical 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.

eEF1A is a novel component of the mammalian nuclear protein export machinery.

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.

Cancer-causing mutations in a novel transcription-dependent nuclear export motif of VHL abrogate oxygen-dependent degradation of hypoxia-inducible factor.

It is thought that degradation of nuclear proteins by the ubiquitylation system requires nuclear-cytoplasmic trafficking of E3 ubiquitin ligases. The von Hippel-Lindau (VHL) tumor suppressor protein is the substrate recognition component of a Cullin-2-containing E3 ubiquitin ligase that recruits hypoxia-inducible factor (HIF) for oxygen-dependent degradation. We demonstrated that VHL engages in nuclear-cytoplasmic trafficking that requires ongoing transcription to promote efficient HIF degradation. Here, we report the identification of a discreet motif, DXGX(2)DX(2)L, that directs transcription-dependent nuclear export of VHL and which is targeted by naturally occurring mutations associated with renal carcinoma and polycythemia in humans. The DXGX(2)DX(2)L motif is also found in other proteins, including poly(A)-binding protein 1, to direct its transcription-dependent nuclear export. We define DXGX(2)DX(2)L as TD-NEM (transcription-dependent nuclear export motif), since inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole abrogates its nuclear export activity. Disease-causing mutations of key residues of TD-NEM restrain the ability of VHL to efficiently mediate oxygen-dependent degradation of HIF by altering its nuclear export dynamics without affecting interaction with its substrate. 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.