RESUMO
Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both by inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of mitochondrial membrane potential is observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial function even in defective mitochondria.
Assuntos
Fosfatos , Saccharomyces cerevisiae , Animais , Potencial da Membrana Mitocondrial , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Respiração , Mamíferos/metabolismoRESUMO
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.
Assuntos
Adenoma/metabolismo , Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ácido Pirúvico/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Drosophila , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2â¼ubiquitin (E2â¼Ub) via their RINGs but canonical RING E3s promote closed E2â¼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2â¼Ub to favor Ub transfer to the E3 active site. This different RING/E2â¼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2â¼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.
Assuntos
Proteínas de Transporte/química , Enzimas de Conjugação de Ubiquitina/química , Sítios de Ligação , Proteínas de Transporte/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Zinco/química , Zinco/metabolismoRESUMO
RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.