RESUMO
In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.
Assuntos
Arabidopsis/metabolismo , Transporte Proteico/fisiologia , Sistema de Translocação de Argininas Geminadas/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Organelas/metabolismo , Transição de Fase , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Sistema de Translocação de Argininas Geminadas/fisiologiaRESUMO
Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
Assuntos
Microscopia Crioeletrônica , Peroxissomos , Complexos Ubiquitina-Proteína Ligase , Citosol/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Peroxinas/química , Peroxinas/metabolismo , Peroxinas/ultraestrutura , Fator 2 da Biogênese de Peroxissomos/química , Fator 2 da Biogênese de Peroxissomos/metabolismo , Fator 2 da Biogênese de Peroxissomos/ultraestrutura , Peroxissomos/enzimologia , Peroxissomos/ultraestrutura , Poliubiquitina , Transporte Proteico , Domínios RING Finger , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/ultraestrutura , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Ubiquitina-Proteína Ligase/ultraestruturaRESUMO
Peroxisomes are membrane-bounded organelles that exist in most eukaryotic cells and are involved in the oxidation of fatty acids and the destruction of reactive oxygen species. Depending on the organism, they house additional metabolic reactions that range from glycolysis in parasitic protozoa to the production of ether lipids in animals and antibiotics in fungi. The importance of peroxisomes for human health is revealed by various disorders - notably the Zellweger spectrum - that are caused by defects in peroxisome biogenesis and are often fatal. Most peroxisomal metabolic enzymes reside in the lumen, but are synthesized in the cytosol and imported into the organelle by mobile receptors. The receptors accompany cargo all the way into the lumen and must return to the cytosol to start a new import cycle. Recycling requires receptor monoubiquitination by a membrane-embedded ubiquitin ligase complex composed of three RING finger (RF) domain-containing proteins: PEX2, PEX10, and PEX12. A recent cryo-electron microscopy (cryo-EM) structure of the complex reveals its function as a retro-translocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that assemble into an open channel. The N terminus of a receptor likely inserts into the pore from the lumenal side, and is then monoubiquitinated by one of the RFs to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitinated by the concerted action of the other two RFs and ultimately degraded. The new data provide mechanistic insight into a crucial step of peroxisomal protein import.
Assuntos
Proteínas de Membrana , Receptores Citoplasmáticos e Nucleares , Animais , Humanos , Peroxinas/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Microscopia Crioeletrônica , Proteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Ubiquitinas/metabolismo , Ligases/metabolismoRESUMO
Cytokinin is an essential phytohormone that controls various biological processes in plants. A number of response regulators are known to be important for cytokinin signal transduction. ARABIDOPSIS RESPONSE REGULATOR 4 (ARR4) mediates the cross-talk between light and cytokinin signaling through modulation of the activity of phytochrome B. However, the mechanism that regulates the activity and stability of ARR4 is unknown. Here we identify an ATP-independent serine protease, degradation of periplasmic proteins 9 (DEG9), which localizes to the nucleus and regulates the stability of ARR4. Biochemical evidence shows that DEG9 interacts with ARR4, thereby targeting ARR4 for degradation, which suggests that DEG9 regulates the stability of ARR4. Moreover, genetic evidence shows that DEG9 acts upstream of ARR4 and regulates the activity of ARR4 in cytokinin and light-signaling pathways. This study thus identifies a role for a ubiquitin-independent selective protein proteolysis in the regulation of the stability of plant signaling components.
Assuntos
Proteínas de Arabidopsis/metabolismo , Citocininas/metabolismo , Luz , Serina Proteases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Light is a major environmental factor regulating flowering time, thus ensuring reproductive success of higher plants. In contrast to our detailed understanding of light quality and photoperiod mechanisms involved, the molecular basis underlying high light-promoted flowering remains elusive. Here we show that, in Arabidopsis, a chloroplast-derived signal is critical for high light-regulated flowering mediated by the FLOWERING LOCUS C (FLC). We also demonstrate that PTM, a PHD transcription factor involved in chloroplast retrograde signaling, perceives such a signal and mediates transcriptional repression of FLC through recruitment of FVE, a component of the histone deacetylase complex. Thus, our data suggest that chloroplasts function as essential sensors of high light to regulate flowering and adaptive responses by triggering nuclear transcriptional changes at the chromatin level.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Flores/genética , Proteínas de Domínio MADS/genética , Dedos de Zinco PHD/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Núcleo Celular/genética , Núcleo Celular/efeitos da radiação , Cloroplastos/genética , Cloroplastos/metabolismo , Cromatina/genética , Cromatina/efeitos da radiação , Flores/crescimento & desenvolvimento , Flores/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Histona Desacetilases/genética , Luz , Transdução de Sinais/genética , Fatores de TranscriçãoRESUMO
Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even import folded proteins, but the underlying mechanism has been a mystery. Recent results reveal how import receptors shuttle cargo into peroxisomes. The cargo-bound receptors move from the cytosol across the peroxisomal membrane completely into the matrix by a mechanism that resembles transport through the nuclear pore. The receptors then return to the cytosol through a separate retrotranslocation channel, leaving the cargo inside the organelle. This cycle concentrates imported proteins within peroxisomes, and the energy for cargo import is supplied by receptor export. Peroxisomal protein import thus fundamentally differs from other previously known mechanisms for translocating proteins across membranes.
RESUMO
Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.
Assuntos
Proteínas de Membrana , Poro Nuclear , Peroxissomos , Humanos , Glicina/química , Glicina/genética , Poro Nuclear/metabolismo , Peroxissomos/metabolismo , Transporte Proteico , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sequência Conservada , Domínios Proteicos , Tirosina/química , Tirosina/genéticaRESUMO
Seedling de-etiolation prepares plants to switch from heterotrophic to photoautotrophic growth, a transition essential for plant survival. This delicate de-etiolation process is precisely controlled by environmental and endogenous signals. Although intracellular plastid-derived retrograde signalling is essential for the de-etiolation process, the molecular nature of these retrograde signals remains elusive(1-3). Here we show that chloroplast and light signals antagonistically fine-tune a suite of developmental and physiological responses associated with de-etiolation through a transcriptional module of ABA INSENSITIVE 4 (ABI4) and ELONGATED HYPOCOTYL 5 (HY5). Moreover, ABI4 and HY5 antagonistically regulate the expression of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and the subsequent greening process. In turn, ABI4 and HY5 are targeted for degradation by COP1 in the light and dark, respectively, to ensure a proper interplay of ABI4 and HY5 actions during seedling de-etiolation. Our study provides a new molecular mechanism for understanding how chloroplast signals converge with light signals to optimize early plant development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Estiolamento , Regulação da Expressão Gênica de Plantas , Proteínas Nucleares/genética , Transdução de Sinais , Fatores de Transcrição/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cloroplastos/fisiologia , Luz , Proteínas Nucleares/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3ω-mediated Ca(2+)-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca(2+) signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain.
Assuntos
Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Complexos de Proteínas Captadores de Luz/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Plastídeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/metabolismo , Arabidopsis/genética , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Biogênese de Organelas , Plantas Geneticamente Modificadas , Transdução de SinaisRESUMO
Intracellular signaling from chloroplast to nucleus followed by a subsequent response in the chloroplast is called retrograde signaling. It not only coordinates the expression of nuclear and chloroplast genes, which is essential for chloroplast biogenesis, but also maintains chloroplast function at optimal levels in response to fluxes in metabolites and changes in environmental conditions. In recent years several putative retrograde signals have been identified and signaling pathways have been proposed. Here we review retrograde signals derived from tetrapyrroles, carotenoids, nucleotides and isoprene precursors in response to abiotic stresses, including oxidative stress. We discuss the responses that these signals elicit and show that they not only modify chloroplast function but also influence other aspects of plant development and adaptation.
Assuntos
Fenômenos Fisiológicos Vegetais , Plantas/metabolismo , Transdução de Sinais , Carotenoides/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Estresse FisiológicoRESUMO
Chloroplast development, maintenance and function depend on the coordinated expression of chloroplast and nuclear genes. The retrograde chloroplast signals are essential in coordinating nuclear gene expression. Although the sources of signals in chloroplasts have been identified and the associated transcription factors in the nucleus extensively studied, the molecular mechanism that relays chloroplast signals to the nucleus remains a mystery. Here we show that PTM, a chloroplast envelope-bound plant homeodomain (PHD) transcription factor with transmembrane domains, functions in multiple retrograde signal pathways. The proteolytic cleavage of PTM occurs in response to retrograde signals and amino-terminal PTM accumulates in the nucleus, where it activates ABI4 transcription in a PHD-dependent manner associated with histone modifications. These results provide a molecular basis for the critical function of PTM in retrograde chloroplast signaling and shed new light on the mechanism whereby chloroplast signals are transmitted to the nucleus through the cytosol.