RESUMO
In this issue of Structure, Oot and Wilkens1 present new mechanistic insights to finally merge the function of V-ATPase and TLDc domain proteins. They show that TLDc proteins directly affect V-ATPase activity and assembly, expanding our understanding of how V-ATPase and TLDc proteins exert a plethora of biological functions.
Assuntos
ATPases Vacuolares Próton-Translocadoras , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Humanos , Domínios ProteicosRESUMO
Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single-particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the proton gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogues synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin-knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin-knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.
Assuntos
Sinaptofisina , ATPases Vacuolares Próton-Translocadoras , Animais , Masculino , Camundongos , Microscopia Crioeletrônica , Camundongos Knockout , Modelos Moleculares , Neurotransmissores/metabolismo , Ligação Proteica , Convulsões/genética , Convulsões/metabolismo , Vesículas Sinápticas/química , Vesículas Sinápticas/enzimologia , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/química , Sinaptofisina/deficiência , Sinaptofisina/metabolismo , Sinaptofisina/ultraestrutura , ATPases Vacuolares Próton-Translocadoras/análise , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/ultraestrutura , Tomografia com Microscopia EletrônicaRESUMO
Intercellular communication in the nervous system occurs through the release of neurotransmitters into the synaptic cleft between neurons. In the presynaptic neuron, the proton pumping vesicular- or vacuolar-type ATPase (V-ATPase) powers neurotransmitter loading into synaptic vesicles (SVs), with the V1 complex dissociating from the membrane region of the enzyme before exocytosis. We isolated SVs from rat brain using SidK, a V-ATPase-binding bacterial effector protein. Single-particle electron cryomicroscopy allowed high-resolution structure determination of V-ATPase within the native SV membrane. In the structure, regularly spaced cholesterol molecules decorate the enzyme's rotor and the abundant SV protein synaptophysin binds the complex stoichiometrically. ATP hydrolysis during vesicle loading results in a loss of the V1 region of V-ATPase from the SV membrane, suggesting that loading is sufficient to induce dissociation of the enzyme.
Assuntos
Vesículas Sinápticas , ATPases Vacuolares Próton-Translocadoras , Animais , Ratos , Proteínas de Bactérias/química , Encéfalo/ultraestrutura , Encéfalo/enzimologia , Colesterol/química , Microscopia Crioeletrônica , Hidrólise , Vesículas Sinápticas/enzimologia , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/isolamento & purificação , ATPases Vacuolares Próton-Translocadoras/ultraestrutura , Conformação ProteicaRESUMO
RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.
Assuntos
Larva , Interferência de RNA , RNA de Cadeia Dupla , Animais , Larva/crescimento & desenvolvimento , Larva/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Hidróxidos/química , Hidróxidos/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Arachis/genética , Arachis/química , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Controle Biológico de Vetores , Besouros/genética , Besouros/crescimento & desenvolvimento , Química Verde , Agentes de Controle Biológico/química , Agentes de Controle Biológico/metabolismo , Nanopartículas/químicaRESUMO
Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.
Assuntos
Proteínas Ativadoras de GTPase , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/química , Coativadores de Receptor Nuclear/metabolismo , Coativadores de Receptor Nuclear/química , Ligação Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas MitocondriaisRESUMO
BACKGROUND: The supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction. RESULTS: Delta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR. CONCLUSIONS: Yeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.
Assuntos
Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos , Prótons , Rodopsina/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Vacuolar H+-ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.
Assuntos
Fosfatos de Fosfatidilinositol , Subunidades Proteicas , ATPases Vacuolares Próton-Translocadoras , Humanos , Sítios de Ligação , Endossomos/enzimologia , Endossomos/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Domínios ProteicosRESUMO
Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
Assuntos
Endossomos , ATPases Vacuolares Próton-Translocadoras , Autofagia , Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , HumanosRESUMO
Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V1-ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V1's activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP's binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Domínio Catalítico , Sítio Alostérico , Modelos Moleculares , ATPases Vacuolares Próton-Translocadoras/química , Trifosfato de Adenosina/química , Sítios de LigaçãoRESUMO
Vacuolar-type ATPases (V-ATPases) are responsible for the acidification of intracellular compartments in almost all eukaryotic cells, while in some specialized cells they acidify the extracellular environment. As ubiquitous proton pumps, these large membrane-embedded enzymes are involved in many fundamental cellular processes that require tight control of pH. Consequently, V-ATPase malfunction or aberrant activity has been linked to numerous diseases. In the past ten years, electron cryomicroscopy (cryoEM) of yeast V-ATPases has revealed the architecture and rotary catalytic mechanism of these macromolecular machines. More recently, studies have revealed the structures of V-ATPases in animals and plants, uncovered aspects of how V-ATPases are assembled and regulated by reversible dissociation, and shown how V-ATPase activity can be modulated by proteins and small molecule inhibitors. In this review, we highlight these recent developments.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Animais , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Microscopia Crioeletrônica , Membrana Celular/metabolismo , Células Eucarióticas/metabolismoRESUMO
Vacuolar/archaeal-type ATPase (V/A-ATPase) is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. However, further characterization is needed to understand the transition from the ground state to the steady state. Here, we identified the cryo-electron microscopy structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure (PNAS NEXUS, pgac116, 2022). In conclusion, our findings provide dynamical information that will enhance the future prospects for studying the initial activation processes of the enzymes, which have unknown intermediate structures in their functional pathway.
Assuntos
Trifosfato de Adenosina , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Ativação Enzimática , Conformação ProteicaRESUMO
Proton pumping ATPases, both F-type and V/A-type ATPases, generate ATP using electrochemical energy or pump protons/sodium ions by hydrolyzing ATP. The enzymatic reaction and proton transport are coupled through subunit rotation, and this unique rotational mechanism (rotational catalysis) has been intensively studied. Single-molecule and thermodynamic analyses have revealed the detailed rotational mechanism, including the catalytically inhibited state and the roles of subunit interactions. In mammals, F- and V-ATPases are involved in ATP synthesis and organelle acidification, respectively. Most bacteria, including anaerobes, have F- and/or A-ATPases in the inner membrane. However, these ATPases are not believed to be essential in anaerobic bacteria since anaerobes generate sufficient ATP without oxidative phosphorylation. Recent studies suggest that F- and A-ATPases perform indispensable functions beyond ATP synthesis in oral pathogenic anaerobes; F-ATPase is involved in acid tolerance in Streptococcus mutans, and A-ATPase mediates nutrient import in Porphyromonas gingivalis. Consistently, inhibitors of oral bacterial F- and A-ATPases, such as phytopolyphenols and bedaquiline, strongly diminish growth and survival. Herein, we discuss rotational catalysis of bacterial F- and A-ATPases, and discuss their physiological roles, focusing on oral bacteria. We also review the effects of ATPase inhibitors on the growth and survival of oral pathogenic bacteria. The features of the catalytic mechanism and unique physiological roles in oral bacteria highlight the potential for proton pumping ATPases to serve as targets for oral antimicrobial agents.
Assuntos
Prótons , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina , Animais , Bactérias/metabolismo , Catálise , Mamíferos/metabolismo , Sódio , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
We used the Legionella pneumophila effector SidK to affinity purify the endogenous vacuolar-type ATPases (V-ATPases) from lemon fruit. The preparation was sufficient for cryoelectron microscopy, allowing structure determination of the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum ΔpH of â¼3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared with yeast and mammalian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual configuration of transmembrane α helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.
Assuntos
Citrus , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismoRESUMO
Progress in structural membrane biology has been significantly accelerated by the ongoing 'Resolution Revolution' in cryo-electron microscopy (cryo-EM). In particular, structure determination by single-particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever-increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo-electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single-particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor lipopolysaccharide peptidoglycan ring (LP ring) from Salmonella enterica.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Microscopia Crioeletrônica/métodos , Lipopolissacarídeos , Peptidoglicano , Complexo de Proteína do Fotossistema I/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in â¼3% of complexes, whereas â¼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.
Assuntos
ATPases Vacuolares Próton-Translocadoras , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Suínos , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Transmembrane ATPases are membrane-bound enzyme complexes and ion transporters that can be divided into F-, V-, and A-ATPases according to their structure. The V-ATPases, also known as H+-ATPases, are large multi-subunit protein complexes composed of a peripheral domain (V1) responsible for the hydrolysis of ATP and a membrane-integrated domain (V0) that transports protons across plasma membrane or organelle membrane. V-ATPases play a fundamental role in maintaining pH homeostasis through lysosomal acidification and are involved in modulating various physiological and pathological processes, such as macropinocytosis, autophagy, cell invasion, and cell death (e.g., apoptosis, anoikis, alkaliptosis, ferroptosis, and lysosome-dependent cell death). In addition to participating in embryonic development, V-ATPase pathways, when dysfunctional, are implicated in human diseases, such as neurodegenerative diseases, osteopetrosis, distal renal tubular acidosis, and cancer. In this review, we summarize the structure and regulation of isoforms of V-ATPase subunits and discuss their context-dependent roles in cancer biology and cell death. Updated knowledge about V-ATPases may enable us to design new anticancer drugs or strategies.
Assuntos
Neoplasias , ATPases Vacuolares Próton-Translocadoras , Humanos , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Membrana Celular/metabolismo , Neoplasias/metabolismo , Morte CelularRESUMO
Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited on disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the regulator of the ATPase of vacuoles and endosomes (RAVE) complex through an unknown mechanism. We used cryogenic-electron microscopy of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex and the V1 complex lacking subunit C. On separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.
Assuntos
Proteínas de Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Endossomos/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , Vacúolos/metabolismoRESUMO
Vacuolar-type adenosine triphosphatases (V-ATPases) are proton pumps found in almost all eukaryotic cells. These enzymes consist of a soluble catalytic V1 region that hydrolyzes ATP and a membrane-embedded VO region responsible for proton translocation. V-ATPase activity leads to acidification of endosomes, phagosomes, lysosomes, secretory vesicles, and the trans-Golgi network, with extracellular acidification occurring in some specialized cells. Small-molecule inhibitors of V-ATPase have played a crucial role in elucidating numerous aspects of cell biology by blocking acidification of intracellular compartments, while therapeutic use of V-ATPase inhibitors has been proposed for the treatment of cancer, osteoporosis, and some infections. Here, we determine structures of the isolated VO complex from Saccharomyces cerevisiae bound to two well-known macrolide inhibitors: bafilomycin A1 and archazolid A. The structures reveal different binding sites for the inhibitors on the surface of the proton-carrying c ring, with only a small amount of overlap between the two sites. Binding of both inhibitors is mediated primarily through van der Waals interactions in shallow pockets and suggests that the inhibitors block rotation of the ring. Together, these structures indicate the existence of a large chemical space available for V-ATPase inhibitors that block acidification by binding the c ring.
Assuntos
Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras , Sítios de Ligação , Microscopia Crioeletrônica , Macrolídeos/farmacologia , Prótons , Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/químicaRESUMO
The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
Assuntos
Proteínas Mitocondriais/química , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Holoenzimas/química , Mutagênese , Ligação Proteica , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved "TLDc" domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.