RESUMO
Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5 , Subunidades Proteicas , Subunidades beta do Canal de Sódio Disparado por Voltagem , Humanos , Potenciais de Ação , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Fosfoproteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades beta do Canal de Sódio Disparado por Voltagem/química , Subunidades beta do Canal de Sódio Disparado por Voltagem/deficiência , Subunidades beta do Canal de Sódio Disparado por Voltagem/genética , Subunidades beta do Canal de Sódio Disparado por Voltagem/metabolismo , MutaçãoRESUMO
Mutations in MPZ (myelin protein zero) can cause demyelinating early-onset Charcot-Marie-Tooth type 1B disease or later onset type 2I/J disease characterized by axonal degeneration, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds apposing membranes of the myelin sheath together, with the adhesion role fulfilled by its extracellular immunoglobulin-like domain (IgMPZ), which oligomerizes. Models for how the IgMPZ might form oligomeric assemblies has been extrapolated from a protein crystal structure in which individual rat IgMPZ subunits are packed together under artificial conditions, forming three weak interfaces. One interface organizes the IgMPZ into tetramers, a second 'dimer' interface links tetramers together across the intraperiod line, and a third hydrophobic interface that mediates binding to lipid bilayers or the same hydrophobic surface on another IgMPZ domain. Presently, there are no data confirming whether the proposed IgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed nuclear magnetic resonance spectroscopy and small angle X-ray scattering analysis of wild-type IgMPZ as well as mutant forms with amino acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates IgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated different types of Charcot-Marie-Tooth disease symptoms to subregions within IgMPZ tetramers. Variants causing axonal late-onset disease (CMT2I/J) map to surface residues of IgMPZ proximal to the transmembrane domain. Variants causing early-onset demyelinating disease (CMT1B) segregate into two groups: one is described by variants that disrupt the stability of the Ig-fold itself and are largely located within the core of the IgMPZ domain; whereas another describes a region on the surface of IgMPZ tetramers, accessible to protein interactions. Computational docking studies predict that this latter disease-relevant subregion may potentially mediate dimerization of IgMPZ tetramers.
Assuntos
Doença de Charcot-Marie-Tooth , Animais , Ratos , Axônios , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/diagnóstico , Domínios de Imunoglobulina , Mutação/genética , Proteína P0 da Mielina/genética , HumanosRESUMO
The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.
Assuntos
Corpos Multivesiculares , Proteínas de Saccharomyces cerevisiae , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Endossomos/metabolismo , Corpos Multivesiculares/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMO
The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 "anti-ligases" that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme (DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the "ubiquitome" of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase-DUb fusions.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Degradação Associada com o Retículo Endoplasmático , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Membrana Celular/metabolismo , Enzimas Desubiquitinantes/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Complexo de Golgi/enzimologia , Lisossomos/enzimologia , Plasmídeos , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
WD40-repeat ß-propellers are found in a wide range of proteins involved in distinct biological activities. We define a large subset of WD40 ß-propellers as a class of ubiquitin-binding domains. Using the ß-propeller from Doa1/Ufd3 as a paradigm, we find the conserved top surface of the Doa1 ß-propeller binds the hydrophobic patch of ubiquitin centered on residues I44, L8, and V70. Mutations that disrupt ubiquitin binding abrogate Doa1 function, demonstrating the importance of this interaction. We further demonstrate that WD40 ß-propellers from a functionally diverse set of proteins bind ubiquitin in a similar fashion. This set includes members of the F box family of SCF ubiquitin E3 ligase adaptors. Using mutants defective in binding, we find that ubiquitin interaction by the F box protein Cdc4 promotes its autoubiquitination and turnover. Collectively, our results reveal a molecular mechanism that may account for how ubiquitin controls a broad spectrum of cellular activities.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas F-Box/metabolismo , Sequências Repetitivas de Aminoácidos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Proteínas F-Box/química , Proteína 7 com Repetições F-Box-WD , Humanos , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Relação Estrutura-Atividade , Propriedades de Superfície , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 approximately Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B approximately Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Estrutura Terciária de Proteína , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ubiquitina-Proteína Ligases Nedd4 , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeastSaccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.
Assuntos
Organelas/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Endocitose , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaçãoRESUMO
Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy-to-use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of Cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers.
Assuntos
Farmacorresistência Fúngica , Integrases/genética , Metotrexato/farmacologia , Plasmídeos/genética , Puromicina/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Códon , Marcadores Genéticos , Integrases/metabolismo , Plasmídeos/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/metabolismoRESUMO
The process in which ubiquitin (Ub) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies (MVBs) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However, Ub-independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna3, the prototypical Ub-independent cargo protein sorted through the MVB pathway in yeast. We show that Sna3 trafficking to the vacuole is critically dependent on Rsp5 ligase activity and ubiquitination. We find Sna3 undergoes Ub-dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna3 as an adaptor protein that recruits Rsp5 to cargo such as the methionine transporter Mup1, resulting in efficient Mup1 delivery to the vacuole.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Proteínas de Membrana/metabolismo , Corpos Multivesiculares/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Membrana/genética , Microscopia de Fluorescência , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.
Assuntos
Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Sequência de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Immunoblotting , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/química , Proteína Adaptadora de Sinalização NOD2/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/química , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Homologia de Sequência de Aminoácidos , Ubiquitina/química , Ubiquitina/genéticaRESUMO
The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood on comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognize ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules.
Assuntos
Membrana Celular/fisiologia , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Ubiquitina/fisiologia , Animais , Endossomos/fisiologia , Humanos , Transporte Proteico/fisiologiaRESUMO
The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.
Assuntos
Corpos Multivesiculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Corpos Multivesiculares/ultraestrutura , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Ubiquitinadas/metabolismoRESUMO
The endosomal-sorting complex required for transport (ESCRT) apparatus has multiple ubiquitin (Ub)-binding domains and participates in a wide variety of cellular processes. Many of these ESCRT-dependent processes are keenly regulated by Ub, which serves as a lysosomal-sorting signal for membrane proteins targeted into multivesicular bodies (MVBs) and which may serve as a mediator of viral budding from the cell surface. Hints that both ESCRTs and Ub work together in the processes such as cytokinesis, transcription and autophagy are beginning to emerge. Here, we explore the relationship between ESCRTs and Ub in MVB sorting and viral budding.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Ubiquitina/fisiologia , Proteínas Virais/metabolismo , Liberação de Vírus/fisiologia , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ligação Proteica , Transporte Proteico , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
BACKGROUND: HIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag. RESULTS: Expressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV's, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not effect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites. CONCLUSIONS: These studies demonstrate a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Ubiquitina/metabolismo , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
PMP22 and MPZ are major myelin proteins in the peripheral nervous system. MPZ is a single pass integral membrane protein with an extracellular immunoglobulin (Ig)-like domain and works as an adhesion protein to hold myelin wraps together across the intraperiod line. Loss of MPZ causes severe demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. PMP22 is an integral membrane tetraspan protein belonging to the Claudin superfamily. Homozygous loss of PMP22 also leads to severe demyelinating neuropathy, and duplication of wildtype PMP22 causes the most common form of CMT, CMT1A. Yet the molecular functions provided by PMP22 and how its alteration causes CMT are unknown. Here we find that these abundant myelin proteins form a strong and specific complex. Mutagenesis and domain swapping experiments reveal that these proteins interact through interfaces within their transmembrane domains. We also find that the PMP22 A67T patient variant that causes an HNPP (Hereditary neuropathy with pressure palsies) phenotype, reflecting a heterozygous loss-of-function, maps to this interface. The PMP22 A67T variant results in the specific loss of MPZ association with PMP22 without affecting PMP22 localization to the plasma membrane or its interactions with other proteins. These data define the molecular basis for the MPZâ¼PMP22 interaction and indicate that the MPZâ¼PMP22 complex fulfills an important function in myelinating cells.
RESUMO
BACKGROUND: The ability to produce the same recombinant protein in both prokaryotic and eukaryotic cells offers many experimental opportunities. However, the cloning of the same gene into multiple plasmids is required, which is time consuming, laborious and still may not produce soluble, stable protein in sufficient quantities. We have developed a set of expression vectors that allows for ligation-independent cloning and rapid functional screening for protein expression in both E. coli and S. cerevisiae. RESULTS: A set of expression vectors was made that can express the same open reading frame in E. coli (via the T7 phage promoter) and in S. cerevisiae (via the CUP1 or MET25 promoter). These plasmids also contain the essential elements for replication and selection in both cell types and have several advantages: they allow for cloning of genes by homologous recombination in yeast, protein expression can be determined before plasmid isolation and sequencing, and a GST-fusion tag is added to aid in soluble expression and purification. We have also included a TEV recognition site that allows for the specific cleavage of the fusion proteins to yield native proteins. CONCLUSIONS: The dual promoter vectors can be used for rapid cloning, expression, and purification of target proteins from both prokaryotic and eukaryotic systems with the ability to study post-translation modifications.
Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Saccharomyces cerevisiae/metabolismo , Endopeptidases/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Fases de Leitura Aberta , Plasmídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca2+-permeable (CP) versus heteromeric Ca2+-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/etiologia , Animais , Endocitose , Mamíferos/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de AMPA/metabolismo , Fatores de RiscoRESUMO
Epithelial Na(+) absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na(+) channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, ß-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.
Assuntos
Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Canais Epiteliais de Sódio/metabolismo , Fosfoproteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Canais Epiteliais de Sódio/genética , Humanos , Síndrome de Liddle/genética , Síndrome de Liddle/metabolismo , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Fosfoproteínas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Ratos Endogâmicos F344 , Ubiquitina-Proteína Ligases/genéticaRESUMO
The trafficking of nutrient transporters is highly regulated and controlled by a variety of signal transduction pathways. A recent study by in Nature Cell Biology demonstrates a role for a novel GTPase-containing complex that allows the amino acid permease Gap1 to recycle out of late endosomal compartments. This complex is likely to play critical roles in allowing animal cells to mobilize their nutrient transporters in response to metabolic demands.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Transporte Biológico Ativo , Endossomos/metabolismo , Genes Fúngicos , Complexos Multiproteicos , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismoRESUMO
Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.