RESUMO
Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO-protein conjugation and cleavage can be regulated. Protein desumoylation is carried out by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period to study these enzymes.
Assuntos
Endopeptidases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Endopeptidases/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
The proteasome is an essential regulator of protein homeostasis. In yeast and many mammalian cells, proteasomes strongly concentrate in the nucleus. Sts1 from the yeast Saccharomyces cerevisiae is an essential protein linked to proteasome nuclear localization. Here, we show that Sts1 contains a non-canonical bipartite nuclear localization signal (NLS) important for both nuclear localization of Sts1 itself and the proteasome. Sts1 binds the karyopherin-α import receptor (Srp1) stoichiometrically, and this requires the NLS. The NLS is essential for viability, and over-expressed Sts1 with an inactive NLS interferes with 26S proteasome import. The Sts1-Srp1 complex binds preferentially to fully assembled 26S proteasomes in vitro Sts1 is itself a rapidly degraded 26S proteasome substrate; notably, this degradation is ubiquitin independent in cells and in vitro and is inhibited by Srp1 binding. Mutants of Sts1 are stabilized, suggesting that its degradation is tightly linked to its role in localizing proteasomes to the nucleus. We propose that Sts1 normally promotes nuclear import of fully assembled proteasomes and is directly degraded by proteasomes without prior ubiquitylation following karyopherin-α release in the nucleus.
Assuntos
Sinais de Localização Nuclear , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In eukaryotes, the ubiquitin-proteasome system is an essential pathway for protein degradation and cellular homeostasis. 26S proteasomes concentrate in the nucleus of budding yeast Saccharomyces cerevisiae due to the essential import adaptor protein Sts1 and the karyopherin-α protein Srp1. Here, we show that Sts1 facilitates proteasome nuclear import by recruiting proteasomes to the karyopherin-α/ß heterodimer. Following nuclear transport, the karyopherin proteins are likely separated from Sts1 through interaction with RanGTP in the nucleus. RanGTP-induced release of Sts1 from the karyopherin proteins initiates Sts1 proteasomal degradation in vitro. Sts1 undergoes karyopherin-mediated nuclear import in the absence of proteasome interaction, but Sts1 degradation in vivo is only observed when proteasomes successfully localize to the nucleus. Sts1 appears to function as a proteasome import factor during exponential growth only, as it is not found in proteasome storage granules (PSGs) during prolonged glucose starvation, nor does it appear to contribute to the rapid nuclear reimport of proteasomes following glucose refeeding and PSG dissipation. We propose that Sts1 acts as a single-turnover proteasome nuclear import factor by recruiting karyopherins for transport and undergoing subsequent RanGTP-initiated ubiquitin-independent proteasomal degradation in the nucleus.
Assuntos
Saccharomyces cerevisiae , Saccharomycetales , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , alfa Carioferinas , beta Carioferinas , Glucose , Carioferinas , Complexo de Endopeptidases do Proteassoma , UbiquitinaRESUMO
Targeted protein degradation (TPD) by PROTAC (proteolysis-targeting chimera) and molecular glue small molecules is an emerging therapeutic strategy. To expand the roster of E3 ligases that can be utilized for TPD, we describe the discovery and biochemical characterization of small-molecule ligands targeting the E3 ligase KLHDC2. Furthermore, we functionalize these KLHDC2-targeting ligands into KLHDC2-based BET-family and AR PROTAC degraders and demonstrate KLHDC2-dependent target-protein degradation. Additionally, we offer insight into the assembly of the KLHDC2 E3 ligase complex. Using biochemical binding studies, X-ray crystallography and cryo-EM, we show that the KLHDC2 E3 ligase assembles into a dynamic tetramer held together via its own C terminus, and that this assembly can be modulated by substrate and ligand engagement.
Assuntos
Ubiquitina-Proteína Ligases , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , LigantesRESUMO
The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.
Assuntos
Adenosina Trifosfatases/metabolismo , Lipídeos/fisiologia , Fusão de Membrana/fisiologia , Chaperonas Moleculares/fisiologia , Proteínas SNARE/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/química , Genes Fúngicos , Lipídeos/química , Lipossomos , Chaperonas Moleculares/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Ligação Proteica , Transporte Proteico , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/fisiologia , Proteínas Qb-SNARE/química , Proteínas Qb-SNARE/fisiologia , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/fisiologia , Proteínas R-SNARE/química , Proteínas R-SNARE/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , Proteínas de Transporte Vesicular/químicaRESUMO
Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.
Assuntos
Fusão de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Adenosina Trifosfatases/metabolismo , Guanosina Trifosfato/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Proteolipídeos/metabolismo , Proteolipídeos/ultraestrutura , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas de Transporte Vesicular/metabolismoRESUMO
Proteolysis-targeting chimera (PROTAC®) protein degraders are heterobifunctional small molecules that bind a specific target protein on one end and a specific ubiquitin ligase enzyme (E3) on the other, thereby driving intracellular degradation of the target protein via the ubiquitin-proteasome system. PROTACs and other small molecule protein degraders are being developed as potential therapeutics for several diseases, with the first PROTACs having entered the clinic for cancer treatments in 2019. While humans express approximately 600 E3s, only a few have been used for protein degrader technology. A major challenge to designing degraders based on additional E3s is the development of quality ligands for other E3s. Most methods to screen for novel ligands employ purified forms of the protein of interest. Ligands discovered in this manner are typically subsequently evaluated in cultured cells. Optimal ligands efficiently cross biological membranes and interact specifically with the protein of interest, which can be assessed by a variety of cell-based methods. Functionality and specificity of ligand-protein interactions can also be evaluated using cell or tissue extracts and affinity beads based on the ligand, as described here. E3 affinity beads described herein are based on conjugation of the potential E3 ligand to biotin and commercially available streptavidin agarose with high affinity for biotin.
Assuntos
Ubiquitina/metabolismo , Biotina , Humanos , Ligantes , Proteínas/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Intracellular proteolysis by the ubiquitin-proteasome system regulates numerous processes and contributes to protein quality control (PQC) in all eukaryotes. Covalent attachment of ubiquitin to other proteins is specified by the many ubiquitin ligases (E3s) expressed in cells. Here we determine the E3s in Saccharomyces cerevisiae that function in degradation of proteins bearing various PQC degradation signals (degrons). The E3 Ubr1 can function redundantly with several E3s, including nuclear-localized San1, endoplasmic reticulum/nuclear membrane-embedded Doa10, and chromatin-associated Slx5/Slx8. Notably, multiple degrons are targeted by more ubiquitylation pathways if directed to the nucleus. Degrons initially assigned as exclusive substrates of Doa10 were targeted by Doa10, San1, and Ubr1 when directed to the nucleus. By contrast, very short hydrophobic degrons-typical targets of San1-are shown here to be targeted by Ubr1 and/or San1, but not Doa10. Thus, distinct types of PQC substrates are differentially recognized by the ubiquitin system in a compartment-specific manner. In human cells, a representative short hydrophobic degron appended to the C-terminus of GFP-reduced protein levels compared with GFP alone, consistent with a recent study that found numerous natural hydrophobic C-termini of human proteins can act as degrons. We also report results of bioinformatic analyses of potential human C-terminal degrons, which reveal that most peptide substrates of Cullin-RING ligases (CRLs) are of low hydrophobicity, consistent with previous data showing CRLs target degrons with specific sequences. These studies expand our understanding of PQC in yeast and human cells, including the distinct but overlapping PQC E3 substrate specificity of the cytoplasm and nucleus.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Sinais Direcionadores de Proteínas , Proteólise , Ubiquitinação , Transporte Ativo do Núcleo Celular , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Like many transcription factors, the yeast protein MATalpha2 (α2) undergoes rapid proteolysis via the ubiquitin-proteasome system (UPS). At least two ubiquitylation pathways regulate α2 degradation: one pathway utilizes the ubiquitin ligase (E3) Doa10 and the other the heterodimeric E3 Slx5/Slx8. Doa10 is a transmembrane protein of the endoplasmic reticulum/inner nuclear membrane, whereas Slx5/Slx8 localizes to the nucleus and binds DNA nonspecifically. While a single protein can often be ubiquitylated by multiple pathways, the reasons for this "division of labor" are not well understood. Here we show that α2 mutants with impaired DNA binding become inaccessible to the Slx5/Slx8 pathway but are still rapidly degraded through efficient shunting to the Doa10 pathway. These results are consistent with the distinct localization of these E3s. We also characterized a novel class of DNA binding-defective α2 variants whose degradation is strongly impaired. Our genetic data suggest that this is due to a gain-of-function interaction that limits their access to Doa10. Together, these results suggest multiple ubiquitin-ligation mechanisms may have evolved to promote rapid destruction of a transcription factor that resides in distinct cellular subcompartments under different conditions. Moreover, gain-of-function mutations, which also occur with oncogenic forms of human transcription factors such as p53, may derail this fail-safe system.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Retículo Endoplasmático/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , UbiquitinaçãoRESUMO
Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mycobacterium/genética , Proteínas Repressoras/metabolismo , Tetraciclinas/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Relação Dose-Resposta a Droga , Macrófagos/microbiologia , Camundongos , Mycobacterium smegmatis/citologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Regiões Operadoras Genéticas , Fagossomos/microbiologia , Regiões Promotoras GenéticasRESUMO
Elaborate control of gene expression by transcription factors is common to all kingdoms of life. In eukaryotes, transcription factor abundance and activity are often regulated by targeted proteolysis via the ubiquitin-proteasome system (UPS). The yeast MATα2 (α2) cell type regulator has long served as a model for UPS-dependent transcription factor degradation. Proteolysis of α2 is complex: it involves at least 2 ubiquitylation pathways and multiple regions of α2 affect its degradation. Such complexity also exists for the degradation of other UPS substrates. Here I review α2 degradation, most notably our recent identification of 2 novel degradation elements within α2 that overlap corepressor binding sites. I discuss possible implications of these findings and consider how principles of α2 proteolysis may be relevant to the degradation of other UPS substrates.
RESUMO
In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2Δ and ulp2Δ slx5Δ cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2Δ,slx5Δ, and ulp2Δ slx5Δ cells. The increase in sumoylation of Net1 and Tof2 in ulp2Δ, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2 Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2Δ cells and both factors largely regain this association in ulp2Δ slx5Δ A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2Δ cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2Δ cells, which is also rescued by elimination of Slx5 Additionally, overexpression of Sir2, another RFB-associated factor, suppresses the growth defect of ulp2Δ cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2Δ cellular defects.
Assuntos
DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/genética , DNA Ribossômico/química , Endopeptidases/metabolismo , Regulação Fúngica da Expressão Gênica , Ligação Proteica , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Especificidade por Substrato , SumoilaçãoRESUMO
The yeast transcription factor MATα2 (α2) is a short-lived protein known to be ubiquitylated by two distinct pathways, one involving the ubiquitin-conjugating enzymes (E2s) Ubc6 and Ubc7 and the ubiquitin ligase (E3) Doa10 and the other operating with the E2 Ubc4 and the heterodimeric E3 Slx5/Slx8. Although Slx5/Slx8 is a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL), it does not require SUMO to target α2 but instead directly recognizes α2. Little is known about the α2 determinants required for its Ubc4- and STUbL-mediated degradation or how these determinants substitute for SUMO in recognition by the STUbL pathway. We describe two distinct degradation elements within α2, both of which are necessary for α2 recognition specifically by the Ubc4 pathway. Slx5/Slx8 can directly ubiquitylate a C-terminal fragment of α2, and mutating one of the degradation elements impairs this ubiquitylation. Surprisingly, both degradation elements identified here overlap specific interaction sites for α2 corepressors: the Mcm1 interaction site in the central α2 linker and the Ssn6 (Cyc8) binding site in the α2 homeodomain. We propose that competitive binding to α2 by the ubiquitylation machinery and α2 cofactors is balanced so that α2 can function in transcription repression yet be short lived enough to allow cell-type switching.
Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Ligação Competitiva , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , UbiquitinaçãoRESUMO
Vacuole homotypic fusion has been reconstituted with all purified components: vacuolar lipids, four soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Sec17p, Sec18p, the Rab Ypt7p, and the hexameric homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a Rab-effector with direct affinity for SNAREs (presumably via its Sec1-Munc18 homologous subunit Vps33p) and for certain vacuolar lipids. Each of these pure vacuolar proteins was required for optimal proteoliposome clustering, raising the question of which was most directly involved. We now present model subreactions of clustering and fusion that reveal that HOPS is the direct agent of tethering. The Rab and vacuole lipids contribute to tethering by supporting the membrane association of HOPS. HOPS indirectly facilitates trans-SNARE complex formation by tethering membranes, because the synthetic liposome tethering factor polyethylene glycol can also stimulate trans-SNARE complex formation and fusion. SNAREs further stabilize the associations of HOPS-tethered membranes. HOPS then protects newly formed trans-SNARE complexes from disassembly by Sec17p/Sec18p.
Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vacúolos/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Yeast vacuole fusion requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids, and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1-46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, although Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.
Assuntos
Fusão de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the K(m) value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Q(c)). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Q(a)) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Trifosfato de Adenosina/farmacologia , Motivos de Aminoácidos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas SNARE/química , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Vacúolos/efeitos dos fármacosRESUMO
SNARE proteins form bundles of four alpha-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q.1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7(Q283R), yet Vam7(Q283R) is preferred by vacuoles with Nyv1(R191Q). Rotation of the position of the arginine in the 0-layer increases the K(m) for Vam7p but does not affect the maximal rate of fusion. Vam7(Q283R) forms stable 2Q.2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion.
Assuntos
Bicamadas Lipídicas/metabolismo , Fusão de Membrana , Mutação , Proteínas SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Clorpromazina/farmacologia , Antagonistas de Dopamina/farmacologia , Estrenos/farmacologia , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Proteínas SNARE/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Vacúolos/genéticaRESUMO
Macrophages respond to several subcellular products of Mycobacterium tuberculosis (Mtb) through TLR2 or TLR4. However, primary mouse macrophages respond to viable, virulent Mtb by pathways largely independent of MyD88, the common adaptor molecule for TLRs. Using microarrays, quantitative PCR, and ELISA with gene-disrupted macrophages and mice, we now show that viable Mtb elicits the expression of inducible NO synthase, RANTES, IFN-inducible protein 10, immune-responsive gene 1, and many other key genes in macrophages substantially independently of TLR2, TLR4, their combination, or the TLR adaptors Toll-IL-1R domain-containing adapter protein and Toll-IL-1R domain-containing adapter inducing IFN-beta. Mice deficient in both TLR2 and TLR4 handle aerosol infection with viable Mtb as well as congenic controls. Viable Mtb also up-regulates inducible NO synthase, RANTES, IFN-inducible protein 10, and IRG1 in macrophages that lack mannose receptor, complement receptors 3 and 4, type A scavenger receptor, or CD40. These MyD88, TLR2/4-independent transcriptional responses require IFN-alphabetaR and STAT1, but not IFN-gamma. Conversely, those genes whose expression is MyD88 dependent do not depend on IFN-alphabetaR or STAT1. Transcriptional induction of TNF is TLR2/4, MyD88, STAT1, and IFN-alphabetaR independent, but TNF protein release requires the TLR2/4-MyD88 pathway. Thus, macrophages respond transcriptionally to viable Mtb through at least three pathways. TLR2 mediates the responses of a numerically minor set of genes that collectively do not appear to affect the course of infection in mice; regulation of TNF requires TLR2/4 for post-transcriptional control, but not for transcriptional induction; and many responding genes are regulated through an unknown, TLR2/4-independent pathway that may involve IFN-alphabetaR and STAT1.