RESUMO
Tight regulation of Rho GTPase-signaling functions requires the proper localization of proteins to the membrane and cytosolic compartments, which can themselves undergo reconfiguration in response to signaling events. The importance of lipid-mediated membrane signal transduction continues to emerge as a critical event in many Rho GTPase-signaling pathways. Here we describe methods for the reconstitution of lipid-modified Rho GTPases with defined lipid vesicles and how this system can be used as a real-time assay for monitoring protein-membrane interactions.
Assuntos
Fluorometria/métodos , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Lipossomos/química , Lipossomos/metabolismo , Lipídeos de Membrana/genética , Ligação Proteica , Transdução de Sinais , Proteínas rho de Ligação ao GTP/isolamento & purificação , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Lipid droplets play a critical role in a variety of metabolic diseases. Numerous proteomic studies have provided detailed information about the protein composition of the droplet, which has revealed that they are functional organelles involved in many cellular processes, including lipid storage and metabolism, membrane traffic, and signal transduction. Thus, the droplet proteome indicates that lipid accumulation is only one of a constellation of organellar functions critical for normal lipid metabolism in the cell. As a result of this new understanding, we suggested the name adiposome for this organelle. The trafficking ability of the adiposome is likely to be very important for lipid uptake, retention, and distribution, as well as membrane biogenesis and lipid signaling. We have taken advantage of the ease of purifying lipid-filled adiposomes to develop a cell-free system for studying adiposome-mediated traffic. Using this approach, we have determined that the interaction between adiposomes and endosomes is dependent on Rab GTPases but is blocked by ATPase. These methods also allowed us to identify multiple proteins that dynamically associate with adiposomes in a nucleotide-dependent manner. An adiposome-endosome interaction in vitro occurs in the absence of cytosolic factors, which simplifies the assay dramatically. This assay will enable researchers to dissect the molecular mechanisms of interaction between these two organelles. This chapter provides a detailed account of the methods developed.
Assuntos
Endossomos/fisiologia , Organelas/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Humanos , Metabolismo dos LipídeosRESUMO
Prenylated Rab proteins exist in the cytosol bound to guanine dissociation inhibitor (GDI). These dimeric complexes contain all of the information needed for accurate membrane delivery. We have shown that membranes contain a proteinaceous activity that is required for Rab delivery, and we named that activity GDI displacement factor (GDF). Biochemical analysis revealed that GDF activity was membrane associated and had a mass of approximately 25 kDa. We therefore used a candidate gene approach and were able to show that pure Yip3/PRA1 protein displays GDF activity. In this chapter, we review key aspects of GDF analysis: our assay and the method by which we purify Yip3/PRA1 in active form.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Transporte Vesicular/isolamento & purificação , Proteínas de Transporte Vesicular/fisiologia , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Inibidores de Dissociação do Nucleotídeo Guanina/química , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/químicaRESUMO
Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their replacement with smaller amino acids lead to effective preparation of X-ray quality crystals of proteins otherwise recalcitrant to crystallization. Furthermore, this technique can also be used to obtain crystals of superior quality as compared to those grown for the wild-type protein, sometimes increasing the effective resolution by as much as 1 A or more. Several recent examples of this new methodology suggest that the method has the potential to become a routine tool in protein crystallography.
Assuntos
Engenharia de Proteínas , Proteínas/isolamento & purificação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Mutação , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/genética , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoAssuntos
Proteínas Ativadoras de GTPase/isolamento & purificação , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Proteínas rab3 de Ligação ao GTP/metabolismo , Animais , Encéfalo/metabolismo , Cromatografia em Camada Fina , Proteínas Ativadoras de GTPase/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , SpodopteraAssuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Adenosina Difosfato Ribose/metabolismo , Sequência de Bases , Expressão Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Nucleotídeos de Guanina/metabolismo , Hidrólise , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/isolamento & purificação , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/isolamento & purificação , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
GTPases of the Rho family play important roles in human leukocyte signal transduction pathways; however, little is known about the function of these proteins in bovine cells. In the present studies, we isolated molecular clones of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP dissociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. These clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences with those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play conserved functional roles in bovine leukocyte signal transduction pathways. Comparative Western blotting of these proteins in human and bovine neutrophil cytosol demonstrated that Rac2 was the predominant Rac species and that D4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the anti-peptide antibody probes prepared against human Rac2 failed to recognize the bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting bovine neutrophils, but is translocated to the plasma membrane after stimulation with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developing more specific probes for investigating the role of these proteins in bovine leukocyte signal transduction pathways and for studying various inflammatory diseases in cattle.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Proteínas rac de Ligação ao GTP/química , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Bovinos , Clonagem Molecular , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Humanos , Dados de Sequência Molecular , Peso Molecular , Ativação de Neutrófilo , Análise de Sequência de DNA , Frações Subcelulares/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/isolamento & purificação , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico , Proteína RAC2 de Ligação ao GTPRESUMO
The yeast two-hybrid system has been useful for identifying many partners and effectors of small GTPases of the Rab family. We describe here such a screen using Rab6, a protein involved in the regulation of intracellular transport at the level of the Golgi apparatus, as bait.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Small GTPases of the Rab family play a key role in controlling vesicular transport, and the Rab GDP-dissociation inhibitor (GDI) is a regulatory protein for the Rab proteins. Here we report the production and characterization of isotype-specific monoclonal antibodies (MAbs) to Rab GDI. Rab GDI was purified from bovine brain in several steps of column chromatography and was injected into BALB/c mice intraperitoneally. The resulting MAbs specifically recognized a single protein band of 55 kDa, which comigrates with purified bovine Rab GDI. To localize Rab GDI, we processed cells from different sources for indirect immunofluorescence microscopy. Interestingly, the MAb stained cytosol and vesicular structures in brain cells, whereas it predominantly stained cytosol in nonbrain cells. Next, we investigated the cross-reactivities of brain Rab GDI from some mammals. The immunoreactive bands on Western blots appeared to be the same in molecular mass, 55 kDa, in all mammalian species tested including human. In summary, we produced a panel of MAbs that are GDI-alpha/1 form-specific and we believe that the MAbs will be valuable tools in elucidating the function of Rab GDI isoforms.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Química Encefálica/imunologia , Bovinos/imunologia , Inibidores de Dissociação do Nucleotídeo Guanina/imunologia , Isotipos de Imunoglobulinas/imunologia , Animais , Especificidade de Anticorpos , Células Cultivadas/química , Células Cultivadas/imunologia , Reações Cruzadas/imunologia , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/análise , Inibidores de Dissociação do Nucleotídeo Guanina/isolamento & purificação , Humanos , Hibridomas/imunologia , Imuno-Histoquímica , Masculino , Mamíferos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Ratos , Especificidade da EspécieRESUMO
Ras-related small GTP-binding proteins execute many cellular functions, such as cell growth, differentiation, cytoskeletal reorganization, membrane trafficking, and membrane fusion. RalA belongs to the superfamily of Ras-related small GTP-binding proteins. Synaptic vesicles (SV) contain small GTP-binding proteins, where RalA, Rab3A, and Rab5A are the major GTP-binding proteins. It has been postulated that a cycling of these proteins between membrane-bound and soluble states is required for regulating cellular functions. Calmodulin (CaM) was found to dissociate Rab3A from SV membranes by forming a 1:1 complex with Ca2+/CaM. RalA was also found to be a Ca2+/CaM-binding protein. Therefore, we examined if Ca2+/CaM can also cause the RalA to dissociate from SV membranes. In this study, we identified that Ca2+/CaM dissociates RalA as well as Rab3A from synaptic vesicles.