RESUMO
Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209-23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Lisossomos/metabolismo , Melanócitos/ultraestrutura , Melanossomas/ultraestrutura , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.
Assuntos
Melanossomas/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Membranas Intracelulares/enzimologia , Melanócitos/enzimologia , Transporte Proteico , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/fisiologia , Proteínas rab27 de Ligação ao GTPRESUMO
We have evaluated the performance of the prototype In Vitro MicroFlow Kit (Litron Laboratories), which offers a flow cytometric method for scoring micronuclei (MN). This method uses sequential staining to differentiate MN from chromatin fragments derived from apoptotic or necrotic cells. Data were generated using the genotoxins methylmethane sulphonate (MMS), dimethylbenzanthracene (DMBA) and vinblastine, and the non-genotoxins dexamethasone and staurosporine, which are known to induce apoptosis in vitro. The results obtained with these agents were compared with conventional microscopy. For short-duration exposures (3-4h) both manual and flow methodologies demonstrated good concordance, with concentration-related increases in the percentage of MN for MMS, DMBA and vinblastine. Statistically significant increases were observed at > or = 20 and 40 microg/mL, for manual and flow analysis, respectively, for MMS; at 0.5 and 0.75 microg/mL for DMBA; and at 0.035 and 0.04 microg/mL, respectively, for vinblastine. Dexamethasone showed clear negative responses by manual and flow cytometric analysis, with comparable results for both methodologies (all <1.7-fold compared with concurrent vehicle controls). Data for staurosporine, however, were less consistent showing significantly higher flow cytometric MN frequencies compared with those seen after manual analysis. Continuous (24 h) treatments were also conducted with MMS, vinblastine, dexamethasone and staurosporine. There was good concordance between the methodologies for MMS, staurosporine and vinblastine. However, dexamethasone generated discordant results, i.e. microscopic analysis was clearly negative at all doses tested, whereas flow cytometry produced significant increases in MN frequency (up to 8.1-fold at 100 microg/mL compared with the concurrent vehicle control). The inconsistencies observed between flow cytometry and standard microscopy, and the differences in assay sensitivity, particularly for apoptosis-inducing compounds, suggest that the prototype In Vitro MicroFlow Kit requires further refinement. Studies to investigate new parameters to address these issues are now under way and will be reported separately.
Assuntos
Citometria de Fluxo/métodos , Micronúcleos com Defeito Cromossômico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de TempoRESUMO
The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.
Assuntos
Melanócitos/metabolismo , Melanossomas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Humanos , Immunoblotting , Camundongos , Microscopia Confocal , Mutação , Ligação Proteica , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.