RESUMEN
UNLABELLED: Ubiquitination and proteasomal degradation have recently emerged as an additional level of regulation of activated forms of Rho GTPases. To characterize this novel regulatory pathway and to gain insight into its biological significance, we studied the ubiquitination of two constitutively activated forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumorigenic splice variant Rac1b, which is defective for several downstream signaling pathways, including JNK activation. Whereas Rac1L61 undergoes polyubiquitination and subsequent proteasomal degradation in HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more resistant to proteasomal degradation than Rac1L61. Mutational analysis of all lysine residues in Rac1 revealed that the major target site for Rac1 ubiquitination is Lys147, a solvent-accessible residue that has a similar conformation in Rac1b. Like Rac1L61, Rac1b was found to be largely associated with plasma membrane, a known prerequisite for Rac1 ubiquitination. Interestingly, Rac1b ubiquitination could be stimulated by coexpression of Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1 downstream signaling. Indeed, ubiquitination of Rac1L61 is critically dependent on JNK activation. IN CONCLUSION: (a) Rac1b appears to be more stable than Rac1L61 with regard to the ubiquitin-proteasome system, and this may be of importance for the expression and tumorigenic capacity of Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK-activated process, which may explain the defective ubiquitination of Rac1b. The JNK-dependent activation of Rac1 ubiquitination would create a regulatory loop allowing the cell to counteract excessive activation of Rac1 GTPase.
Asunto(s)
Lisina/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Western Blotting , Línea Celular , Activación Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Isoformas de Proteínas/química , Proteína de Unión al GTP rac1/químicaRESUMEN
Rnd proteins are atypical members of the Rho small G protein family that inhibit the formation of actomyosin contractile fibers via activation of RhoGAPs and inhibition of a Rho effector, the Ser/Thr kinase Rock. These mechanisms might be used to fine-tune Rho GTPase inhibition locally at sites where particular actin structures need to be made.
Asunto(s)
Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Animales , Activación Enzimática , Proteínas Activadoras de GTPasa/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rhoRESUMEN
The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell-cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four-point-one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two-hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1-dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP-bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N-terminal NPAY motif interacts with its C-terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)-1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP-1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP-1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N- and C-termini and that Rap1 and ICAP-1 inhibit Krit1 binding to microtubules. Consistently, YFP-Krit1 localizes on cyan fluorescent protein-labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5-P(2)-containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP-1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Sitios de Unión , Cricetinae , Membranas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.
Asunto(s)
Adhesiones Focales/metabolismo , Fosfolipasa D/metabolismo , Fibras de Estrés/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Línea Celular , Células HeLa , Humanos , Inmunoprecipitación , Fosfolipasa D/efectos de los fármacos , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP rhoA/farmacologíaRESUMEN
We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin-mediated cell adhesion, EFA6 is recruited to a Triton X-100-insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Elongación de Péptidos/metabolismo , Uniones Estrechas/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Cadherinas/metabolismo , Calcio/metabolismo , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Polaridad Celular/fisiología , Células Cultivadas , Perros , Endocitosis/fisiología , Proteínas de la Membrana/metabolismo , Microscopía Confocal , OcludinaRESUMEN
Aluminum fluoride (AlFx) is known to activate directly the alpha subunit of G-proteins but not the homologous small GTP-binding proteins. However, AlFx can stabilize complexes formed between Ras, RhoA or Cdc42 and their corresponding GTPase-activating proteins (GAPs). Here, we demonstrate that Arf1GDP can be converted into an active conformation by AlFx to form a complex with the Arf-GAP ASAP1 in vitro and in vivo. Within this complex ASAP1, which GAP activity is inoperative, can still alter the recruitment of paxillin to the focal complexes, thus indicating that ASAP1 interferes with focal complexes independently of its GAP activity.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Compuestos de Aluminio/farmacología , Fluoruros/farmacología , Proteínas Activadoras de GTPasa/metabolismo , Factores de Ribosilacion-ADP/efectos de los fármacos , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Cultivadas , Cricetinae , Proteínas Activadoras de GTPasa/efectos de los fármacos , Proteínas Activadoras de GTPasa/genética , TransfecciónRESUMEN
Today, gorillas and chimpanzees live in tropical forests, where acid soils do not favor fossilization. It is thus widely believed that there are no fossils of chimpanzees or gorillas. However, four teeth of a 0.5-million-year (Ma)-old chimpanzee were discovered in the rift valley of Kenya (McBrearty and Jablonski, 2005), and a handful of teeth of a 10-Ma-old gorilla-like creature were found in Ethiopia (Suwa et al., 2007), close to the major sites of Homo discoveries. These discoveries indicate that chimpanzees and gorillas once shared their range with early Homo. However, the thousands of hominin fossils discovered in the past century have all been attributed to the Homo line. Thus far, our family tree looks like a bush with many dead-branches. If one admits the possibility that the australopithecines can also be the ancestors of African great apes, one can place Paranthropus on the side of gorilla ancestors and divide the remaining Australopithecus based on the brain size into the two main lines of humans and chimpanzees, thereby resulting in a coherent family tree.
Asunto(s)
Encéfalo/anatomía & histología , Hominidae/anatomía & histología , Hominidae/clasificación , Cráneo/anatomía & histología , África , Animales , Evolución Biológica , Fósiles , Gorilla gorilla/anatomía & histología , Gorilla gorilla/clasificación , Humanos , Modelos Biológicos , Tamaño de los Órganos , Pan troglodytes/anatomía & histología , Pan troglodytes/clasificación , DienteAsunto(s)
Proteínas Fúngicas/química , Glicoproteínas de Membrana/química , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Saccharomyces cerevisiae , Animales , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Fúngicas/genética , Factores de Intercambio de Guanina Nucleótido , Humanos , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Pliegue de Proteína , Estructura Secundaria de Proteína/fisiología , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular , LevadurasRESUMEN
Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.
Asunto(s)
Factor 1 de Ribosilacion-ADP/antagonistas & inhibidores , Factor 1 de Ribosilacion-ADP/química , Factores de Intercambio de Guanina Nucleótido/química , Factor 1 de Ribosilacion-ADP/metabolismo , Compuestos de Anilina/farmacología , Animales , Bencimidazoles/farmacología , Brefeldino A/farmacología , Línea Celular , Movimiento Celular/efectos de los fármacos , Simulación por Computador , Perros , Relación Dosis-Respuesta a Droga , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas/antagonistas & inhibidores , ARN Interferente Pequeño/metabolismo , Sensibilidad y Especificidad , Relación Estructura-Actividad , Factores de TiempoRESUMEN
The Rnd proteins, which form a distinct sub-group of the Rho family of small GTP-binding proteins, have been shown to regulate the organization of the actin cytoskeleton in several tissues. In the brain, they participate in neurite extension, whereas in smooth muscle, they modulate contractility. Recent evidence has shown that Rnd3 (RhoE) is also involved in the regulation of cell-cycle progression and transformation, indicating that these proteins might have other, as yet unexplored roles.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Axones/fisiología , Axones/ultraestructura , Ciclo Celular , Humanos , Neuritas/fisiología , Neuritas/ultraestructuraRESUMEN
We have analyzed both biochemically and functionally a series of Arf6 mutants, providing new insights into the molecular mode of action of the small G protein Arf6. First, by comparing a fast-cycling mutant (Arf6(T157N)) and a GTPase-deficient mutant (Arf6(Q67L)), we established the necessity for completion of the Arf6 GDP/GTP cycle for recycling of major histocompatibility complex molecules to the plasma membrane. Second, we found that aluminum fluoride (AlF), known for inducing membrane protrusion in cells expressing exogenous wild-type Arf6, stabilized a functional wild-type Arf6.AlF(x) . GTPase-activating protein (GAP) complex in vitro and in vivo. We also found that the tandem mutation Q37E/S38I prevented the binding of two Arf GAPs, but not the effector ARHGAP10, and blocked the formation of membrane protrusion and actin reorganization. Together, our results with AlF(x) and Arf6(Q37E/S38I) demonstrate the critical role of the Arf6 GAPs as effectors for Arf6-regulated actin cytoskeleton remodeling. Finally, competition experiments conducted in vivo suggest the existence of a membrane receptor for GDP-bound Arf6.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Actinas/metabolismo , Proteínas Activadoras de GTPasa/química , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Actinas/química , Compuestos de Aluminio/farmacología , Animales , Transporte Biológico , Línea Celular , Cricetinae , Citoesqueleto/metabolismo , Fluoruros/farmacología , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Difosfato/química , Células HeLa , Humanos , Mutación , Fosfolípidos/química , Unión Proteica , Proteínas Recombinantes/químicaRESUMEN
The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.
Asunto(s)
Factores de Ribosilacion-ADP/fisiología , Proteínas de Unión al ADN/química , Factores de Transcripción/química , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Encéfalo/metabolismo , Membrana Celular/metabolismo , Clatrina/química , Cricetinae , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Endocitosis , Glutatión Transferasa/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Cinética , Liposomas/química , Liposomas/metabolismo , Microscopía Confocal , Mutación , Fosfatidilinositoles/química , Unión Proteica , Transporte de Proteínas , Receptores de Transferrina/metabolismo , Factores de Tiempo , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo , Transfección , Transferrina/metabolismoRESUMEN
Small G proteins of the Rho/Rac/Cdc42 family are associated with lipid membranes through their prenylated C termini. Alternatively, these proteins form soluble complexes with GDI proteins. To assess how this membrane partitioning influences the activation of Rac by guanine nucleotide exchange factors, GDP-to-GTP exchange reactions were performed in the presence of liposomes using different forms of Rac-GDP. We show that both non-prenylated Rac-GDP and the soluble complex between prenylated Rac-GDP and GDI are poorly activated by the Dbl homology-pleckstrin homology (DH-PH) domain of the exchange factor Tiam1, whereas prenylated Rac-GDP bound to liposomes is activated about 10 times more rapidly. Sedimentation experiments with liposomes reveal that the DH-PH region of Tiam1 forms, with nucleotide-free prenylated Rac, a membrane-bound complex from which GDI is excluded. Taken together, these experiments demonstrate that the dissociation of Rac-GDP from GDI and its translocation to membrane lipids favor DH-PH-catalyzed nucleotide exchange because the steric hindrance caused by GDI is relieved and because the membrane environment favors functional interaction between the DH-PH domain and the small G protein.
Asunto(s)
Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Proteínas/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas Sanguíneas/genética , Regulación de la Expresión Génica , Inhibidores de Disociación de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido , Humanos , Liposomas , Fosfoproteínas/genética , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas , Proteínas Oncogénicas de Retroviridae/genética , Saccharomyces cerevisiae , Homología de Secuencia , Transducción de Señal/genética , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Transfección , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rho/metabolismo , Inhibidores de la Disociación del Nucleótido Guanina rho-EspecíficoRESUMEN
The function of Arf6 has been investigated largely by using the T27N and the Q67L mutants, which are thought to be blocked in GDP- and GTP-bound states, respectively. However, these mutants have been poorly characterized biochemically. Here, we found that Arf6(T27N) is not an appropriate marker of the inactive GDP-bound form because it has a high tendency to lose its nucleotide in vitro and to denature. As a consequence, most of the protein is aggregated in vivo and localizes to detergent-insoluble structures. However, a small proportion of Arf6(T27N) is able to form a stable complex with its exchange factor EFA6 at the plasma membrane, accounting for its dominant-negative phenotype. To define the cellular localization of Arf6-GDP, we designed a new mutant, Arf6(T44N). In vitro, this mutant has a 30-fold decreased affinity for GTP. In vivo, it is mostly GDP bound and, in contrast to the wild type, does not switch to the active conformation when expressed with EFA6. This GDP-locked mutant is found at the plasma membrane, where it localizes with EFA6 and Ezrin in actin- and phosphatidylinositol (4,5)-bisphosphate-enriched domains. From these results, we conclude that the Arf6 GDP-GTP cycle takes place at the plasma membrane.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Membrana Celular/metabolismo , Guanosina Difosfato/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Actinas/metabolismo , Animales , Sitios de Unión , Línea Celular , Membrana Celular/enzimología , Cricetinae , Proteínas del Citoesqueleto , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Trifosfato/metabolismo , Mutación/genética , Factores de Elongación de Péptidos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Treonina/genética , Treonina/metabolismo , TransfecciónRESUMEN
The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP -null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2-MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Espermátides/metabolismo , Espermatocitos/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Hibridación in Situ , Masculino , Ratones , Espermatogonias/metabolismo , Testículo/metabolismoRESUMEN
A phenotypic screen was used to search for drug-like molecules that can interfere with specific steps in membrane traffic. 2-(4-Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), identified in this screen, induces a rapid collapse of the Golgi to the endoplasmic reticulum, thus acutely inhibiting the traffic emanating from the endoplasmic reticulum. Like Brefeldin A (BFA), Exo1 induces the rapid release of ADP-ribosylation factor (ARF) 1 from Golgi membranes but has less effect on the organization of the trans-Golgi network. Our data indicate that Exo1 acts by a different mechanism from BFA. Unlike BFA, Exo1 does not induce the ADP-ribosylation of CtBP/Bars50 and does not interfere with the activity of guanine nucleotide exchange factors specific for Golgi-based ARFs. Thus, Exo1 allows the fatty acid exchange activity of Bars50 to be distinguished from ARF1 activity in the control of Golgi tubulation.