scholarly journals Rab1b Interacts with GBF1 and Modulates both ARF1 Dynamics and COPI Association

2007 ◽  
Vol 18 (7) ◽  
pp. 2400-2410 ◽  
Author(s):  
Pablo Monetta ◽  
Ileana Slavin ◽  
Nahuel Romero ◽  
Cecilia Alvarez
Keyword(s):  
Time Lapse ◽  
Limited Range ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Golgi Membranes ◽  
Golgi Transport ◽  
Nucleotide Exchange Factor ◽  
Er Exit Sites ◽  
Interfering Rna

Assembly of the cytosolic coat protein I (COPI) complex at the ER–Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t1/2 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.

scholarly journals Coordinated regulation of AP2 uncoating from clathrin-coated vesicles by rab5 and hRME-6

2008 ◽  
Vol 183 (3) ◽  
pp. 499-511 ◽  
Author(s):  
Sophia Semerdjieva ◽  
Barry Shortt ◽  
Emma Maxwell ◽  
Sukhdeep Singh ◽  
Paul Fonarev ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Coated Vesicles ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Steady State Levels ◽  
Endocytic Vesicles ◽  
Interfering Rna

Here we investigate the role of rab5 and its cognate exchange factors rabex-5 and hRME-6 in the regulation of AP2 uncoating from endocytic clathrin-coated vesicles (CCVs). In vitro, we show that the rate of AP2 uncoating from CCVs is dependent on the level of functional rab5. In vivo, overexpression of dominant-negative rab5S34N, or small interfering RNA (siRNA)–mediated depletion of hRME-6, but not rabex-5, resulted in increased steady-state levels of AP2 associated with endocytic vesicles, which is consistent with reduced uncoating efficiency. hRME-6 guanine nucleotide exchange factor activity requires hRME-6 binding to α-adaptin ear, which displaces the ear-associated μ2 kinase AAK1. siRNA-mediated depletion of hRME-6 increases phospho-μ2 levels, and expression of a phosphomimetic μ2 mutant increases levels of endocytic vesicle-associated AP2. Depletion of hRME-6 or rab5S35N expression also increases the levels of phosphoinositide 4,5-bisphosphate (PtdIns(4,5)P2) associated with endocytic vesicles. These data are consistent with a model in which hRME-6 and rab5 regulate AP2 uncoating in vivo by coordinately regulating μ2 dephosphorylation and PtdIns(4,5)P2 levels in CCVs.

scholarly journals Concentration of Sec12 at ER exit sites via interaction with cTAGE5 is required for collagen export

2014 ◽  
Vol 206 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Kota Saito ◽  
Koh Yamashiro ◽  
Noriko Shimazu ◽  
Tomoya Tanabe ◽  
Kenji Kontani ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Secretory Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Transport Vesicles ◽  
Nucleotide Exchange Factor ◽  
Receptor Component ◽  
Er Exit Sites ◽  
Guanosine Triphosphatase ◽  
Trimeric Structure

Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

scholarly journals Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1

2003 ◽  
Vol 160 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Metello Innocenti ◽  
Emanuela Frittoli ◽  
Isabella Ponzanelli ◽  
John R. Falck ◽  
Saskia M. Brachmann ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Downstream Target ◽  
Signaling Complex ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

scholarly journals A conserved and regulated mechanism drives endosomal Rab transition

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lars Langemeyer ◽  
Ann-Christin Borchers ◽  
Eric Herrmann ◽  
Nadia Füllbrunn ◽  
Yaping Han ◽  
...  
Keyword(s):  
Underlying Mechanism ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Molecular Determinants ◽  
Key Factor ◽  
Membrane Bound ◽  
Endosome Maturation

Endosomes and lysosomes harbor Rab5 and Rab7 on their surface as key proteins involved in their identity, biogenesis, and fusion. Rab activation requires a guanine nucleotide exchange factor (GEF), which is Mon1-Ccz1 for Rab7. During endosome maturation, Rab5 is replaced by Rab7, though the underlying mechanism remains poorly understood. Here, we identify the molecular determinants for Rab conversion in vivo and in vitro, and reconstitute Rab7 activation with yeast and metazoan proteins. We show (i) that Mon1-Ccz1 is an effector of Rab5, (ii) that membrane-bound Rab5 is the key factor to directly promote Mon1-Ccz1 dependent Rab7 activation and Rab7-dependent membrane fusion, and (iii) that this process is regulated in yeast by the casein kinase Yck3, which phosphorylates Mon1 and blocks Rab5 binding. Our study thus uncovers the minimal feed-forward machinery of the endosomal Rab cascade and a novel regulatory mechanism controlling this pathway.

scholarly journals cTAGE5 acts as a Sar1 GTPase regulator for collagen export

2018 ◽  
Author(s):  
Norito Sasaki ◽  
Masano Shiraiwa ◽  
Miharu Maeda ◽  
Tomohiro Yorimitsu ◽  
Ken Sato ◽  
...  
Keyword(s):  
Small Gtpase ◽  
Coated Vesicles ◽  
Secretory Proteins ◽  
Efficient Production ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanine Nucleotide Exchange ◽  
Collagen Secretion ◽  
Nucleotide Exchange Factor ◽  
Er Exit Sites

AbstractSecretory proteins synthesized within the endoplasmic reticulum (ER) are exported via coat protein complex II (COPII)-coated vesicles. The formation of the COPII-coated vesicles is initiated by activation of the small GTPase, Sar1. cTAGE5 directly interacts with a guanine-nucleotide exchange factor (GEF), Sec12, and a GTPase-activating protein (GAP) of Sar1, Sec23. We have previously shown that cTAGE5 recruits Sec12 to the ER exit sites for efficient production of activated Sar1 for collagen secretion. However, the functional significance of the interaction between cTAGE5 and Sec23 has not been fully elucidated. In this study, we showed that cTAGE5 enhances the GAP activity of Sec23 toward Sar1. In addition, the interaction of cTAGE5 with Sec23 is necessary for collagen exit from the ER. Our data suggests that cTAGE5 acts as a Sar1 GTPase regulator for collagen secretion.

Abstract 303: HDL Mimetic Peptide 4F Rescues Pre-existing Pulmonary Hypertension via MicroRNA 193-3p

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Salil Sharma ◽  
Soban Umar ◽  
Gabe Wong ◽  
Denise Mai ◽  
Mohamad Navab ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Intratracheal Instillation ◽  
Nucleotide Exchange ◽  
Mimetic Peptide ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Human Pulmonary Artery ◽  
Proliferation In Vitro

Pulmonary hypertension (PH) is a chronic lung disease associated with severe vascular disorders leading to right ventricular(RV) failure. An HDL mimetic peptide, 4F, has been shown to be effective for the treatment of atherosclerosis and a number of inflammatory disorders. Here, we explored whether 4F can rescue advanced PH by controlling the expression of specific microRNAs (miRs). PH was induced in rats by a single injection of monocrotaline (MCT, 60mg/kg, s.c .) or by placing mice in hypoxia chamber(O2≤10%) for 21 days. MCT-rats or hypoxic mice received 4F therapy (50mg/kg/day, s.c .,days 21-30 in MCT model and days 14-21 in hypoxia model). We performed microRNA microarray analysis (non-affymetrix) in lung tissues of CTRL, PH and 4F-rescued groups. OE of miR193 was achieved by intratracheal instillation of 20nM dose at days 16, 21 and 26 in MCT model or at days 14 and 18 in hypoxia model. 4F therapy starting after the establishment of PH in both MCT and hypoxia models improved significantly RV pressure (RVP) and RV hypertrophy index (RVP=46±3 vs RVP=74±1 mmHg in PH; RV/LV+IVS=0.38±0.02 vs RV/(LV+IVS)=0.68±0.05 in PH, p<0.05 vs PH and in hypoxia model RVP=22±3.8 vs. 36.91±5.74 in PH and 20.93±2.52mmHg in ctrl, p<0.05 vs PH ). Microarray and qPCR showed downregulation of miR-193 by ~3 fold in MCT model. 4F therapy normalized miR-193 to ctrl levels. MiR-193 OE in both MCT-rats and hypoxic-mice rescued PH (RVP=38±5.5mmHg, RV/LV+IVS=0.37±0.034 in MCT-rats and RVP=25.48±0.88mmHg in hypoxic-mice). Lung sections showed increased arteriolar muscularization and ox-LDL deposition in the PH group, prevented by miR-193 therapy. In vivo, OE of miR-193 suppressed transcription of in-silico targets ALOX5, a lipoxygenase; IGF1R, insulin-like growth factor 1 receptor and ARHGEF12, a Rho guanine nucleotide exchange factor and decreased human pulmonary artery smooth muscle cell (HPASMC) proliferation in vitro in the presence of serum or 12-HETE by >2 folds whereas miR-193 KD increased proliferation. In conclusion, 4F rescues pre-existing severe PH by targeting genes associated with HETEs and HODEs production, inflammation and growth via inducing miR-193.

scholarly journals Ccpg1, a Novel Scaffold Protein That Regulates the Activity of the Rho Guanine Nucleotide Exchange Factor Dbs

2006 ◽  
Vol 26 (23) ◽  
pp. 8964-8975 ◽  
Author(s):  
Elena V. Kostenko ◽  
Oyenike O. Olabisi ◽  
Sutapa Sahay ◽  
Pedro L. Rodriguez ◽  
Ian P. Whitehead
Keyword(s):  
Family Member ◽  
Guanine Nucleotide Exchange Factor ◽  
Scaffold Protein ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Novel Scaffold ◽  
Exchange Activity

ABSTRACT Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.

scholarly journals The eps8 Family of Proteins Links Growth Factor Stimulation to Actin Reorganization Generating Functional Redundancy in the Ras/Rac Pathway

2004 ◽  
Vol 15 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Nina Offenhäuser ◽  
Alessandro Borgonovo ◽  
Andrea Disanza ◽  
Pascale Romano ◽  
Isabella Ponzanelli ◽  
...  
Keyword(s):  
Growth Factor ◽  
Functional Redundancy ◽  
Nucleotide Exchange ◽  
Lipid Kinase ◽  
Actin Remodeling ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Phosphoinositide 3 Kinase ◽  
Related Proteins

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice.

scholarly journals Different Levels of Bfa1/Bub2 GAP Activity Are Required to Prevent Mitotic Exit of Budding Yeast Depending on the Type of Perturbations

2008 ◽  
Vol 19 (10) ◽  
pp. 4328-4340 ◽  
Author(s):  
Junwon Kim ◽  
Selma Sun Jang ◽  
Kiwon Song
Keyword(s):  
Budding Yeast ◽  
Mitotic Exit ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Surveillance Mechanism ◽  
Spindle Position ◽  
First Time ◽  
Different Levels

In budding yeast, Tem1 is a key regulator of mitotic exit. Bfa1/Bub2 stimulates Tem1 GTPase activity as a GTPase-activating protein (GAP). Lte1 possesses a guanine-nucleotide exchange factor (GEF) domain likely for Tem1. However, recent observations showed that cells may control mitotic exit without either Lte1 or Bfa1/Bub2 GAP activity, obscuring how Tem1 is regulated. Here, we assayed BFA1 mutants with varying GAP activities for Tem1, showing for the first time that Bfa1/Bub2 GAP activity inhibits Tem1 in vivo. A decrease in GAP activity allowed cells to bypass mitotic exit defects. Interestingly, different levels of GAP activity were required to prevent mitotic exit depending on the type of perturbation. Although essential, more Bfa1/Bub2 GAP activity was needed for spindle damage than for DNA damage to fully activate the checkpoint. Conversely, Bfa1/Bub2 GAP activity was insufficient to delay mitotic exit in cells with misoriented spindles. Instead, decreased interaction of Bfa1 with Kin4 was observed in BFA1 mutant cells with a defective spindle position checkpoint. These findings demonstrate that there is a GAP-independent surveillance mechanism of Bfa1/Bub2, which, together with the GTP/GDP switch of Tem1, may be required for the genomic stability of cells with misaligned spindles.

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