scholarly journals GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis

2016 ◽  
Vol 215 (4) ◽  
pp. 499-513 ◽  
Author(s):  
Laura L. Thomas ◽  
J. Christopher Fromme
Keyword(s):  
Cross Talk ◽  
Vesicle Formation ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Direct Role ◽  
Definitive Evidence ◽  
Nucleotide Exchange Factor ◽  
Vesicle Biogenesis ◽  
Simultaneous Activation ◽  
Bona Fide

Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering.

scholarly journals Plekhg4 Is a Novel Dbl Family Guanine Nucleotide Exchange Factor Protein for Rho Family GTPases

2013 ◽  
Vol 288 (20) ◽  
pp. 14522-14530 ◽  
Author(s):  
Meghana Gupta ◽  
Elena Kamynina ◽  
Samantha Morley ◽  
Stacey Chung ◽  
Nora Muakkassa ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Developmentally Regulated ◽  
Nih3t3 Cells ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Bona Fide

Mutations in the PLEKHG4 (puratrophin-1) gene are associated with the heritable neurological disorder autosomal dominant spinocerebellar ataxia. However, the biochemical functions of this gene product have not been described. We report here that expression of Plekhg4 in the murine brain is developmentally regulated, with pronounced expression in the newborn midbrain and brainstem that wanes with age and maximal expression in the cerebellar Purkinje neurons in adulthood. We show that Plekhg4 is subject to ubiquitination and proteasomal degradation, and its steady-state expression levels are regulated by the chaperones Hsc70 and Hsp90 and by the ubiquitin ligase CHIP. On the functional level, we demonstrate that Plekhg4 functions as a bona fide guanine nucleotide exchange factor (GEF) that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. Overexpression of Plekhg4 in NIH3T3 cells induces rearrangements of the actin cytoskeleton, specifically enhanced formation of lamellopodia and fillopodia. These findings indicate that Plekhg4 is an aggregation-prone member of the Dbl family GEFs and that regulation of GTPase signaling is critical for proper cerebellar function.

scholarly journals In Vitro Formation of Recycling Vesicles from Endosomes Requires Adaptor Protein-1/Clathrin and Is Regulated by Rab4 and the Connector Rabaptin-5

2004 ◽  
Vol 15 (11) ◽  
pp. 4990-5000 ◽  
Author(s):  
Adriana Pagano ◽  
Pascal Crottet ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Brefeldin A ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Vesicle Formation ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and γ-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

scholarly journals LET-413/Erbin acts as a RAB-5 effector to promote RAB-10 activation during endocytic recycling

2017 ◽  
Vol 217 (1) ◽  
pp. 299-314 ◽  
Author(s):  
Hang Liu ◽  
Shimin Wang ◽  
Weijian Hang ◽  
Jinghu Gao ◽  
Wenjuan Zhang ◽  
...  
Keyword(s):  
Interacting Proteins ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Binding Partner ◽  
C Elegans ◽  
Intestinal Epithelia ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

RAB-10/Rab10 is a master regulator of endocytic recycling in epithelial cells. To better understand the regulation of RAB-10 activity, we sought to identify RAB-10(GDP)–interacting proteins. One novel RAB-10(GDP)–binding partner that we identified, LET-413, is the Caenorhabditis elegans homologue of Scrib/Erbin. Here, we focus on the mechanistic role of LET-413 in the regulation of RAB-10 within the C. elegans intestine. We show that LET-413 is a RAB-5 effector and colocalizes with RAB-10 on endosomes, and the overlap of LET-413 with RAB-10 is RAB-5 dependent. Notably, LET-413 enhances the interaction of DENN-4 with RAB-10(GDP) and promotes DENN-4 guanine nucleotide exchange factor activity toward RAB-10. Loss of LET-413 leads to cytosolic dispersion of the RAB-10 effectors TBC-2 and CNT-1. Finally, we demonstrate that the loss of RAB-10 or LET-413 results in abnormal overextensions of lateral membrane. Hence, our studies indicate that LET-413 is required for DENN-4–mediated RAB-10 activation, and the LET-413–assisted RAB-5 to RAB-10 cascade contributes to the integrity of C. elegans intestinal epithelia.

scholarly journals FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis

2016 ◽  
Vol 113 (37) ◽  
pp. E5519-E5527 ◽  
Author(s):  
Jia Chen ◽  
Feng Yu ◽  
Ying Liu ◽  
Changqing Du ◽  
Xiushan Li ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Cross Talk ◽  
Stress Responses ◽  
Peptide Hormone ◽  
Negative Regulator ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Nucleotide Exchange Factor ◽  
Stress Signals ◽  
Aba Insensitive

Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)–A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals.

scholarly journals The small GTPase Rap1b regulates the cross talk between platelet integrin α2β1 and integrin αIIbβ3

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2728-2735 ◽  
Author(s):  
Bruno Bernardi ◽  
Gianni F. Guidetti ◽  
Francesca Campus ◽  
Jill R. Crittenden ◽  
Ann M. Graybiel ◽  
...  
Keyword(s):  
Cross Talk ◽  
Platelet Adhesion ◽  
Second Messengers ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Integrin Αiibβ3 ◽  
Exchange Factor ◽  
Fibrinogen Binding ◽  
Nucleotide Exchange Factor ◽  
The Cross

AbstractThe involvement of the small GTPase Rap1b in platelet integrin α2β1-dependent outside-in signaling was investigated. Platelet adhesion to 4 different specific ligands for integrin α2β1, monomeric collagen, decorin, and collagen-derived peptides CB8(II) and CB11(II), induced a robust and rapid activation of Rap1b. This process did not require secreted ADP or thromboxane A2 production but was critically regulated by phospholipase C (PLC)–derived second messengers. Both Ca2+ and protein kinase C were found to organize independent but additive pathways for Rap1b activation downstream of integrin-α2β1, which were completely blocked by inhibition of PLC with U73122. Moreover, integrin α2β1 engagement failed to trigger Rap1b activation in murine platelets lacking CalDAG-GEFI, a guanine nucleotide exchange factor regulated by Ca2+ and diacylglycerol, despite normal phosphorylation and activation of PLCγ2. In addition, CalDAG-GEFI–deficient platelets showed defective integrin α2β1-dependent adhesion and spreading. We found that outside-in signaling through integrin α2β1 triggered inside-out activation of integrin αIIbβ3 and promoted fibrinogen binding. Similarly to Rap1b stimulation, this process occurred downstream of PLC activation and was dramatically impaired in murine platelets lacking the Rap1 exchange factor CalDAG-GEFI. These results demonstrate that Rap1b is an important element in integrin-dependent outside-in signaling during platelet adhesion and regulates the cross talk between adhesive receptors.

scholarly journals Structural basis for the initiation of COPII vesicle biogenesis

2020 ◽  
Author(s):  
Aaron M.N. Joiner ◽  
J. Christopher Fromme
Keyword(s):  
Secretory Pathway ◽  
Membrane Surface ◽  
Structural Basis ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Copii Vesicle ◽  
Cargo Proteins ◽  
Vesicle Biogenesis ◽  
Copii Vesicles ◽  
Gtpase Activation

AbstractThe first stage of the eukaryotic secretory pathway is the packaging of cargo proteins into COPII vesicles exiting the endoplasmic reticulum (ER). The cytoplasmic COPII vesicle coat machinery is recruited to the ER membrane by the activated, GTP-bound, form of the conserved Sar1 GTPase. Activation of Sar1 on the surface of the ER by Sec12, a membrane-anchored GEF (guanine nucleotide exchange factor), is therefore the initiating step of the secretory pathway. Here we report the structure of the complex between Sar1 and the cytoplasmic GEF domain of Sec12, both from Saccharomyces cerevisiae. This structure, representing the key nucleotide-free activation intermediate, reveals how the potassium ion-binding K-loop disrupts the nucleotide binding site of Sar1. We describe an unexpected orientation of the GEF domain relative to the membrane surface and propose a mechanism for how Sec12 facilitates membrane insertion of the amphipathic helix exposed by Sar1 upon GTP-binding.

scholarly journals Modeling the dynamic behaviors of the COPI vesicle formation regulators, the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

2021 ◽  
pp. mbc.E20-09-0587
Author(s):  
Garrett Sager ◽  
Tomasz Szul ◽  
Eunjoo Lee ◽  
Ryoichi Kawai ◽  
John F. Presley ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpase ◽  
Vesicle Formation ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Golgi Membranes ◽  
Nucleotide Exchange Factor ◽  
Copi Vesicle ◽  
Sec7 Domain

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1. This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used Fluorescence Recovery After Photobleaching data and kinetic Monte Carlo simulation to assess behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1, and suggests GBF1/E794K is stabilized on the membrane independently of complex formation.

scholarly journals SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity

2018 ◽  
Vol 217 (6) ◽  
pp. 2121-2139 ◽  
Author(s):  
Dan Chen ◽  
Chao Yang ◽  
Sha Liu ◽  
Weijian Hang ◽  
Xianghong Wang ◽  
...  
Keyword(s):  
Negative Regulator ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Concentration Dependent Manner ◽  
Guanosine Diphosphate ◽  
Guanine Nucleotide Exchange ◽  
Phosphoinositide Phosphatase ◽  
Nucleotide Exchange Factor

Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1’s catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1.

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