scholarly journals Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Lars Langemeyer ◽  
Ricardo Nunes Bastos ◽  
Yiying Cai ◽  
Aymelt Itzen ◽  
Karin M Reinisch ◽  
...  
Keyword(s):  
Active Site ◽  
Free Form ◽  
Release Mechanism ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Active Site Residues ◽  
Gtp Hydrolysis ◽  
Activation Mechanisms ◽  
Gtpase Activation

Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases.

Who’s in control? Principles of Rab GTPase activation in endolysosomal membrane trafficking and beyond

2021 ◽  
Vol 220 (9) ◽  
Author(s):  
Ann-Christin Borchers ◽  
Lars Langemeyer ◽  
Christian Ungermann
Keyword(s):  
Membrane Trafficking ◽  
Transport Processes ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Organelle Biogenesis ◽  
Nucleotide Exchange Factor ◽  
Recent Developments ◽  
Gtpase Activation

The eukaryotic endomembrane system consists of multiple interconnected organelles. Rab GTPases are organelle-specific markers that give identity to these membranes by recruiting transport and trafficking proteins. During transport processes or along organelle maturation, one Rab is replaced by another, a process termed Rab cascade, which requires at its center a Rab-specific guanine nucleotide exchange factor (GEF). The endolysosomal system serves here as a prime example for a Rab cascade. Along with endosomal maturation, the endosomal Rab5 recruits and activates the Rab7-specific GEF Mon1-Ccz1, resulting in Rab7 activation on endosomes and subsequent fusion of endosomes with lysosomes. In this review, we focus on the current idea of Mon1-Ccz1 recruitment and activation in the endolysosomal and autophagic pathway. We compare identified principles to other GTPase cascades on endomembranes, highlight the importance of regulation, and evaluate in this context the strength and relevance of recent developments in in vitro analyses to understand the underlying foundation of organelle biogenesis and maturation.

scholarly journals Rab GTPase localization and Rab cascades in Golgi transport

2012 ◽  
Vol 40 (6) ◽  
pp. 1373-1377 ◽  
Author(s):  
Suzanne R. Pfeffer
Keyword(s):  
Effector Proteins ◽  
Membrane Microdomains ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Golgi Transport ◽  
Guanine Nucleotide Dissociation Inhibitor ◽  
Membrane Bound ◽  
Effector Binding ◽  
Endocytic Pathways

Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.

Regulation of RabGAPs involved in insulin action

2018 ◽  
Vol 46 (3) ◽  
pp. 683-690 ◽  
Author(s):  
Samaneh Mafakheri ◽  
Alexandra Chadt ◽  
Hadi Al-Hasani
Keyword(s):  
Insulin Action ◽  
Glucose Transporter ◽  
Rab Gtpase ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Glucose Transporter Glut4 ◽  
Related Proteins

Rab (Ras-related proteins in brain) GTPases are key proteins responsible for a multiplicity of cellular trafficking processes. Belonging to the family of monomeric GTPases, they are regulated by cycling between their active GTP-bound and inactive GDP-bound conformations. Despite possessing a slow intrinsic GTP hydrolysis activity, Rab proteins rely on RabGAPs (Rab GTPase-activating proteins) that catalyze GTP hydrolysis and consequently inactivate the respective Rab GTPases. Two related RabGAPs, TBC1D1 and TBC1D4 (=AS160) have been described to be associated with obesity-related traits and type 2 diabetes in both mice and humans. Inactivating mutations of TBC1D1 and TBC1D4 lead to substantial changes in trafficking and subcellular distribution of the insulin-responsive glucose transporter GLUT4, and to subsequent alterations in energy substrate metabolism. The activity of the RabGAPs is controlled through complex phosphorylation events mediated by protein kinases including AKT and AMPK, and by putative regulatory interaction partners. However, the dynamics and downstream events following phosphorylation are not well understood. This review focuses on the specific role and regulation of TBC1D1 and TBC1D4 in insulin action.

scholarly journals Stochastic activation and bistability in a Rab GTPase regulatory network

2019 ◽  
Author(s):  
Urban Bezeljak ◽  
Hrushikesh Loya ◽  
Beata Kaczmarek ◽  
Timothy E. Saunders ◽  
Martin Loose
Keyword(s):  
Positive Feedback ◽  
Regulatory Network ◽  
Signaling Networks ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Intracellular Traffic ◽  
Bistable Switching ◽  
In Vitro Reconstitution

AbstractRab GTPases are the central regulators of intracellular traffic. Their function relies on a conformational change triggered by nucleotide exchange and hydrolysis. While this switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate a biochemical signal in space and time is unclear. Here, we combine in vitro reconstitution experiments with theoretical modeling to study a minimal Rab5 activation network. We find that positive feedback in this network gives rise to bistable switching of Rab5 activation and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Together, our findings reveal new insights into the non-equilibrium properties and general principles of biochemical signaling networks underlying the spatiotemporal organization of the cell.

scholarly journals Sbf/MTMR13 coordinates PI(3)P and Rab21 regulation in endocytic control of cellular remodeling

2012 ◽  
Vol 23 (14) ◽  
pp. 2723-2740 ◽  
Author(s):  
Steve Jean ◽  
Sarah Cox ◽  
Eric J. Schmidt ◽  
Fred L. Robinson ◽  
Amy Kiger
Keyword(s):  
Endosomal Trafficking ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Membrane Compartment ◽  
Endosomal Pathway ◽  
Gtpase Activation ◽  
Phosphatidylinositol 3

Cells rely on the coordinated regulation of lipid phosphoinositides and Rab GTPases to define membrane compartment fates along distinct trafficking routes. The family of disease-related myotubularin (MTM) phosphoinositide phosphatases includes catalytically inactive members, or pseudophosphatases, with poorly understood functions. We found that Drosophila MTM pseudophosphatase Sbf coordinates both phosphatidylinositol 3-phosphate (PI(3)P) turnover and Rab21 GTPase activation in an endosomal pathway that controls macrophage remodeling. Sbf dynamically interacts with class II phosphatidylinositol 3-kinase and stably recruits Mtm to promote turnover of a PI(3)P subpool essential for endosomal trafficking. Sbf also functions as a guanine nucleotide exchange factor that promotes Rab21 GTPase activation associated with PI(3)P endosomes. Of importance, Sbf, Mtm, and Rab21 function together, along with Rab11-mediated endosomal trafficking, to control macrophage protrusion formation. This identifies Sbf as a critical coordinator of PI(3)P and Rab21 regulation, which specifies an endosomal pathway and cortical control.

scholarly journals Rab GTPases: master regulators that establish the secretory and endocytic pathways

2017 ◽  
Vol 28 (6) ◽  
pp. 712-715 ◽  
Author(s):  
Suzanne R. Pfeffer
Keyword(s):  
Membrane Trafficking ◽  
Membrane Microdomains ◽  
Rab Gtpase ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Open Questions ◽  
Endocytic Pathways

Several of the most important discoveries in the field of membrane traffic have come from studies of Rab GTPases by Marino Zerial and Peter Novick and their colleagues. Zerial was the first to discover that Rab GTPases represent identity markers for different membrane-bound compartments, and each Rab organizes a collection of specific effectors into function-specifying membrane microdomains to carry out receptor trafficking. Novick discovered that the order (and thus polarity) of Rab GTPases along the secretory and endocytic pathways are established by their specific, cognate guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which partner with one Rab to regulate the subsequent- and prior-acting Rabs. Such so-called Rab cascades have evolved to establish domains that contain unique Rab proteins and their cognate effectors, which drive all steps of membrane trafficking. These findings deserve much broader recognition by the biomedical research community and are highlighted here, along with open questions that require serious attention for full understanding of the molecular basis of Rab GTPase-regulated membrane trafficking in eukaryotic cells.

scholarly journals Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase

2008 ◽  
Vol 182 (6) ◽  
pp. 1141-1151 ◽  
Author(s):  
Christopher L. Brett ◽  
Rachael L. Plemel ◽  
Braden T. Lobingier ◽  
Marissa Vignali ◽  
Stanley Fields ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signal Propagation ◽  
Rab Gtpase ◽  
Nucleotide Exchange ◽  
Gtpase Activating Protein ◽  
Guanosine Triphosphate ◽  
Casein Kinase I ◽  
Gtp Hydrolysis ◽  
Nucleotide Exchange Factor

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance from an inhibitory casein kinase I, Yck3. Yck3 mediates phosphorylation of at least two Ypt7 signaling targets: a tether, the Vps-C/homotypic fusion and vacuole protein sorting (HOPS) subunit Vps41, and a SNARE, Vam3. Phosphorylation of both substrates is opposed by Ypt7-guanosine triphosphate (GTP). We further demonstrate that Ypt7 binds not one but two Vps-C/HOPS subunits: Vps39, a putative Ypt7 nucleotide exchange factor, and Vps41. Gyp7-stimulated GTP hydrolysis on Ypt7 therefore appears to trigger both passive termination of Ypt7 signaling and active kinase-mediated inhibition of Ypt7's downstream targets. We propose that signal propagation through the Ypt7 pathway is controlled by integrated feedback and feed-forward loops. In this model, Yck3 enforces a requirement for the activated Rab in docking and fusion.

scholarly journals Novel peptide recognized by RhoA GTPase.

2006 ◽  
Vol 53 (3) ◽  
pp. 515-524
Author(s):  
Dominika Drulis-Fajdasz ◽  
Filip Jelen ◽  
Arkadiusz Oleksy ◽  
Jacek Otlewski
Keyword(s):  
Disulfide Bridge ◽  
Free Form ◽  
High Signal ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Rhoa Gtpase ◽  
Switch Regions ◽  
Kinetics Of ◽  
Micromolar Range

A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.

scholarly journals Functional Synergy between Rab5 Effector Rabaptin-5 and Exchange Factor Rabex-5 When Physically Associated in a Complex

2001 ◽  
Vol 12 (7) ◽  
pp. 2219-2228 ◽  
Author(s):  
Roger Lippé ◽  
Marta Miaczynska ◽  
Vladimir Rybin ◽  
Anja Runge ◽  
Marino Zerial
Keyword(s):  
Complex Formation ◽  
Rho Gtpases ◽  
Endocytic Pathway ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Physical Association ◽  
Nucleotide Exchange Factor ◽  
Early Endosomes ◽  
Gtpase Activation

Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.

scholarly journals The two TRAPP complexes of metazoans have distinct roles and act on different Rab GTPases

2017 ◽  
Vol 217 (2) ◽  
pp. 601-617 ◽  
Author(s):  
Falko Riedel ◽  
Antonio Galindo ◽  
Nadine Muschalik ◽  
Sean Munro
Keyword(s):  
Drosophila Melanogaster ◽  
Guanine Nucleotide Exchange Factors ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Core Set ◽  
Protein Particle

Originally identified in yeast, transport protein particle (TRAPP) complexes are Rab GTPase exchange factors that share a core set of subunits. TRAPPs were initially found to act on Ypt1, the yeast orthologue of Rab1, but recent studies have found that yeast TRAPPII can also activate the Rab11 orthologues Ypt31/32. Mammals have two TRAPP complexes, but their role is less clear, and they contain subunits that are not found in the yeast complexes but are essential for cell growth. To investigate TRAPP function in metazoans, we show that Drosophila melanogaster have two TRAPP complexes similar to those in mammals and that both activate Rab1, whereas one, TRAPPII, also activates Rab11. TRAPPII is not essential but becomes so in the absence of the gene parcas that encodes the Drosophila orthologue of the SH3BP5 family of Rab11 guanine nucleotide exchange factors (GEFs). Thus, in metazoans, Rab1 activation requires TRAPP subunits not found in yeast, and Rab11 activation is shared by TRAPPII and an unrelated GEF that is metazoan specific.

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