Identification and Verification of Sro7p as an Effector of the Sec4p Rab GTPase

The eukaryotic endomembrane system is controlled by small GTPases of the Rab family, which are activated at defined times and locations in a switch-like manner. While this switch is well understood for an individual protein, how regulatory networks produce intracellular activity patterns is currently not known. Here, we combine in vitro reconstitution experiments with computational modeling to study a minimal Rab5 activation network. We find that the molecular interactions in this system give rise to a positive feedback and bistable collective switching of Rab5. Furthermore, we find that switching near the critical point is intrinsically stochastic and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Notably, we demonstrate that collective switching can spread on the membrane surface as a traveling wave of Rab5 activation. Together, our findings reveal how biochemical signaling networks control vesicle trafficking pathways and how their nonequilibrium properties define the spatiotemporal organization of the cell.

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The Hsp90 Chaperone Complex Regulates GDI-dependent Rab Recycling

Molecular Biology of the Cell ◽

10.1091/mbc.e05-12-1096 ◽

2006 ◽

Vol 17 (8) ◽

pp. 3494-3507 ◽

Cited By ~ 33

Author(s):

Christine Y. Chen ◽

William E. Balch

Keyword(s):

Rab Gtpase ◽

Rab Gtpases ◽

Coding System ◽

Golgi Transport ◽

Guanine Nucleotide Dissociation Inhibitor ◽

Hsp90 Activity ◽

Hsp90 Chaperone ◽

Chaperone Complex ◽

Hsp 90 ◽

Synaptic Vesicle Fusion

Rab GTPase regulated hubs provide a framework for an integrated coding system, the membrome network, that controls the dynamics of the specialized exocytic and endocytic membrane architectures found in eukaryotic cells. Herein, we report that Rab recycling in the early exocytic pathways involves the heat-shock protein (Hsp)90 chaperone system. We find that Hsp90 forms a complex with guanine nucleotide dissociation inhibitor (GDI) to direct recycling of the client substrate Rab1 required for endoplasmic reticulum (ER)-to-Golgi transport. ER-to-Golgi traffic is inhibited by the Hsp90-specific inhibitors geldanamycin (GA), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), and radicicol. Hsp90 activity is required to form a functional GDI complex to retrieve Rab1 from the membrane. Moreover, we find that Hsp90 is essential for Rab1-dependent Golgi assembly. The observation that the highly divergent Rab GTPases Rab1 involved in ER-to-Golgi transport and Rab3A involved in synaptic vesicle fusion require Hsp90 for retrieval from membranes lead us to now propose that the Hsp90 chaperone system may function as a general regulator for Rab GTPase recycling in exocytic and endocytic trafficking pathways involved in cell signaling and proliferation.

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TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0947 ◽

2010 ◽

Vol 21 (13) ◽

pp. 2285-2296 ◽

Cited By ~ 51

Author(s):

Laëtitia Chotard ◽

Ashwini K. Mishra ◽

Marc-André Sylvain ◽

Simon Tuck ◽

David G. Lambright ◽

...

Keyword(s):

Rab Gtpase ◽

Endosomal Trafficking ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Late Endosomes ◽

Arginine Finger ◽

Endosome Maturation ◽

Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

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Yeast vacuolar HOPS, regulated by its kinase, exploits affinities for acidic lipids and Rab:GTP for membrane binding and to catalyze tethering and fusion

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1298 ◽

2015 ◽

Vol 26 (2) ◽

pp. 305-315 ◽

Cited By ~ 20

Author(s):

Amy Orr ◽

William Wickner ◽

Scott F. Rusin ◽

Arminja N. Kettenbach ◽

Michael Zick

Keyword(s):

Protein Sorting ◽

Rab Gtpase ◽

Attachment Protein ◽

Functional Relationships ◽

Sensitive Factor ◽

Protein Receptors ◽

Membrane Tethering ◽

Rab Effector ◽

Supporting Membrane ◽

Acidic Lipids

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide–sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.

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Interdependent assembly of specific regulatory lipids and membrane fusion proteins into the vertex ring domain of docked vacuoles

The Journal of Cell Biology ◽

10.1083/jcb.200409068 ◽

2004 ◽

Vol 167 (6) ◽

pp. 1087-1098 ◽

Cited By ~ 170

Author(s):

Rutilio A. Fratti ◽

Youngsoo Jun ◽

Alexey J. Merz ◽

Nathan Margolis ◽

William Wickner

Keyword(s):

Membrane Fusion ◽

Protein Interactions ◽

Fusion Proteins ◽

Membrane Microdomains ◽

Rab Gtpase ◽

Protein Protein Interactions ◽

Px Domain ◽

Protein Partitioning ◽

Membrane Fusion Proteins ◽

Rab Effector

Membrane microdomains are assembled by lipid partitioning (e.g., rafts) or by protein–protein interactions (e.g., coated vesicles). During docking, yeast vacuoles assemble “vertex” ring-shaped microdomains around the periphery of their apposed membranes. Vertices are selectively enriched in the Rab GTPase Ypt7p, the homotypic fusion and vacuole protein sorting complex (HOPS)–VpsC Rab effector complex, SNAREs, and actin. Membrane fusion initiates at vertex microdomains. We now find that the “regulatory lipids” ergosterol, diacylglycerol and 3- and 4-phosphoinositides accumulate at vertices in a mutually interdependent manner. Regulatory lipids are also required for the vertex enrichment of SNAREs, Ypt7p, and HOPS. Conversely, SNAREs and actin regulate phosphatidylinositol 3-phosphate vertex enrichment. Though the PX domain of the SNARE Vam7p has direct affinity for only 3-phosphoinositides, all the regulatory lipids which are needed for vertex assembly affect Vam7p association with vacuoles. Thus, the assembly of the vacuole vertex ring microdomain arises from interdependent lipid and protein partitioning and binding rather than either lipid partitioning or protein interactions alone.

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Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

Biochemical Journal ◽

10.1042/bcj20160557 ◽

2016 ◽

Vol 473 (17) ◽

pp. 2671-2685 ◽

Cited By ~ 80

Author(s):

Genta Ito ◽

Kristina Katsemonova ◽

Francesca Tonelli ◽

Pawel Lis ◽

Marco A.S. Baptista ◽

...

Keyword(s):

Autosomal Dominant ◽

Mouse Lung ◽

Rab Gtpase ◽

Leucine Rich Repeat ◽

Serine Residue ◽

Mouse Embryonic Fibroblasts ◽

Embryonic Fibroblasts ◽

The Impact ◽

Effector Binding

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

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