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The biochemical mechanism of Rho GTPase membrane binding, activation and retention in activity patterning.

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Název: The biochemical mechanism of Rho GTPase membrane binding, activation and retention in activity patterning.
Autoři: Armstrong, Michael C, Weiß, Yannic R, Hoachlander-Hobby, Lila E, Roy, Ankit A, Visco, Ilaria, Moe, Alison, Golding, Adriana E, Hansen, Scott D, Bement, William M, Bieling, Peter
Zdroj: EMBO Journal; May2025, Vol. 44 Issue 9, p2620-2657, 38p
Témata: GUANINE nucleotide exchange factors, GTPASE-activating protein, RHO GTPases, CELL polarity, GUANOSINE triphosphatase
Abstrakt: Rho GTPases form plasma membrane-associated patterns that control the cytoskeleton during cell division, morphogenesis, migration, and wound repair. Their patterning involves transitions between inactive cytosolic and active membrane-bound states, regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). However, the relationships between these transitions and role of different regulators remain unclear. We developed a novel reconstitution approach to study Rho GTPase patterning with all major GTPase regulators in a biochemically defined system. We show that Rho GTPase dissociation from RhoGDI is rate-limiting for its membrane association. Rho GTPase activation occurs after membrane insertion, which is unaffected by GEF activity. Once activated, Rho GTPases are retained at the membrane through effector interactions, essential for their enrichment at activation sites. Thus, high cytosolic levels of RhoGDI-bound GTPases ensure a constant supply of inactive GTPases for the membrane, where GEF-mediated activation and effector binding stabilize them. These results delineate the route by which Rho GTPase patterns are established and define stage-dependent roles of its regulators. Synopsis: Formation of Rho GTPase activity patterns on the plasma membrane is key to numerous morphogenic processes in eukaryotic cells. This study employs a reconstitution approach to reveal the biochemical principles of Rho GTPase pattern formation and show that these signaling proteins enrich at locations where they are activated by effector-mediated membrane retention. A biochemically defined reconstitution system allows investigation of Rho GTPase patterning dynamics in the presence of all major GTPase regulators. Rho GTPase membrane binding is limited by dissociation from RhoGDI. Activation of Rho GTPases occurs after membrane binding and GEFs do not recruit Rho GTPases to membranes. Activation prolongs retention on the membrane by effector-mediated stabilization. A biochemically defined in vitro reconstitution approach reveals mechanistic principles of Rho GTPase patterning at cellular membranes and the stage-dependent role of regulators. [ABSTRACT FROM AUTHOR]
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Abstrakt:Rho GTPases form plasma membrane-associated patterns that control the cytoskeleton during cell division, morphogenesis, migration, and wound repair. Their patterning involves transitions between inactive cytosolic and active membrane-bound states, regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). However, the relationships between these transitions and role of different regulators remain unclear. We developed a novel reconstitution approach to study Rho GTPase patterning with all major GTPase regulators in a biochemically defined system. We show that Rho GTPase dissociation from RhoGDI is rate-limiting for its membrane association. Rho GTPase activation occurs after membrane insertion, which is unaffected by GEF activity. Once activated, Rho GTPases are retained at the membrane through effector interactions, essential for their enrichment at activation sites. Thus, high cytosolic levels of RhoGDI-bound GTPases ensure a constant supply of inactive GTPases for the membrane, where GEF-mediated activation and effector binding stabilize them. These results delineate the route by which Rho GTPase patterns are established and define stage-dependent roles of its regulators. Synopsis: Formation of Rho GTPase activity patterns on the plasma membrane is key to numerous morphogenic processes in eukaryotic cells. This study employs a reconstitution approach to reveal the biochemical principles of Rho GTPase pattern formation and show that these signaling proteins enrich at locations where they are activated by effector-mediated membrane retention. A biochemically defined reconstitution system allows investigation of Rho GTPase patterning dynamics in the presence of all major GTPase regulators. Rho GTPase membrane binding is limited by dissociation from RhoGDI. Activation of Rho GTPases occurs after membrane binding and GEFs do not recruit Rho GTPases to membranes. Activation prolongs retention on the membrane by effector-mediated stabilization. A biochemically defined in vitro reconstitution approach reveals mechanistic principles of Rho GTPase patterning at cellular membranes and the stage-dependent role of regulators. [ABSTRACT FROM AUTHOR]
ISSN:02614189
DOI:10.1038/s44318-025-00418-z