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Regulation of the actin cytoskeleton by ROPs in plants

Fachliche Zuordnung Zellbiologie
Förderung Förderung von 2003 bis 2009
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 5408517
 
Erstellungsjahr 2011

Zusammenfassung der Projektergebnisse

Rho-related ROP proteins are molecular switches that essentially regulate the cytoskeleton and thereby affect vital processes in plants like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors eventually impacting on actin filaments and microtubules. Although some general principles of Rho proteins and cytoskeletal regulation can be deduced from homologous systems in animals and fungi, the ROPs and their regulators, as well as their downstream targets are rather special: We have resolved 3D-structures of ROPs and determined the specific characteristics which distinguish various ROP isoforms from each other and from their non-plant Rho homologs. Moreover, we have identified and characterized the plant-specific Rop guanine nucleotide exchange factors (RopGEFs) which catalyze the GTP-loading and activation of ROPs via a unique catalytic domain called PRONE. Through the crystal structure of two successive intermediates of the RopGEF reaction and additional mutation analyses we finally elucidated the catalytic mechanism of ROP activation and the molecular basis for substrate specificity of the plant GEFs. Our studies have also expanded to further levels of ROP activation suggesting an involvement of upstream receptor-like kinases and putative regulatory feedback mechanisms. Inactivation of ROPs is catalyzed by GTPase activating proteins (RopGAPs). Many of those dimeric enzymes are characterized by a plant-specific combination of a classical RhoGAP domain and a Cdc42/Rac interactive binding (CRIB) motif, which, in animal and fungi, has never been found in GAPs but in effector proteins. Our analyses revealed that both regions are essential for effective catalysis, involving a conserved catalytic arginine in the GAP domain and the CRIB motif which mediates the high affinity and specificity of ROP binding. A more precise picture of the RopGAP function still requires some structural analyses and further data on the control of RopGAP activity by diverse interaction partners. As concerns the downstream targets of ROPs, relatively few effectors with a direct impact on the cytoskeleton have been identified in plants. Instead plants often use unusual effectors with an adapter function connecting ROPs to their actual targets on the route to cytoskeletal elements. We have isolated and characterized members of those plant-specific effector families, known as RICs and RIPs. RICs were proposed to link ROPs to plant formins which are known as actin nucleators, but our extensive interaction assays using various RIC and formin isoforms revealed no interaction, even in the presence of activated ROPs. While the connection of the RICs to the cytoskeleton remains ambiguous, we found that a member of the RIP family (RIP3 from A. thaliana) is indeed linked to microtubules where it’s interaction with kinesin-13A would lead to mircotubule fragmentation, suggesting a role for RIP3 in microtubule dynamics.

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