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Yaowen Wu - One of the best experts on this subject based on the ideXlab platform.
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the role of the hypervariable c terminal domain in rab gtpases Membrane Targeting
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Fu Li, Long Yi, Lei Zhao, Aymelt Itzen, Roger S Goody, Yaowen WuAbstract:Intracellular Membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable Membrane association. A model asserting HVD-directed Targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab Membrane Targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in Membrane Targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for Membrane Targeting and appears to function simply as a linker between the GTPase domain and the Membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma Membrane (PM) Targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab Membrane Targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with Membranes to varying extents, and possibly other binding partners.
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rabgefs are a major determinant for specific rab Membrane Targeting
Journal of Cell Biology, 2013Co-Authors: Julia Blumer, Roger S Goody, Yaowen Wu, Leif Dehmelt, Tomas Mazel, Philippe I H Bastiaens, Aymelt ItzenAbstract:Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular Membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct Membrane, but it is unclear how this specific Membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal Targeting machinery for recruiting Rabs from the cytosol to the correct Membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific misTargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab Membrane Targeting.
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Membrane Targeting mechanism of rab gtpases elucidated by semisynthetic protein probes
Nature Chemical Biology, 2010Co-Authors: Yaowen Wu, Lena K Oesterlin, Herbert Waldmann, Kirill Alexandrov, Roger S GoodyAbstract:Semisynthetic versions of the small G protein Rab7 in the GDP-bound form have 1,000-fold higher affinity for regulators REP1 and RabGDI because of faster dissociation rates from Rab7-GTP, directly linking nucleotide exchange to Rab Membrane Targeting.
Roger S Goody - One of the best experts on this subject based on the ideXlab platform.
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the role of the hypervariable c terminal domain in rab gtpases Membrane Targeting
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Fu Li, Long Yi, Lei Zhao, Aymelt Itzen, Roger S Goody, Yaowen WuAbstract:Intracellular Membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable Membrane association. A model asserting HVD-directed Targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab Membrane Targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in Membrane Targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for Membrane Targeting and appears to function simply as a linker between the GTPase domain and the Membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma Membrane (PM) Targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab Membrane Targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with Membranes to varying extents, and possibly other binding partners.
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rabgefs are a major determinant for specific rab Membrane Targeting
Journal of Cell Biology, 2013Co-Authors: Julia Blumer, Roger S Goody, Yaowen Wu, Leif Dehmelt, Tomas Mazel, Philippe I H Bastiaens, Aymelt ItzenAbstract:Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular Membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct Membrane, but it is unclear how this specific Membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal Targeting machinery for recruiting Rabs from the cytosol to the correct Membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific misTargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab Membrane Targeting.
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Membrane Targeting mechanism of rab gtpases elucidated by semisynthetic protein probes
Nature Chemical Biology, 2010Co-Authors: Yaowen Wu, Lena K Oesterlin, Herbert Waldmann, Kirill Alexandrov, Roger S GoodyAbstract:Semisynthetic versions of the small G protein Rab7 in the GDP-bound form have 1,000-fold higher affinity for regulators REP1 and RabGDI because of faster dissociation rates from Rab7-GTP, directly linking nucleotide exchange to Rab Membrane Targeting.
Mark A. Lemmon - One of the best experts on this subject based on the ideXlab platform.
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The Dbs PH domain contributes independently to Membrane Targeting and regulation of guanine nucleotide-exchange activity.
Biochemical Journal, 2006Co-Authors: Mark A. Baumeister, Kent L. Rossman, John Sondek, Mark A. LemmonAbstract:Dbl family GEFs (guanine nucleotide-exchange factors) for the Rho GTPases almost invariably contain a PH (pleckstrin homology) domain adjacent to their DH (Dbl homology) domain. The DH domain is responsible for GEF activity, and the PH domain plays a regulatory role that remains poorly understood. We demonstrated previously that Dbl family PH domains bind phosphoinositides with low affinity and cannot function as independent Membrane Targeting modules. In the present study, we show that dimerization of a Dbs (Dbl's big sister) DH/PH domain fragment is sufficient to drive it to the plasma Membrane through a mechanism involving PH domain-phosphoinositide interactions. Thus, the Dbs PH domain could play a significant role in Membrane Targeting if it co-operates with other domains in the protein. We also show that mutations that prevent phosphoinositide binding by the Dbs PH domain significantly impair cellular GEF activity even in chimaeric proteins that are robustly Membrane targeted by farnesylation or by the PH domain of phospholipase C-delta1. This finding argues that the Dbs PH domain plays a regulatory role that is independent of its ability to aid Membrane Targeting. Thus, we suggest that the PH domain plays dual roles, contributing independently to Membrane localization of Dbs (as part of a multi-domain interaction) and allosteric regulation of the DH domain.
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genome wide analysis of Membrane Targeting by s cerevisiae pleckstrin homology domains
Molecular Cell, 2004Co-Authors: Jong W Yu, Diana Murray, Jeannine M Mendrola, Anjon Audhya, Shaneen Singh, David Keleti, Daryll B Dewald, Mark A. LemmonAbstract:Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive Membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are Membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently Membrane targeted. Our proteome-wide analysis argues that Membrane Targeting is important for only ∼30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.
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signal dependent Membrane Targeting by pleckstrin homology ph domains
Biochemical Journal, 2000Co-Authors: Mark A. Lemmon, Kathryn M FergusonAbstract:Pleckstrin homology (PH) domains are small protein modules of around 120 amino acids found in many proteins involved in cell signalling, cytoskeletal rearrangement and other processes. Although several different protein ligands have been proposed for PH domains, their only clearly demonstrated physiological function to date is to bind Membrane phosphoinositides. The PH domain from phospholipase C-δ 1 binds specifically to PtdIns(4,5) P 2 and its headgroup, and has become a valuable tool for studying cellular PtdIns(4,5) P 2 functions. More recent developments have demonstrated that a subset of PH domains recognizes the products of agonist-stimulated phosphoinositide 3-kinases. Fusion of these PH domains to green fluorescent protein has allowed dramatic demonstrations of their independent ability to drive signal-dependent recruitment of their host proteins to the plasma Membrane. We discuss the structural basis for this 3-phosphoinoistide recognition and the role that it plays in cellular signalling. PH domains that bind specifically to phosphoinositides comprise only a minority (perhaps 15%) of those known, raising questions as to the physiological role of the remaining 85% of PH domains. Most (if not all) PH domains bind weakly and non-specifically to phosphoinositides. Studies of dynamin-1 have indicated that oligomerization of its PH domain may be important in driving Membrane association. We discuss the possibility that Membrane Targeting by PH domains with low affinity for phosphoinositides could be driven by alteration of their oligomeric state and thus the avidity of their Membrane binding.
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activation of phospholipase cγ by pi 3 kinase induced ph domain mediated Membrane Targeting
The EMBO Journal, 1998Co-Authors: Marco Falasca, Mark A. Lemmon, Susan K Logan, V P Lehto, G Baccante, Joseph SchlessingerAbstract:Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase Cγ (PLCγ) and phosphatidylinositol (PI) 3‐kinase. While it is well established that tyrosine phosphorylation of PLCγ is necessary for its activation, we show here that PLCγ is regulated additionally by the lipid products of PI 3‐kinase. We demonstrate that the pleckstrin homology (PH) domain of PLCγ binds to phosphatidylinositol 3,4,5‐trisphosphate [PdtIns(3,4,5)P3], and is targeted to the Membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not Membrane targeted. Consistent with these observations, activation of PI 3‐kinase causes PLCγ PH domain‐mediated Membrane Targeting and PLCγ activation. By contrast, either the inhibition of PI 3‐kinase by overexpression of a dominant‐negative mutant or the prevention of PLCγ Membrane Targeting by overexpression of the PLCγ PH domain prevents growth factor‐induced PLCγ activation. These experiments reveal a novel mechanism for cross‐talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.
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Activation of phospholipase Cγ by PI 3‐kinase‐induced PH domain‐mediated Membrane Targeting
The EMBO Journal, 1998Co-Authors: Marco Falasca, Mark A. Lemmon, Susan K Logan, V P Lehto, G Baccante, Joseph SchlessingerAbstract:Signaling via growth factor receptors frequently results in the concomitant activation of phospholipase Cγ (PLCγ) and phosphatidylinositol (PI) 3‐kinase. While it is well established that tyrosine phosphorylation of PLCγ is necessary for its activation, we show here that PLCγ is regulated additionally by the lipid products of PI 3‐kinase. We demonstrate that the pleckstrin homology (PH) domain of PLCγ binds to phosphatidylinositol 3,4,5‐trisphosphate [PdtIns(3,4,5)P3], and is targeted to the Membrane in response to growth factor stimulation, while a mutated version of this PH domain that does not bind PdtIns(3,4,5)P3 is not Membrane targeted. Consistent with these observations, activation of PI 3‐kinase causes PLCγ PH domain‐mediated Membrane Targeting and PLCγ activation. By contrast, either the inhibition of PI 3‐kinase by overexpression of a dominant‐negative mutant or the prevention of PLCγ Membrane Targeting by overexpression of the PLCγ PH domain prevents growth factor‐induced PLCγ activation. These experiments reveal a novel mechanism for cross‐talk and mutual regulation of activity between two enzymes that participate in the control of phosphoinositide metabolism.
Aymelt Itzen - One of the best experts on this subject based on the ideXlab platform.
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the role of the hypervariable c terminal domain in rab gtpases Membrane Targeting
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Fu Li, Long Yi, Lei Zhao, Aymelt Itzen, Roger S Goody, Yaowen WuAbstract:Intracellular Membrane trafficking requires correct and specific localization of Rab GTPases. The hypervariable C-terminal domain (HVD) of Rabs is posttranslationally modified by isoprenyl moieties that enable Membrane association. A model asserting HVD-directed Targeting has been contested in previous studies, but the role of the Rab HVD and the mechanism of Rab Membrane Targeting remain elusive. To elucidate the function of the HVD, we have substituted this region with an unnatural polyethylenglycol (PEG) linker by using oxime ligation. The PEGylated Rab proteins undergo normal prenylation, underlining the unique ability of the Rab prenylation machinery to process the Rab family with diverse C-terminal sequences. Through localization studies and functional analyses of semisynthetic PEGylated Rab1, Rab5, Rab7, and Rab35 proteins, we demonstrate that the role of the HVD of Rabs in Membrane Targeting is more complex than previously understood. The HVD of Rab1 and Rab5 is dispensable for Membrane Targeting and appears to function simply as a linker between the GTPase domain and the Membrane. The N-terminal residues of the Rab7 HVD are important for late endosomal/lysosomal localization, apparently due to their involvement in interaction with the Rab7 effector Rab-interacting lysosomal protein. The C-terminal polybasic cluster of the Rab35 HVD is essential for plasma Membrane (PM) Targeting, presumably because of the electrostatic interaction with negatively charged lipids on the PM. Our findings suggest that Rab Membrane Targeting is dictated by a complex mechanism involving GEFs, GAPs, effectors, and C-terminal interaction with Membranes to varying extents, and possibly other binding partners.
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rabgefs are a major determinant for specific rab Membrane Targeting
Journal of Cell Biology, 2013Co-Authors: Julia Blumer, Roger S Goody, Yaowen Wu, Leif Dehmelt, Tomas Mazel, Philippe I H Bastiaens, Aymelt ItzenAbstract:Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular Membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct Membrane, but it is unclear how this specific Membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal Targeting machinery for recruiting Rabs from the cytosol to the correct Membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific misTargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab Membrane Targeting.
Robert C Liddington - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of Membrane Targeting by the Dock180 family of Rho family guanine exchange factors (Rho-GEFs)
Journal of Biological Chemistry, 2010Co-Authors: Lakshmanane Premkumar, Jean François Côté, Manishha Patel, Lukasz Jaroszewski, Laurie A. Bankston, Boguslaw Stec, Andrey A Bobkov, Kristiina Vuori, Robert C LiddingtonAbstract:The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific Membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic Membrane Targeting of the Rho-GEF activity of Dock180.