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Pamela J. Yao - One of the best experts on this subject based on the ideXlab platform.
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Reduction of Ap180 and CALM Produces Defects in Synaptic Vesicle Size and Density
NeuroMolecular Medicine, 2013Co-Authors: Ronald S. Petralia, Mark P. Mattson, Ya-xian Wang, Fred E. Indig, Ittai Bushlin, Pamela J. YaoAbstract:Clathrin assembly proteins Ap180 and CALM regulate the assembly of clathrin-coated vesicles (CCVs), which mediate diverse intracellular trafficking processes, including synaptic vesicle (SV) recycling at the synapse. Although studies using several invertebrate model systems have indicated a role for Ap180 in SV recycling, less is known about Ap180’s or CALM’s function in the synapse of mammalian neurons. In this study, we examined synapses of rat hippocampal neurons in which the level of Ap180 or CALM had been reduced by RNA interference (RNAi). Using light microscopy, we visualized synaptic puncta in these Ap180- or CALM-reduced neurons by co-expressing Synaptophysin::EGFP (Syp::EGFP). We found that neurons with reduced Ap180 or reduced CALM had smaller Syp::EGFP-illuminated puncta. Using electron microscopy, we further examined the ultrastructure of the Ap180- or CALM-reduced presynaptic terminals. We found that SVs became variably enlarged in both the Ap180-reduced and CALM-reduced presynaptic terminals. Lower Ap180 and CALM also reduced the density of SVs and the size of SV clusters. Our findings demonstrate that in the presynaptic terminals of hippocampal neurons, Ap180 and CALM have a similar role in regulating synaptic vesicles. This overlapping activity may be necessary for high-precision and high-efficacy SV formation during endocytosis.
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Clathrin assembly proteins Ap180 and CALM in the embryonic rat brain
The Journal of comparative neurology, 2010Co-Authors: Catherine M. Schwartz, Mark P. Mattson, Mohamed R. Mughal, Aiwu Cheng, Pamela J. YaoAbstract:Clathrin-coated vesicles are known to play diverse and pivotal roles in cells. The proper formation of clathrin-coated vesicles is dependent on, and highly regulated by, a large number of clathrin assembly proteins. These assembly proteins likely determine the functional specificity of clathrin-coated vesicles, and together they control a multitude of intracellular trafficking pathways, including those involved in embryonic development. In this study, we focus on two closely related clathrin assembly proteins, Ap180 and CALM (clathrin assembly lymphoid myeloid leukemia protein), in the developing embryonic rat brain. We find that Ap180 begins to be expressed at embryonic day 14 (E14), but only in postmitotic cells that have acquired a neuronal fate. CALM, on the other hand, is expressed as early as E12, by both neural stem cells and postmitotic neurons. In vitro loss-of-function studies using RNA interference (RNAi) indicate that Ap180 and CALM are dispensable for some aspects of embryonic neurogenesis but are required for the growth of postmitotic neurons. These results identify the developmental stage of Ap180 and CALM expression and suggest that each protein has distinct functions in neural development.
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Neuronal activity and the expression of clathrin-assembly protein Ap180.
Biochemical and Biophysical Research Communications, 2010Co-Authors: Mark P. Mattson, Pamela J. YaoAbstract:The clathrin-assembly protein Ap180 is known to promote the assembly of clathrin-coated vesicles in the neuron. However, it is unknown whether the expression of Ap180 is influenced by neuronal activity. In this study, we report that chronic depolarization results in a reduction of Ap180 from hippocampal neurons, while acute depolarization causes a dispersed synaptic distribution of Ap180. Activity-induced effects are observed only for Ap180, but not for the structurally-related clathrin-assembly proteins CALM, epsin1, or HIP1. These findings suggest that Ap180 levels and synaptic distribution are highly sensitive to neuronal activity.
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The clathrin assembly protein Ap180 regulates the generation of amyloid-β peptide
Biochemical and biophysical research communications, 2009Co-Authors: Yasuji Matsuoka, Mark P. Mattson, Pamela J. YaoAbstract:Abstract The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer’s disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar clathrin assembly proteins, Ap180 and CALM. We show that RNA interference-mediated knockdown of Ap180 reduces the generation of Aβ1–40 and Aβ1–42, whereas CALM knockdown has no effect on Aβ generation. Thus Ap180 is among the traffic controllers that oversee and regulate amyloid precursor protein processing pathways. Our results also suggest that Ap180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.
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Clathrin Assembly Protein Ap180 and CALM Differentially Control Axogenesis and Dendrite Outgrowth in Embryonic Hippocampal Neurons
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2008Co-Authors: Ittai Bushlin, Mark P. Mattson, Ronald S. Petralia, Asaff Harel, Mohamed R. Mughal, Pamela J. YaoAbstract:Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins Ap180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that Ap180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of Ap180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress Ap180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does Ap180. Functional analysis shows that endocytosis is reduced in both Ap180-deficient and CALM-deficient neurons. Additionally, CALM-deficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for Ap180 and CALM in intracellular trafficking that are essential in the growth of neurons.
Volker Haucke - One of the best experts on this subject based on the ideXlab platform.
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vesicular synaptobrevin vamp2 levels guarded by Ap180 control efficient neurotransmission
Neuron, 2015Co-Authors: Seong Joo Koo, Dmytro Puchkov, Gaga Kochlamazashvili, Benjamin R. Rost, Niclas Gimber, Martin Lehmann, Georgi Tadeus, Jan Schmoranzer, Christian Rosenmund, Volker HauckeAbstract:Summary Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor Ap180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in Ap180 −/− / Syb2 +/− mice results in perinatal lethality, whereas Syb2 +/− mice partially phenocopy loss of Ap180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by Ap180 is crucial to sustain efficient neurotransmission and SV reformation.
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Vesicular Synaptobrevin/VAMP2 Levels Guarded by Ap180 Control Efficient Neurotransmission
Neuron, 2015Co-Authors: Seong Joo Koo, Dmytro Puchkov, Gaga Kochlamazashvili, Benjamin R. Rost, Niclas Gimber, Martin Lehmann, Georgi Tadeus, Jan Schmoranzer, Christian Rosenmund, Volker HauckeAbstract:Summary Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor Ap180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in Ap180 −/− / Syb2 +/− mice results in perinatal lethality, whereas Syb2 +/− mice partially phenocopy loss of Ap180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by Ap180 is crucial to sustain efficient neurotransmission and SV reformation.
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Turning CALM into excitement: Ap180 and CALM in endocytosis and disease.
Biology of the cell, 2012Co-Authors: Tanja Maritzen, Seong Joo Koo, Volker HauckeAbstract:Dynamic flux of membrane between intracellular compartments is a key feature of all eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in membrane dynamics by facilitating membrane fusion, for example at synapses where small synaptic vesicles (SVs) undergo activity-regulated neuroexocytosis, followed by the endocytic re-cycling of SV proteins and lipids. Recent work shows that the assembly protein 180 (Ap180) N-terminal homology (ANTH) domain containing proteins Ap180 and clathrin assembly lymphoid myeloid leukaemia (CALM) not only regulate the assembly of the endocytic machinery but also act as sorters for a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs), most notably VAMP/synaptobrevin 2 at synapses. In this review, we summarise the current state of knowledge about the roles of Ap180 and CALM family members in clathrin-dependent membrane traffic, the molecular mechanistic basis for their activities and their potential involvement in human disease.
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snare motif mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia calm and Ap180 at synapses
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Seong Joo Koo, Carsten C Mahrenholz, Stefan Markovic, Figen Becerenbraun, Tanja Maritzen, Jens Dernedde, Rudolf Volkmer, Dmytro Puchkov, Hartmut Oschkinat, Volker HauckeAbstract:Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors Ap180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or Ap180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and Ap180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins Ap180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and Ap180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.
Seong Joo Koo - One of the best experts on this subject based on the ideXlab platform.
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vesicular synaptobrevin vamp2 levels guarded by Ap180 control efficient neurotransmission
Neuron, 2015Co-Authors: Seong Joo Koo, Dmytro Puchkov, Gaga Kochlamazashvili, Benjamin R. Rost, Niclas Gimber, Martin Lehmann, Georgi Tadeus, Jan Schmoranzer, Christian Rosenmund, Volker HauckeAbstract:Summary Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor Ap180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in Ap180 −/− / Syb2 +/− mice results in perinatal lethality, whereas Syb2 +/− mice partially phenocopy loss of Ap180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by Ap180 is crucial to sustain efficient neurotransmission and SV reformation.
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Vesicular Synaptobrevin/VAMP2 Levels Guarded by Ap180 Control Efficient Neurotransmission
Neuron, 2015Co-Authors: Seong Joo Koo, Dmytro Puchkov, Gaga Kochlamazashvili, Benjamin R. Rost, Niclas Gimber, Martin Lehmann, Georgi Tadeus, Jan Schmoranzer, Christian Rosenmund, Volker HauckeAbstract:Summary Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor Ap180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in Ap180 −/− / Syb2 +/− mice results in perinatal lethality, whereas Syb2 +/− mice partially phenocopy loss of Ap180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by Ap180 is crucial to sustain efficient neurotransmission and SV reformation.
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Turning CALM into excitement: Ap180 and CALM in endocytosis and disease.
Biology of the cell, 2012Co-Authors: Tanja Maritzen, Seong Joo Koo, Volker HauckeAbstract:Dynamic flux of membrane between intracellular compartments is a key feature of all eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a crucial role in membrane dynamics by facilitating membrane fusion, for example at synapses where small synaptic vesicles (SVs) undergo activity-regulated neuroexocytosis, followed by the endocytic re-cycling of SV proteins and lipids. Recent work shows that the assembly protein 180 (Ap180) N-terminal homology (ANTH) domain containing proteins Ap180 and clathrin assembly lymphoid myeloid leukaemia (CALM) not only regulate the assembly of the endocytic machinery but also act as sorters for a subset of SNAREs, the vesicle-associated membrane proteins (VAMPs), most notably VAMP/synaptobrevin 2 at synapses. In this review, we summarise the current state of knowledge about the roles of Ap180 and CALM family members in clathrin-dependent membrane traffic, the molecular mechanistic basis for their activities and their potential involvement in human disease.
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snare motif mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia calm and Ap180 at synapses
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Seong Joo Koo, Carsten C Mahrenholz, Stefan Markovic, Figen Becerenbraun, Tanja Maritzen, Jens Dernedde, Rudolf Volkmer, Dmytro Puchkov, Hartmut Oschkinat, Volker HauckeAbstract:Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors Ap180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or Ap180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and Ap180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins Ap180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and Ap180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.
Bing Zhang - One of the best experts on this subject based on the ideXlab platform.
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Ap180 couples protein retrieval to clathrin mediated endocytosis of synaptic vesicles
Traffic, 2014Co-Authors: Phillip A. Vanlandingham, Bing Zhang, Hong Bao, Mojgan Padash Barmchi, Suzanne M. Royer, Rebekah Green, Noreen E. ReistAbstract:How clathrin-mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin-coated vesicle formation and regulating SV size, the clathrin assembly protein Ap180 has been identified as a key player in retrieving SV proteins. The mechanisms by which Ap180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of Ap180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1-43 uptake and synaptic ultrastructure. More dramatically, Ap180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that Ap180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in Ap180 mutant flies. Finally, Ap180 co-immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which Ap180 couples protein retrieval to CME of SVs. Ap180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of Ap180 dysfunction in Alzheimer's disease.
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Ap180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles
Traffic (Copenhagen Denmark), 2014Co-Authors: Phillip A. Vanlandingham, Hong Bao, Mojgan Padash Barmchi, Suzanne M. Royer, Rebekah Green, Noreen E. Reist, Bing ZhangAbstract:How clathrin-mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin-coated vesicle formation and regulating SV size, the clathrin assembly protein Ap180 has been identified as a key player in retrieving SV proteins. The mechanisms by which Ap180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of Ap180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1-43 uptake and synaptic ultrastructure. More dramatically, Ap180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that Ap180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in Ap180 mutant flies. Finally, Ap180 co-immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which Ap180 couples protein retrieval to CME of SVs. Ap180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of Ap180 dysfunction in Alzheimer's disease.
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Ap180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis
Journal of neurophysiology, 2005Co-Authors: Hong Bao, Richard W. Daniels, Gregory T. Macleod, Milton P. Charlton, Harold L. Atwood, Bing ZhangAbstract:Ap180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP18...
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A novel all helix fold of the Ap180 amino-terminal domain for phosphoinositide binding and clathrin assembly in synaptic vesicle endocytosis.
Cell, 2001Co-Authors: Yuxin Mao, Bing Zhang, Jue Chen, Jennifer A. Maynard, Florante A. QuiochoAbstract:Clathrin-mediated endocytosis plays a major role in retrieving synaptic vesicles from the plasma membrane following exocytosis. This endocytic process requires Ap180 (or a homolog), which promotes the assembly and restricts the size of clathrin-coated vesicles. The highly conserved 33 kDa amino-terminal domain of Ap180 plays a critical role in binding to phosphoinositides and in regulating the clathrin assembly activity of Ap180. The crystal structure of the amino-terminal domain reported herein reveals a novel fold consisting of a large double layer of sheets of ten alpha helices and a unique site for binding phosphoinositides. The finding that the clathrin-box motif is mostly buried and lies in a helix indicates a different site and mechanism for binding of the domain to clathrins than previously assumed.
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synaptic vesicle size and number are regulated by a clathrin adaptor protein required for endocytosis
Neuron, 1998Co-Authors: Bing Zhang, Young Ho Koh, Robert B Beckstead, Vivian Budnik, Barry Ganetzky, Hugo J BellenAbstract:Clathrin-mediated endocytosis is thought to involve the activity of the clathrin adaptor protein Ap180. However, the role of this protein in endocytosis in vivo remains unknown. Here, we show that a mutation that eliminates an Ap180 homolog (LAP) in Drosophila severely impairs the efficiency of synaptic vesicle endocytosis and alters the normal localization of clathrin in nerve terminals. Most importantly, the size of both synaptic vesicles and quanta is significantly increased in lap mutants. These results provide novel insights into the molecular mechanism of endocytosis and reveal a role for Ap180 in regulating vesicle size through a clathrin-dependent reassembly process.
Mark E Graham - One of the best experts on this subject based on the ideXlab platform.
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The interaction of assembly protein Ap180 and clathrin is inhibited by multi-site phospho-mimetics.
Neurochemistry international, 2019Co-Authors: Lia Moshkanbaryans, Phillip J. Robinson, Ling Shan Chan, Kasper Engholm-keller, Jesse Ray Wark, Mark E GrahamAbstract:Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (Ap180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, Ap180 was mutated to determine the effect on binding. N-terminally truncated Ap180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant Ap180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) Ap180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in Ap180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length Ap180. Furthermore, phospho-mimetic Ap180 was deficient at assembling clathrin cages. These latter discoveries support a model where Ap180 phosphorylation inhibits clathrin binding and assembly.
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A Novel Sequence in Ap180 and CALM Promotes Efficient Clathrin Binding and Assembly.
PloS one, 2016Co-Authors: Lia Moshkanbaryans, Phillip J. Robinson, Jesse Ray Wark, Jing Xue, Mark E GrahamAbstract:The clathrin heavy chain N-terminal domain interacts with endocytic adapter proteins via clathrin binding motifs to assemble clathrin triskelia into cages. However, the precise mechanism of clathrin assembly is not yet known. Clathrin assembly protein Ap180 has more clathrin binding motifs than any other endocytic protein and has a major role in the assembly of the clathrin coat during synaptic vesicle biogenesis. We now demonstrate that some of the previously identified binding motifs in Ap180 may be non-functional and that a non-conventional clathrin binding sequence has a major influence on Ap180 function. The related protein, clathrin assembly lymphoid myeloid leukemia protein (CALM), has fewer clathrin binding motifs and functions ubiquitously in clathrin-mediated endocytosis. The C-terminal ~16 kDa sub-domain in Ap180, which has relatively high similarity with CALM, was shown in earlier work to have an unexplained role in clathrin binding. We identified the specific sequences in this sub-domain that bind to clathrin. Evidence for a role for these sequences in promoting clathrin binding was examined using in vitro and ex vivo experiments that compared the clathrin binding ability of site mutants with the wild type sequence. A sequence conserved in both Ap180 and CALM (LDSSLA[S/N]LVGNLGI) was found to be the major interaction site and mutation caused a deficit in clathrin assembly, which is the first example of a mutation having this effect. In contrast, single or double mutation of DL(L/F) motifs in full length Ap180 had no significant effect on clathrin binding, despite higher clathrin affinity for isolated peptides containing these motifs. We conclude that the novel clathrin interaction sites identified here in CALM and Ap180 have a major role in how these proteins interface with clathrin. This work advances the case that Ap180 and CALM are required to use a combination of standard clathrin N-terminal domain binding motifs and the sequence identified here for optimal binding and assembling clathrin.
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The ∼ 16 kDa C-terminal sequence of clathrin assembly protein Ap180 is essential for efficient clathrin binding.
PloS one, 2014Co-Authors: Ling Shan Chan, Lia Moshkanbaryans, Jing Xue, Mark E GrahamAbstract:Brain-specific Ap180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both Ap180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of Ap180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ~16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.
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the 16 kda c terminal sequence of clathrin assembly protein Ap180 is essential for efficient clathrin binding
PLOS ONE, 2014Co-Authors: Ling Shan Chan, Lia Moshkanbaryans, Jing Xue, Mark E GrahamAbstract:Brain-specific Ap180 is present in clathrin coats at equal concentration to the adapter complex, AP2, and assembles clathrin faster than any other protein in vitro. Both Ap180 and its ubiquitously expressed homolog clathrin assembly lymphoid myeloid leukemia protein (CALM) control vesicle size and shape in clathrin mediated endocytosis. The clathrin assembly role of Ap180 is mediated by a long disordered C-terminal assembly domain. Within this assembly domain, a central acidic clathrin and adapter binding (CLAP) sub-domain contains all of the known short binding motifs for clathrin and AP2. The role of the remaining ~16 kDa C-terminal sequence has not been clear. We show that this sequence has a separate function in ensuring efficient binding of clathrin, based on in vitro binding and ex vivo transferrin uptake assays. Sequence alignment suggests the C-terminal sub-domain is conserved in CALM.
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The Biochemical Properties and Functions of CALM and Ap180 in Clathrin Mediated Endocytosis.
Membranes, 2014Co-Authors: Lia Moshkanbaryans, Ling Shan Chan, Mark E GrahamAbstract:Clathrin-mediated endocytosis (CME) is a fundamental process for the regulated internalization of transmembrane cargo and ligands via the formation of vesicles using a clathrin coat. A vesicle coat is initially created at the plasma membrane by clathrin assembly into a lattice, while a specific cargo sorting process selects and concentrates proteins for inclusion in the new vesicle. Vesicles formed via CME traffic to different parts of the cell and fuse with target membranes to deliver cargo. Both clathrin assembly and cargo sorting functions are features of the two gene family consisting of assembly protein 180 kDa (Ap180) and clathrin assembly lymphoid myeloid leukemia protein (CALM). In this review, we compare the primary structure and domain organization of CALM and Ap180 and relate these properties to known functions and roles in CME and disease.