NECAP 1 Regulates AP-2 Interactions to Control Vesicle Size, Number, and Cargo During Clathrin-Mediated Endocytosis

Ritter, Brigitte; Murphy, S. M. C.; Dokainish, Hatem; Girard, Martine; Gudheti, Manasa V.; Kozlov, Guennadi; Halin, Marilene; Philie, Jacynthe; Jørgensen, Erik M.; Gehring, Kalle; McPherson, Peter S.

doi:10.1371/journal.pbio.1001670

Cited by 65 publications

(92 citation statements)

References 60 publications

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“…1A,B). The immunofluorescence signal intensity of the coat proteins AP-2 and clathrin directly correlates to the size of the forming structure (Antonescu et al, 2011;Ehrlich et al, 2004;Mettlen et al, 2010;Ritter et al, 2013). As expected, NECAP1 knockdown decreased the number and increased the intensity of AP-2-labeled puncta, whereas NECAP2 depletion has no effect ( Fig.…”

Section: Necap2 Does Not Regulate the Size And Number Of Endocytic CLsupporting

confidence: 65%

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NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling

Chamberland

Antonow

Santos

et al. 2016

Journal of Cell Science

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Endocytic recycling returns receptors to the plasma membrane following internalization and is essential to maintain receptor levels on the cell surface, re-sensitize cells to extracellular ligands and for continued nutrient uptake. Yet, the protein machineries and mechanisms that drive endocytic recycling remain ill-defined. Here, we establish that NECAP2 regulates the endocytic recycling of EGFR and transferrin receptor. Our analysis of the recycling dynamics revealed that NECAP2 functions in the fast recycling pathway that directly returns cargo from early endosomes to the cell surface. In contrast, NECAP2 does not regulate the clathrin-mediated endocytosis of these cargos, the degradation of EGFR or the recycling of transferrin along the slow, Rab11-dependent recycling pathway. We show that protein knockdown of NECAP2 leads to enlarged early endosomes and causes the loss of the clathrin adapter AP-1 from the organelle. Through structure-function analysis, we define the protein-binding interfaces in NECAP2 that are crucial for AP-1 recruitment to early endosomes. Together, our data identify NECAP2 as a pathway-specific regulator of clathrin coat formation on early endosomes for fast endocytic recycling.

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Section: Necap2 Does Not Regulate the Size And Number Of Endocytic CLsupporting

confidence: 65%

“…Furthermore, each NECAP protein failed to rescue the knockdown phenotype of the other family member (Fig. 7A,B; Ritter et al, 2013). Taken together, these data demonstrate that NECAP1 and NECAP2 have functionally diverged to selectively regulate clathrin-mediated sorting at distinct cellular locations.…”

Section: Discussionmentioning

confidence: 70%

NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling

Chamberland

Antonow

Santos

et al. 2016

Journal of Cell Science

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“…Blots were probed with affinity-purified antibodies directed against FCHO1 or FCHO2 or with a mAb (C-8) the α subunit of AP-2. FCHO1, FCHO2 and AP-2 all show dose-dependent interactions with the Necap 1 PHear domain (Ritter et al, 2004, 2007, 2013), although FCHO1 clearly displays the highest apparent affinity. In general, FCHO2 shows a weaker capacity to correct the clathrin distribution phenotype in HeLa clone #64/1.E cells, which is correlated with poorer binding to AP-2 and Necap 1. DOI: http://dx.doi.org/10.7554/eLife.04137.017…”

Section: Resultsmentioning

confidence: 99%

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

Umasankar

Thieman

et al. 2014

eLife

125

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Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.DOI: http://dx.doi.org/10.7554/eLife.04137.001

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“…Importantly, a number of physiological interactions, particular in the regulation of synaptic vesicle exocytosis and endocytosis are known to be sensitive to salt [for example, synapsin binding to synaptic vesicles (Huttner et al, 1983) is completely disrupted by 50 mM NaCl, and the ability of AP-2 to bind to NECAP1 or clathrin, both undoubtedly important physiological interactions, are sensitive to salt in the 30-50 mM range (Ritter et al, 2013)]. …”

Section: Syt1jxm Interacts Directly With the Dynamin 1 Ph Domainmentioning

confidence: 99%

“…Lentiviruses were produced and titered as described (Ritter et al, 2013). For chromaffin cell transduction, several hours after plating, 3-5 viral particles per cell were applied to the cells for 3 h and patch-clamp measurements were performed 12-24 h later.…”

Section: Lentivirus Experimentsmentioning

confidence: 99%

The juxtamembrane region of synaptotagmin 1 interacts with dynamin 1 and regulates vesicle fission during compensatory endocytosis in endocrine cells

McAdam

Varga

Jiang

et al. 2015

Journal of Cell Science

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Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that is important for the kinetics of both exocytosis and endocytosis, and is thus a candidate molecule to link these two processes. Although the tandem Ca 2+ -binding C2 domains of Syt1 have important roles in exocytosis and endocytosis, the function of the conserved juxtamembrane ( jxm) linker region has yet to be determined. We now demonstrate that the jxm region of Syt1 interacts directly with the pleckstrin homology (PH) domain of the endocytic protein dynamin 1. By using cell-attached capacitance recordings with millisecond time resolution to monitor clathrin-mediated endocytosis of single vesicles in neuroendocrine chromaffin cells, we find that loss of this interaction prolongs the lifetime of the fission pore leading to defects in the dynamics of vesicle fission. These results indicate a previously undescribed interaction between two major regulatory proteins in the secretory vesicle cycle and that this interaction regulates endocytosis.

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NECAP 1 Regulates AP-2 Interactions to Control Vesicle Size, Number, and Cargo During Clathrin-Mediated Endocytosis

Abstract: The endocytic protein NECAP 1 cooperates with the endocytic adapter protein AP-2 to modulate interactions with accessory proteins and clathrin and to control the size, number, and cargo content of clathrin-coated vesicles.

Cited by 65 publications

References 60 publications

NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling

NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

The juxtamembrane region of synaptotagmin 1 interacts with dynamin 1 and regulates vesicle fission during compensatory endocytosis in endocrine cells

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