Quantification of cytosolic interactions identifies <scp>E</scp>de1 oligomers as key organizers of endocytosis

Boeke, Dominik; Trautmann, Susanne; Meurer, Matthias; Wachsmuth, Malte; Godlee, Camilla; Knop, Michael; Kaksonen, Marko

doi:10.15252/msb.20145422

Cited by 48 publications

(71 citation statements)

References 71 publications

(103 reference statements)

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“…The N-and C-terminal regions of Ede1 mediate the known protein-protein interactions between Ede1 and endocytic adaptor proteins, while the core region mediates Ede1's selfassembly (Aguilar et al, 2003;Boeke et al, 2014;Reider et al, 2009). Both the terminal protein-protein interactions and the self-interacting core are needed for the function of Ede1 in endocytic initiation (Lu and Drubin, 2017).…”

Section: Molecular Mechanisms Of Ede1's Functionmentioning

confidence: 99%

Phase separation of Ede1 promotes the initiation of endocytic events

Kozak

Kaksonen

2019

Preprint

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Clathrin-mediated endocytosis is a major pathway that eukaryotic cells use to produce transport vesicles from the plasma membrane. The assembly of the endocytic coat is initiated by a dynamic network of weakly interacting proteins, but the exact mechanism of initiation is unknown. Ede1, the yeast homologue of mammalian Eps15, is one of the early-arriving endocytic proteins and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and the frequency of endocytic initiation is decreased. We show here that in mutants with increased amounts of cytoplasmic Ede1, the excess protein forms large condensates which exhibit properties of phase separated liquid protein droplets. These Ede1 condensates recruit many other early-arriving endocytic proteins. Their formation depends on the core region of Ede1 that contains a coiled coil and a low-complexity domain. We demonstrate that Ede1 core region is essential for the endocytic function of Ede1. The core region can also promote clustering of a heterologous lipid-binding domain into discrete sites on the plasma membrane that initiate endocytic events. We propose that the clustering of the early endocytic proteins and cargo depend on phase separation mediated by Ede1.

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Section: Molecular Mechanisms Of Ede1's Functionmentioning

confidence: 99%

Phase separation of Ede1 promotes the initiation of endocytic events

Kozak

Kaksonen

2019

Preprint

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“…Fluorescence cross‐correlation spectroscopy has been recently used as a powerful experimental tool to study protein–protein interactions in living cells, such as those between early module EPs . A cytoplasmic physical interaction map was created with this technique, covering multiple modules of EPs including coat formation, adaptor function, actin assembly and scission . The spatiotemporal regulation of EPs starts from the early coat proteins, Ede1 and Syp1.…”

Section: Actin‐mediated Endocytosis and Endosomal Transportmentioning

confidence: 99%

“…The FEBS Journal 285 (2018) 2762-2784 ª 2018 Federation of European Biochemical Societies formation, adaptor function, actin assembly and scission [84]. The spatiotemporal regulation of EPs starts from the early coat proteins, Ede1 and Syp1.…”

mentioning

confidence: 99%

Phospho‐regulation of intrinsically disordered proteins for actin assembly and endocytosis

et al. 2018

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Actin filament assembly contributes to the endocytic pathway pleiotropically, with active roles in clathrin-dependent and clathrin-independent endocytosis as well as subsequent endosomal trafficking. Endocytosis comprises a series of dynamic events, including the initiation of membrane curvature, bud invagination, vesicle abscission and subsequent vesicular transport. The ultimate success of endocytosis requires the coordinated activities of proteins that trigger actin polymerization, recruit actin-binding proteins (ABPs) and organize endocytic proteins (EPs) that promote membrane curvature through molecular crowding or scaffolding mechanisms. A particularly interesting phenomenon is that multiple EPs and ABPs contain a substantial percentage of intrinsically disordered regions (IDRs), which can contribute to protein coacervation and phase separation. In addition, intrinsically disordered proteins (IDPs) frequently contain sites for post-translational modifications (PTMs) such as phosphorylation, and these modifications exhibit a high preference for IDR residues [Groban ES et al. (2006) PLoS Comput Biol 2, e32]. PTMs are implicated in regulating protein function by modulating the protein conformation, protein-protein interactions and the transition between order and disorder states of IDPs. The molecular mechanisms by which IDRs of ABPs and EPs fine-tune actin assembly and endocytosis remain mostly unexplored and elusive. In this review, we analyze protein sequences of budding yeast EPs and ABPs, and discuss the potential underlying mechanisms for regulating endocytosis and actin assembly through the emerging concept of IDR-mediated protein multivalency, coacervation, and phase transition, with an emphasis on the phospho-regulation of IDRs. Finally, we summarize the current understanding of how these mechanisms coordinate actin cytoskeleton assembly and membrane curvature formation during endocytosis in budding yeast.

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“…We tested if our proximity screen could also identify cytoplasmic subcomplexes of endocytic coat-associated proteins similarly to FCCS. Indeed, we detected all three cytoplasmic interactions between Syp1-Ede1, Pan1-End3, and Las17-Sla1 found by FCCS (Boeke et al, 2014) by their cytoplasmic FRET (Table 1). Strikingly, while FRET between Syp1-Ede1 and Pan1-End3 pairs persisted at endocytic sites, FRET between Las17 and Sla1 was specifically detected only in the cytoplasm.…”

Section: Fret Proximity Screen Detects Cytoplasmic Assemblies Of Endomentioning

confidence: 82%

“…Fluorescence cross-correlation spectroscopy (FCCS), another powerful microscopy technique for detection of molecular interactions in vivo, was recently applied to search for assemblies of endocytic proteins in the yeast cytoplasm (Boeke et al, 2014). We tested if our proximity screen could also identify cytoplasmic subcomplexes of endocytic coat-associated proteins similarly to FCCS.…”

Section: Fret Proximity Screen Detects Cytoplasmic Assemblies Of Endomentioning

confidence: 99%

The protein architecture of the endocytic coat analyzed by FRET

Skružný

Pohl

Gnoth

et al. 2018

Preprint

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Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast. We assessed proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

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Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis

Cited by 48 publications

References 71 publications

Phase separation of Ede1 promotes the initiation of endocytic events

Phase separation of Ede1 promotes the initiation of endocytic events

Phospho‐regulation of intrinsically disordered proteins for actin assembly and endocytosis

The protein architecture of the endocytic coat analyzed by FRET

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