Deep-etch visualization of proteins involved in clathrin assembly.

Heuser, John E.; Keen, James H.

doi:10.1083/jcb.107.3.877

Cited by 155 publications

(101 citation statements)

References 34 publications

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“…This autoinhibition could induce a functional switch by a conformational change that indicates cargo uptake and initiates clathrin coat formation. This model would also explain why empty clathrin cages are never observed in vivo (Kirchhausen, 1999) and correlate with previous observations by electron microscopy that show various dispositions of the appendage domain relative to the trunk portion (Heuser and Keen, 1988). Interestingly, autoinhibition by an internal nuclear localization signal was discovered for Importin ␣ and found to explain the regulatory switch between the cytoplasmic, high-affinity form, and the nuclear, low-affinity form for NLS binding of the Importin (Kobe, 1999).…”

Section: Molecular Biology Of the Cell 2052supporting

confidence: 69%

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

Geyer

Fackler

Peterlin

2002

MBoC

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The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the ␤-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the ␤-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For ␤-adaptins, this fragment binds to cytoplasmic di-leucine-based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine-based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold. INTRODUCTIONTargeting of transmembrane proteins to different compartments of the endocytic and late secretory pathways depends largely on sorting signals contained within their cytosolic domains (Letourneur and Klausner, 1992;Mellman, 1996;Kirchhausen et al., 1997). These signals are thought to interact with specific recognition molecules, which are components of membrane-bound transport intermediates (Schmid, 1997;Hirst and Robinson, 1998;Bonifacino and Dell'Angelica, 1999;Kirchhausen, 1999;Marsh and McMahon, 1999). Most internalized proteins contain sorting signals such as the tyrosine-based motif, Yxx , where x is any amino acid and is a bulky hydrophobic side chain (Lobel et al., 1989;Collawn et al., 1990;Boll et al., 1996) or the di-leucine-based motif, LL (Letourneur and Klausner, 1992). For the tyrosine-based motif, the interaction is mediated by the medium chains of adaptor protein (AP) complexes (Ohno et al., 1995). Cocrystallization of the signal binding domain of 2 with two different Yxx peptides provided a structural explanation for this binding specificity (Owen and Evans, 1998).The Nef protein of primate lentiviruses is the only nontransmembrane protein known to traffic via a di-leucinebased motif (Kirchhausen, 1999). It is required for the internalization of CD4 from the cell surface to endosomes and lysosomes (Craig et al., 1998;Greenberg et al., 1998). Several proteins have been proposed to direct the sorting of Nef. They include the ␤-chain of AP-1 complexes (Bresnahan et al., 1998;Greenberg et al., 1998), the ␤-subunit of the COP-I coatomer (Piguet et al., 1999;Janvier et al., 2001), and the regulatory subunit H (V1H) of the vacuolar ATPase (VATPase), previously named NBP1 for Nef binding protein 1 (Lu et al., 1998;Mandic et al., 2001). Although the precise sites of these interactions varied, all protein complexes were reported to bind ...

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Section: Molecular Biology Of the Cell 2052supporting

confidence: 69%

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

Geyer

Fackler

Peterlin

2002

MBoC

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“…We have recently shown that Eps15 forms parallel dimers and anti-parallel tetramers of ϳ31 nm in length (11), and so this result is not surprising. Presumably this result indicates that the globular AP-2 complexes (ϳ60-nm diameter) (17) do not bind at the extreme ends of the molecule.…”

Section: Resultsmentioning

confidence: 94%

“…Alternatively, it could be due to steric or competitive interactions between clathrin and Eps15 on AP-2 upon binding to each other that results in the disruption of the contact between AP-2 and Eps15. Eps15 and clathrin bind to sites in AP-2 that are very close to each other (17): the ␣ ear, the binding site for Eps15 (7,8), is only 2-10 nm from the ␤ hinge, the binding site for clathrin (5). Eps15, at 31 nm in length (11), could easily span this distance, and the terminal domain of clathrin is ϳ7 nm in diameter (19).…”

Section: Disruption Of Eps15⅐ap-2 Complexes By Clathrin Coats 1848mentioning

confidence: 99%

Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Cupers

Jadhav

Kirchhausen

1998

Journal of Biological Chemistry

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Eps15 is a phosphorylation substrate of the epidermal growth factor receptor kinase. In vivo, it is largely found in complex with AP-2, the plasma membrane clathrin adaptor protein complex. Although AP-2 is uniformly distributed across the surface of clathrin-coated pits and vesicles, Eps15 is preferentially found in the rims of endocytic clathrin-coated pits (1). This observation suggests that Eps15 may disengage from AP-2 during coat formation. Here we use two new anti-Eps15 antibodies to show that, contrary to our own earlier suggestion, coated vesicles isolated from brain do not contain detectable amounts of Eps15. Furthermore, when AP-2 complexes that are saturated with Eps15 are used for in vitro assembly of clathrin-AP-2 coats, normal structures are formed that contain the expected amounts of clathrin and AP-2, but the amount of Eps15 present is dramatically lower than that of AP-2. We propose that during coated pit formation, addition of clathrin to the growing edge at the rim of the pit releases Eps15 from AP-2.During the past decade, several groups have extensively studied the protein components and the mechanism responsible for the formation of clathrin-coated pits. Clathrin and its tetrameric adaptors AP-1 and AP-2 are among the best characterized structural elements of the coat (for recent reviews, see Refs. 2-4). The APs recruit clathrin, and they facilitate coat formation through its association with clathrin. The interaction involves a short segment located in the hinge region of the ␤ chain of AP-2 (5) and the terminal domain of clathrin. 1 Recently, it was suggested that Eps15 (6), a ϳ100-kDa molecule that forms complexes with AP-2 (7-9), is involved in the clathrin-mediated internalization pathway. Eps15-AP-2 complexes are found both in the cytosol and at the plasma membrane (1, 9, 10). In solution, the interaction between Eps15 and AP-2 involves a short segment toward the C terminus of Eps15 and the C-terminal ear domain of the ␣ chain of AP-2 (7, 8).Most of the Eps15 in the cell is bound to AP-2, whereas ϳ80% of AP-2 is free (1). The membrane-bound Eps15/AP-2 complexes are mainly found within clathrin-coated pits (1, 10), usually at the rims and growing edges of coated pits (1).The formation of coated pits at the plasma membrane requires AP-2 complexes and clathrin to be recruited to the membrane and a poorly understood nucleation event that initiates clathrin coat assembly. After nucleation, new clathrin and AP-2 complexes presumably add to the free edges of the pit, so that an AP-2 complex that is added to the pit early on will move progressively deeper into the pit. How is it, then, that Eps15 is concentrated at the edges of pits, regardless of the state of completion of the pit? We previously examined whether Eps15 is present in coated vesicles (1) and found a band of the approximately correct molecular weight in gels of purified bovine brain vesicle preparations. However, this band was not conclusively identified as Eps15, and in the absence of purified Eps15 quantitation was impossible...

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“…An extended hinge segment of about 80 -100 residues connects the stacked HEAT-repeats to an "appendage" domain at the carboxy-terminal end of each of the large chains (Fig. 4B,C) (Heuser and Keen 1988).…”

Section: Heterotetrameric Clathrin Adaptorsmentioning

confidence: 99%

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

Kirchhausen

Owen

Harrison

2014

Cold Spring Harbor Perspectives in Biology

433

442

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Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.

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Deep-etch visualization of proteins involved in clathrin assembly.

Cited by 155 publications

References 34 publications

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

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