共查询到20条相似文献,搜索用时 31 毫秒
1.
Ybe JA Perez-Miller S Niu Q Coates DA Drazer MW Clegg ME 《Traffic (Copenhagen, Denmark)》2007,8(8):1101-1110
The self-assembly of clathrin into lattices relies on the ability of heavy chain legs to form a three-legged pinwheel structure. We investigated the role of light chains in clathrin trimerization by challenging recombinant hub (plus and minus light chain) with an anionic detergent. The binding of light chain increases the amount of detergent needed to induce detrimerization, suggesting light chains reinforced hub trimers. We also show that light chain C-terminal residues are important for enhancing the in vitro assembly of hub at low pH. We assessed how much the C-terminus of light chain contributed to the stability of the trimerization domain by adding full-length and truncated light chains to trimer-defective hub mutants, C1573S and C1573A. Adding full-length LCb to C1573S caused some retrimerization, but little activity was restored, suggesting the majority of oligomeric C1573S was nonnative. A larger percentage of monomeric C1573A could be retrimerized into an assembly-competent form by adding intact LCb. We also discovered that C-terminally deleted light chains produced a heterogeneous population of hubs that were smaller than native hubs, but were assembly active. We propose a model showing how light chains reinforce the puckered clathrin triskelion. Finally, the ability of light chains to retrimerize C1573A hub suggests that the structural role of light chain may be conserved in yeast and mammals. 相似文献
2.
Joel A. Ybe Sarah N. Fontaine Todd Stone Jay Nix Xiaoyan Lin Sanjay Mishra 《FEBS letters》2013,587(2):142-149
Clathrin is a trimeric protein involved in receptor-mediated-endocytosis, but can function as a non-trimer outside of endocytosis. We have discovered that the subcellular distribution of a clathrin cysteine mutant we previously studied is altered and a proportion is also localized to nuclear spaces. MALS shows C1573A hub is a mixture of trimer-like and detrimerized molecules. The X-ray structure of the trimerization domain reveals that without light chains, a helix harboring cysteine-1573 is reoriented. We propose clathrin has a detrimerization switch, which suggests clathrin topology can be altered naturally for new functions. 相似文献
3.
Yi Xing Till Böcking Matthias Wolf Nikolaus Grigorieff Tomas Kirchhausen Stephen C Harrison 《The EMBO journal》2010,29(3):655-665
The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J‐domain containing co‐chaperone, auxilin, associates with a freshly budded clathrin‐coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy‐chain‐binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6‐barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C‐terminus of the heavy chain, with a stoichiometry of about one per three‐fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J‐domain, splits ATP, it clamps firmly onto its heavy‐chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly. 相似文献
4.
Compromise of clathrin function and membrane association by clathrin light chain deletion 总被引:2,自引:1,他引:1
While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA– mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium . In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA– mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA– mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo . 相似文献
5.
The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function. 相似文献
6.
New faces of the familiar clathrin lattice 总被引:1,自引:1,他引:0
The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues. 相似文献
7.
Clathrin light chain: importance of the conserved carboxy terminal domain to function in living cells 总被引:1,自引:1,他引:0
Clathrin triskelions assemble into coats capable of packaging membrane and receptors for transport to intracellular destinations. A triskelion is formed from three heavy chains bound to three light chains. All clathrin light chains (clc) contain an acidic amino terminal domain, a central coiled segment, and a carboxy terminal domain conserved in amino acid sequence. To assess their functional contribution in vivo, we expressed tagged segments of the Dictyostelium clcA in clc-minus Dictyostelium (clc null) cells. We examined the ability of these clcA fragments to rescue clathrin phenotypic deficiencies, to cluster into punctae on membranes, and to bind to the heavy chain. When expressed in clc null cells, a clcA fragment containing the amino terminal domain and the central coiled domain bound heavy chain but was dispensable for clathrin function. Instead, the carboxy terminal domain of clcA was a critical determinant for association with punctae, for clathrin function and for robust binding to the heavy chain. A 70 amino acid carboxy terminal fragment was necessary and sufficient for full function, and for localization into punctae on intracellular membranes. A shorter 49 amino acid carboxy terminal fragment could distribute into punctae but failed to rescue developmental deficiencies. These results reveal the importance of the carboxy terminal domain of the light chain in vivo. 相似文献
8.
Tahara H Yokota E Igarashi H Orii H Yao M Sonobe S Hashimoto T Hussey PJ Shimmen T 《Protoplasma》2007,230(1-2):1-11
Summary. We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid
sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2
cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis,
clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression
of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the
suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the
observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle
and phragmoplast, as well as in endocytosis.
Correspondence and reprints: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science
Park City, Hyogo 678-1297, Japan.
Present address: RIKEN Plant Science Center, Yokohama, Kanagawa, Japan. 相似文献
9.
Folding and trimerization of clathrin subunits at the triskelion hub. 总被引:11,自引:0,他引:11
The triskelion shape of the clathrin molecule enables it to form the polyhedral protein network that covers clathrin-coated pits and vesicles. Domains within the clathrin heavy chain that are responsible for maintaining triskelion shape and function were identified and localized. Sequences that mediate trimerization are distal to the carboxyl terminus and are adjacent to a domain that mediates both light chain binding and clathrin assembly. Structural modeling predicts that within this domain, the region of heavy chain-light chain interaction is a bundle of three or four alpha helices. These studies establish a low resolution model of clathrin subunit folding in the central portion (hub) of the triskelion, thus providing a basis for future mutagenesis experiments. 相似文献
10.
Guruprasad R. Medigeshi Maria Krikunova Karthikeyan Radhakrishnan Dirk Wenzel Jürgen Klingauf Peter Schu 《Traffic (Copenhagen, Denmark)》2008,9(1):121-132
The adaptor protein complex AP-1 mediates vesicular protein sorting between the trans Golgi network and endosomes. AP-1 recycles between membranes and the cytoplasm together with clathrin during transport vesicle formation and vesicle uncoating. AP-1 recycles independent of clathrin, indicating binding to unproductive membrane domains and premature termination of vesicle budding. Membrane recruitment requires ADP ribosylation factor-1-GTP, a transmembrane protein containing an AP-1-binding motif and phosphatidyl-inositol phosphate (PI-4-P). Little is known about the regulation of AP-1 membrane–cytoplasm recycling. We identified the N-terminal domain of μ1A-adaptin as being involved in the regulation of AP-1 membrane–cytoplasm recycling by constructing chimeras of μ1A and its homologue μ2. The AP-1* complex containing this μ2–μ1A chimera had slowed down recycling kinetics, resulting in missorting of mannose 6-phosphate receptors. The N-terminal domain is only accessible from the cytoplasmic AP-1 surface. None of the proteins known to influence AP-1 membrane recycling bound to this μ1A domain, indicating the regulation of AP-1 membrane–cytoplasm recycling by an yet unidentified cytoplasmic protein. 相似文献
11.
Heymann JB Iwasaki K Yim YI Cheng N Belnap DM Greene LE Eisenberg E Steven AC 《The Journal of biological chemistry》2005,280(8):7156-7161
Clathrin assembly into coated pits and vesicles is promoted by accessory proteins such as auxilin and AP180, and disassembly is effected by the Hsc70 ATPase. These interactions may be mimicked in vitro by the assembly and disassembly of clathrin "baskets." The chimera C58J is a minimal construct capable of supporting both reactions; it consists of the C58 moiety of AP180, which facilitates clathrin assembly, fused with the J domain of auxilin, which recruits Hsc70 to baskets. We studied the process of disassembly by using cryo-electron microscopy to identify the initial binding site of Hsc70 on clathrin-C58J baskets at pH 6, under which conditions disassembly does not proceed further. Hsc70 interactions involve two sites: (i) its major interaction is with the sides of spars of the clathrin lattice, close to the triskelion hubs and (ii) there is another interaction at a site at the N-terminal hooks of the clathrin heavy chains, presumably via the J domain of C58J. We propose that individual triskelions may be extricated from the clathrin lattice by the concerted action of up to six Hsc70 molecules, which intercalate between clathrin leg segments, prying them apart. Three Hsc70s remain bound to the dissociated triskelion, close to its trimerization hub. 相似文献
12.
Clathrin self-assembly involves coordinated weak interactions favorable for cellular regulation
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The clathrin triskelion self-assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C-termini, extending into proximal and distal leg segments ending in a globular N-terminal domain. In the clathrin coat, leg segments entwine into parallel and anti-parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo- or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self-assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin-coated vesicles. 相似文献
13.
Kohji Nishimura Syouta Ishikawa Erika Matsunami Junji Yamauchi Keiichi Homma Christine Faulkner 《Bioscience, biotechnology, and biochemistry》2013,77(12):1995-2006
Protein–protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC–CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation. 相似文献
14.
AAK1, the adaptor-associated kinase 1, phosphorylates the μ2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the μ2 subunit in vitro , μ2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates μ2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization. 相似文献
15.
The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922-20929). Here we used deletion and site- directed mutagenesis to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871-881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits. 相似文献
16.
Wu X Zhao X Baylor L Kaushal S Eisenberg E Greene LE 《The Journal of cell biology》2001,155(2):291-300
During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate. 相似文献
17.
The clathrin triskelion is composed of three light chain (LC) and three heavy chain (HC) subunits. Cellular control of clathrin function is thought to be aimed at the LC subunit, mainly on the basis of structural information. To test this hypothesis in vivo, we used evanescent-wave photobleaching recovery to study clathrin exchange from single pits using LC (LCa and LCb) and HC enhanced green fluorescent protein fusion constructs. The recovery signal was corrected for cytosolic diffusional background, yielding the pure exchange reaction times. For LCa, we measured an unbinding time constant tau(LEa) = 18.9 +/- 1.0 seconds at room temperature, faster than previously published; for LCb, we found tau(LCb) = 10.6 +/- 1.9 seconds and for HC tau(HC) = 15.9 +/- 1.0 seconds. Sucrose treatment, ATP or Ca(2+) depletion blocked exchange of LCa completely, but only partially of HC, lowering its time constant to tau = 10.0 +/- 0.9 seconds, identical to the one for LCb exchange. The latter was also not blocked by Ca(2+) depletion or sucrose. We conclude that HCs bound both to LCa and to LCb contribute side by side to pit formation in vivo, but the affinity of LCa-free HC in pits is reduced, and the Ca(2+)- and ATP-mediated control of clathrin function is lost. 相似文献
18.
19.
Two regions on the clathrin heavy chain that are involved in triskelion interactions during assembly have been localized on the triskelion structure. These regions were previously identified with anti-heavy chain monoclonal antibodies X19 and X35, which disrupt clathrin assembly (Blank, G. S., and F. M. Brodsky, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:2087-2095). Antibody-binding sites were determined based on their reactivity with truncated triskelions, and were mapped to an 8-kD region in the middle of the proximal portion of the triskelion arm (X19) and a 6-kD region at the triskelion elbow (X35). The elbow site implicated in triskelion assembly was also shown to be included within a heavy chain region involved in binding the light chains and to constitute part of the light chain-binding site. We postulate that this region of the heavy chain binds to the interaction site identified on the light chains that has homology to intermediate filament proteins (Brodsky, F. M., C. J. Galloway, G. S. Blank, A. P. Jackson, H.-F. Seow, K. Drickamer, and P. Parham, 1987, Nature (Lond.), 326:203-205). These findings suggest the existence of a heavy chain site, near the triskelion elbow, which is involved in both intramolecular and intermolecular interactions during clathrin assembly. 相似文献
20.
Zhang CX Engqvist-Goldstein AE Carreno S Owen DJ Smythe E Drubin DG 《Traffic (Copenhagen, Denmark)》2005,6(12):1103-1113
Cyclin G-associated kinase (GAK), also known as auxilin 2, is a potential regulator of clathrin-mediated membrane trafficking. It possesses a kinase domain at its N-terminus that can phosphorylate the clathrin adaptors AP-1 and AP-2 in vitro. The GAK C-terminus can act as a cochaperaone in vitro for Hsc70, a heat-shock protein required for clathrin uncoating. Here we show that the specificity of GAK is very similar to that of adaptor-associated kinase 1, another mammalian adaptor kinase. We used siRNA to investigate GAK's in vivo function. We discovered that early stages of clathrin-mediated endocytosis (CME) were partially inhibited when GAK expression was knocked down. This defect was specifically caused by GAK knockdown because it could be rescued by expressing a rat GAK gene that could not be silenced by one of the siRNAs. To identify the GAK activity required during CME, we mutated the kinase domain and the J domain of the rat gene. Only GAK with a functional J domain could rescue the defect, suggesting that GAK is important for clathrin uncoating. Furthermore, we demonstrated that GAK plays a role in the clathrin-dependent trafficking from the trans Golgi network. 相似文献