Nathke, I.S. et al. Folding and trimerization of clathrin subunits at the triskelion hub. Cell 68, 899-910
ABSTRACT 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.
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- "To confirm protein loading, nitrocellulose was stained with 0.1% amido black in 10% acetic acid, 45% methanol and destained with 10% acetic acid, 45% methanol. Clathrin was detected using mouse monoclonal antibody TD1 . Chicken transferrin was detected using mouse monoclonal antibodies . "
ABSTRACT: We have previously deleted both endogenous copies of the clathrin heavy-chain gene in the chicken pre B-cell-line DT40 and replaced them with clathrin under the control of a tetracycline-regulatable promoter (Tet-Off). The originally derived cell-line DKO-S underwent apoptosis when clathrin expression was repressed. We have also described a cell-line DKO-R derived from DKO-S cells that was less sensitive to clathrin-depletion. Here we show that the restriction of transferrin uptake, resulting in iron deprivation, is responsible for the lethal consequence of clathrin-depletion. We further show that the DKO-R cells have up-regulated an anti-apoptotic survival pathway based on the chemokine SDF-1 and its receptor CXCR4. Our work clarifies several puzzling features of clathrin-depleted DT40 cells and reveals an example of how SDF-1/CXCR4 signalling can abrogate pro-apoptotic pathways and increase cell survival. We propose that the phenomenon described here has implications for the therapeutic approach to a variety of cancers.PLoS ONE 08/2014; 9(8):e106278. DOI:10.1371/journal.pone.0106278 · 3.23 Impact Factor
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- "The clathrin-H-link domain is short and often appears directly downstream from the clathrin heavy-chain linker . Clathrin and VPS repeat regions are approximately 140 amino acids long, composed of multiple alpha helical repeats and occur in the arm regions of the clathrin heavy chain [23,24], as well as in VPS proteins . "
ABSTRACT: Background Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins. Results Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex. Conclusion Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.BMC Cell Biology 06/2014; 15(1):23. DOI:10.1186/1471-2121-15-23 · 2.34 Impact Factor
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- "A 2.9 Å resolution structure of the proximal leg revealed CHC repeats (CHCRs) comprising 10 helices of 10–12 residues each connected by loops, creating two helical and two loop faces to the triskelion leg (Ybe et al., 1999). Mapping CLC position along CHC by electron microscopy and immunolabeling has generated conflicting evidence for a bent or an extended CLC conformation, suggesting possible conformational lability (Kirchhausen and Toyoda, 1993; Nathke et al., 1992). A reconstructed image of the assembled clathrin lattice between 7.9 and 12 Å resolution based on cryo-electron microscopy (cryoEM) showed the backbone of the central third of the CLC as an alpha helix bound to the loop face of the CHC (Fotin et al., 2004), as predicted by mutagenesis and modeling studies (Chen et al., 2002). "
ABSTRACT: Clathrin-coated vesicle formation is responsible for membrane traffic to and from the endocytic pathway during receptor-mediated endocytosis and organelle biogenesis, influencing how cells relate to their environment. Generating these vesicles involves self-assembly of clathrin molecules into a latticed coat on membranes that recruits receptors and organizes protein machinery necessary for budding. Here we define a molecular mechanism regulating clathrin lattice formation by obtaining structural information from co-crystals of clathrin subunits. Low resolution X-ray diffraction data (7.9-9.0 A) was analyzed using a combination of molecular replacement with an energy-minimized model and noncrystallographic symmetry averaging. Resulting topological information revealed two conformations of the regulatory clathrin light chain bound to clathrin heavy chain. Based on protein domain positions, mutagenesis, and biochemical assays, we identify an electrostatic interaction between the clathrin subunits that allows the observed conformational variation in clathrin light chains to alter the conformation of the clathrin heavy chain and thereby regulates assembly.Developmental Cell 05/2010; 18(5):841-8. DOI:10.1016/j.devcel.2010.04.007 · 9.71 Impact Factor