Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins

Department of Pathology, Institute of Ophthalmology, London, U.K.
Biochemical Journal (Impact Factor: 4.4). 11/1996; 319 ( Pt 1)(1):103-8. DOI: 10.1042/bj3190103
Source: PubMed


The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Although we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.

Download full-text


Available from: Mike Cheetham,
  • Source
    • "In DKO-R cells treated with doxycycline for 96 h, clathrin expression was undetectable by immunoblotting (Fig. 3 b). Using a more sensitive clathrin triskelion ELISA (Cheetham et al., 1996), residual expression corresponding to ∼0.5% of the unrepressed level was detected under these conditions (unpublished data). As judged by immunoblotting, clathrin depletion did not affect the level of μ2 (Fig. 3 b) or, in separate experiments, α- and β-adaptin (unpublished data). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of mu2 phosphorylation. We identify clathrin as a specific activator of the mu2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of mu2 phosphorylation. Furthermore, we show that AP2 containing phospho-mu2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-mu2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-mu2 levels.
    The Journal of Cell Biology 11/2003; 163(2):231-6. DOI:10.1083/jcb.200304079 · 9.83 Impact Factor
  • Source
    • "Since other DnaJ homologues cannot substitute for auxilin [121] [122], the clathrin-binding domain of auxilin is apparently crucial to support uncoating by Hsc70. While auxilin shares many properties with other J-proteins such as stimulating ATP hydrolysis and ATP-dependent polymerization of Hsc70 [117] [119] [123] [124], it also shows two unique differences . "
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulated neurotransmitter release depends on a precise sequence of events that lead to repeated cycles of exocytosis and endocytosis. These events are mediated by a series of molecular interactions among vesicular, plasma membrane, and cytosolic proteins. An emerging theme has been that molecular chaperones may guide the sequential restructuring of stable or transient protein complexes to promote a temporal and spatial regulation of the endo- and exocytotic machinery and to ensure a vectorial passage through the vesicle cycle. Chaperones, specialized for a few substrates, are ideally suited to participate in regulatory processes that require some molecular dexterity to rearrange conformational or oligomeric protein structures. This article emphasizes the significance of three molecular chaperone systems in regulated neurotransmitter release: the regulation of soluble NSF attachment protein receptor (SNARE) complexes by N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment protein (SNAP), the uncoating of clathrin-coated vesicles by the 70 kDa heat-shock cognate protein (Hsc70), and the regulation of SNARE complex-associated protein interactions by cysteine-string protein and Hsc70.
    Biochemical Pharmacology 08/2001; 62(1):1-11. DOI:10.1016/S0006-2952(01)00648-7 · 5.01 Impact Factor
  • Source
    • "and inhibits heat shock cognate 70 protein (hsc70)-cata- lyzed clathrin uncoating (Cheetham et al., 1996). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.
    The Journal of Cell Biology 01/1998; 139(5):1089-95. · 9.83 Impact Factor
Show more