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A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo

Nature Cell Biology volume 2, pages 958–963 (2000)Cite this article

Abstract

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission1,2,3 (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly4. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

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Figure 1: Aux1 is a functional homologue of auxilin.
Figure 2: aux1Δ cells accumulate clathrin-coated vesicles.
Figure 3: Aux1 depletion impairs clathrin-dependent protein transport and uncoating of clathrin-coated vesicles.
Figure 4: In situ impact of Aux1 depletion on intracellular clathrin distribution.
Figure 5: The Aux1 J domain is required for uncoating.

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Acknowledgements

We thank A. Nakamura for construction of the GST–Aux1 fusion, A. Rajasekaran for assistance with confocal microscopy, and A. van der Bliek and J. Hutton for comments on the manuscript. We also thank T. Graham for sharing unpublished results. This study was supported by NIH grant RO1 GM39040 to G.S.P.

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Authors and Affiliations

  1. Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, 90024, California, USA

    Babak Pishvaee, Giancarlo Costaguta, Bonny G. Yeung, Sergey Ryazantsev & Gregory S. Payne

  2. Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Tsvika Greener, Lois E. Greene & Evan Eisenberg

  3. Department of Biology, Integrated Imaging Center, Johns Hopkins University, Baltimore, 21218 , Maryland, USA

    J. Michael McCaffery

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  1. Babak Pishvaee
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Correspondence to Babak Pishvaee or Gregory S. Payne.

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Pishvaee, B., Costaguta, G., Yeung, B. et al. A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo. Nat Cell Biol 2, 958–963 (2000). https://doi.org/10.1038/35046619

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