The adaptor protein complex-1 (AP-1) sorts and packages membrane proteins into clathrin-coated vesicles (CCVs) at the TGN and endosomes. Here we show that this process is highly regulated by phosphorylation of AP-1 subunits. Cell fractionation studies revealed that membrane-associated AP-1 differs from cytosolic AP-1 in the phosphorylation status of its β1 and μ1 subunits. AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)–mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated μ1. Once on the membrane, the μ1 subunit undergoes phosphorylation, which results in a conformation change, as revealed by increased sensitivity to trypsin. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of μ1 (and μ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Immunodepletion and okadaic acid inhibition studies demonstrate that PP2A is the cytosolic cofactor for Hsc-70–mediated adaptor uncoating. A model is proposed where cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.
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3 March 2003
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February 25 2003
AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation
Pradipta Ghosh,
Pradipta Ghosh
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
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Stuart Kornfeld
Stuart Kornfeld
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
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Pradipta Ghosh
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
Stuart Kornfeld
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
Address correspondence to Stuart Kornfeld, Washington University School of Medicine, Department of Internal Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel: (314)-362-8803. Fax: (314)-362-8826. E-mail: [email protected]
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Abbreviations used in this paper: AP-1, adaptor protein complex-1; CCV, clathrin-coated vesicle; CD-MPR, cation-dependent MPR; CI-MPR, cation-independent MPR; CK2, casein kinase-2; GAK, cyclin G–associated kinase; GGA, Golgi-localized, γ ear–containing, adenosine 5′ diphosphate-ribosylation factor–binding protein; MPR, mannose 6-phosphate receptor; PP2A, protein phosphatase 2A.
Received:
November 19 2002
Revision Received:
January 09 2003
Accepted:
January 10 2003
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2003
J Cell Biol (2003) 160 (5): 699–708.
Article history
Received:
November 19 2002
Revision Received:
January 09 2003
Accepted:
January 10 2003
Citation
Pradipta Ghosh, Stuart Kornfeld; AP-1 binding to sorting signals and release from clathrin-coated vesicles is regulated by phosphorylation . J Cell Biol 3 March 2003; 160 (5): 699–708. doi: https://doi.org/10.1083/jcb.200211080
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