Abstract
Clathrin-coated vesicles mediate endocytosis and transport between the trans-Golgi network (TGN) and endosomes in eukaryotic cells. Clathrin adaptors play central roles in coat assembly, interacting with clathrin, cargo and membranes. Two main types of clathrin adaptor act in TGN–endosome traffic: GGA proteins and the AP-1 complex. Here we characterize the relationship between GGA proteins, AP-1 and other TGN clathrin adaptors using live-cell and super-resolution microscopy in yeast. We present evidence that GGA proteins and AP-1 are recruited sequentially in two waves of coat assembly at the TGN. Mutations that decrease phosphatidylinositol 4-phosphate (PtdIns(4)P) levels at the TGN slow or uncouple AP-1 coat assembly from GGA coat assembly. Conversely, enhanced PtdIns(4)P synthesis shortens the time between adaptor waves. Gga2p binds directly to the TGN PtdIns(4)-kinase Pik1p and contributes to Pik1p recruitment. These results identify a PtdIns(4)P-based mechanism for regulating progressive assembly of adaptor-specific clathrin coats at the TGN.
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Acknowledgements
We thank J.Thorner, S. Emr and T. Levine for plasmids and strains, J. Atkins for help with SIM, and members of the laboratory for helpful discussions. We are especially grateful to K. Kilborn for advice and assistance with confocal microscopy and K. Martin, G. Weinmaster and A. van der Bliek for comments on the manuscript. This work was supported by NIH NRSA T32 GM-007104 and a UCLA Dissertation Year Fellowship to L.D. and NIH GM39040 to G.P.
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L.D. and G.S.P. conceived the experiments. L.D. carried out all microscopy, protein interaction and CPY experiments. G.C. carried out the α-factor experiments, and generated recombinant expression and integration constructs. L.D. and G.S.P. wrote the manuscript.
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Daboussi, L., Costaguta, G. & Payne, G. Phosphoinositide-mediated clathrin adaptor progression at the trans-Golgi network. Nat Cell Biol 14, 239–248 (2012). https://doi.org/10.1038/ncb2427
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DOI: https://doi.org/10.1038/ncb2427
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