Abstract
How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a "wave" of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP-Ribosylation Factor 1 / metabolism
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ADP-Ribosylation Factors / metabolism
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Animals
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Chlorocebus aethiops
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Coat Protein Complex I / metabolism*
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Coatomer Protein / metabolism
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Cytoplasm / metabolism
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GTPase-Activating Proteins / metabolism
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Golgi Apparatus / metabolism
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Ligands
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Membrane Proteins / metabolism
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Peptide Fragments / metabolism
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Protein Binding / physiology
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Protein Transport / physiology*
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Receptors, Peptide / metabolism*
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Recombinant Fusion Proteins / metabolism
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Reproducibility of Results
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Spectrometry, Fluorescence / standards
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Vero Cells
Substances
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Coat Protein Complex I
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Coatomer Protein
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GTPase-Activating Proteins
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KDEL receptor
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Ligands
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Membrane Proteins
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Peptide Fragments
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Receptors, Peptide
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Recombinant Fusion Proteins
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ADP-Ribosylation Factor 1
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ADP-Ribosylation Factors