KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET

Dev Cell. 2001 Jul;1(1):139-53. doi: 10.1016/s1534-5807(01)00004-1.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 1 / metabolism
  • ADP-Ribosylation Factors / metabolism
  • Animals
  • Chlorocebus aethiops
  • Coat Protein Complex I / metabolism*
  • Coatomer Protein / metabolism
  • Cytoplasm / metabolism
  • GTPase-Activating Proteins / metabolism
  • Golgi Apparatus / metabolism
  • Ligands
  • Membrane Proteins / metabolism
  • Peptide Fragments / metabolism
  • Protein Binding / physiology
  • Protein Transport / physiology*
  • Receptors, Peptide / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Reproducibility of Results
  • Spectrometry, Fluorescence / standards
  • Vero Cells

Substances

  • Coat Protein Complex I
  • Coatomer Protein
  • GTPase-Activating Proteins
  • KDEL receptor
  • Ligands
  • Membrane Proteins
  • Peptide Fragments
  • Receptors, Peptide
  • Recombinant Fusion Proteins
  • ADP-Ribosylation Factor 1
  • ADP-Ribosylation Factors