EpsinR: an ENTH domain-containing protein that interacts with AP-1.
We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.
Hirst
Jennifer
J
University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, United Kingdom.
Motley
Alison
A
Harasaki
Kouki
K
Peak Chew
Sew Y
SY
Robinson
Margaret S
MS
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018808:Transcription Factor AP-1 / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D033942:Adaptor Proteins, Vesicular Transport; C475910:CLINT1 protein, human; D002352:Carrier Proteins; D018808:Transcription Factor AP-1; D020126:Brefeldin A; D005982:Glutathione Transferase