The gamma/sigma1 and alpha/sigma2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site

Mol Biol Cell. 2007 May;18(5):1887-96. doi: 10.1091/mbc.e07-01-0012. Epub 2007 Mar 14.

Abstract

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma1 or alpha/sigma2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta1/mu1 and beta2/mu2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma1 and alpha/sigma2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma1 hemicomplex, whereas minimally affecting binding to alpha/sigma2. There was an excellent correlation between binding to the alpha/sigma2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Protein Complex 1 / chemistry*
  • Adaptor Protein Complex 1 / genetics
  • Adaptor Protein Complex 1 / metabolism
  • Adaptor Protein Complex 2 / chemistry*
  • Adaptor Protein Complex 2 / genetics
  • Adaptor Protein Complex 2 / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Dipeptides / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • In Vitro Techniques
  • Mice
  • Multiprotein Complexes / chemistry
  • Protein Sorting Signals / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Adaptor Protein Complex 1
  • Adaptor Protein Complex 2
  • Dipeptides
  • Multiprotein Complexes
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • leucylleucine