Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes.
We recently determined that fusion proteins containing tyrosine-based endocytic signals bind to the mu 2 subunit of AP-2, the complex that drives clathrin coat formation and mediates endocytosis from the plasma membrane. Here we analyze the selectivity of peptide recognition by mu 2 and by AP-2 using combinatorial selection methods and surface plasmon resonance. Both mu 2 and AP-2 are shown to interact with various sequences of the form tyrosine-polar-polar-hydrophobic (Yppø) found on receptors that follow the clathrin pathway. The optimal sequence for interaction with mu 2 and with AP-2 has tyrosine as an anchor and prefers arginine at position Y + 2 and leucine at position Y + 3. In contrast, no preferred sequence is detected surrounding the Yppø signal, indicating that recognition of the Yppø endocytic signal does not require a prefolded structure. We conclude that sorting into the endocytic pathway is governed by a surprisingly simple interaction between the mu 2 chain and a tyrosine-containing tetrapeptide sequence.
Boll
W
W
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Ohno
H
H
Songyang
Z
Z
Rapoport
I
I
Cantley
L C
LC
Bonifacino
J S
JS
Kirchhausen
T
T
D000595:Amino Acid Sequence; D002966:Clathrin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D004705:Endocytosis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D014443:Tyrosine / Q000737:chemistry
D002966:Clathrin; D004268:DNA-Binding Proteins; D019151:Peptide Library; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D014443:Tyrosine