Sequence requirements for the recognition of tyrosine-based endocytic signals by clathrin AP-2 complexes.

Abstract

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.

β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3

“…Finally, the analogous subunits might play similar roles. For instance, the p47A ( 3A) subunit of AP-3 is capable of binding tyrosine-based sorting signals (22,30), like its relatives 1 and 2 (16,18,22). The comparable domain organization of ␤1-, ␤2-, and ␤3A-adaptins suggests that they might fulfill similar roles in the interaction of the adaptor complexes with scaffolding proteins.…”

“…The adaptors are also responsible for the recognition of sorting signals present in the cytosolic domains of integral membrane proteins (10 -21), an event that leads to the concentration of these proteins within clathrincoated areas of the trans-Golgi network and the plasma membrane. Recent evidence suggests that the 1 subunit of AP-1 and the 2 subunit of AP-2 are directly involved in signal recognition (16,18,22).…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…The sorting mechanism is best characterized for the medium (m) subunit, which is known to recognize the YXXF motif (F represents Leu, Ile, Phe, Met, or Val) that is present in the cytosolic domains of cargo proteins (Ohno et al, 1995). Mutations of the YXXF motif abolish the interaction with m (Ohno et al, 1995;Boll et al, 1996;Stephens et al, 1997) and alter the subcellular localization of the cargo proteins (Bos et al, 1993;Humphrey et al, 1993). The interaction of m with cargo proteins through the YXXF motif is disrupted by the structural Tyr analog Tyrphostin A23 (TyrA23), which is an efficient inhibitor of clathrin-mediated endocytosis (Banbury et al, 2003).…”

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

“…These signals are thought to interact with specific recognition molecules, which are components of membrane-bound transport intermediates (Schmid, 1997;Hirst and Robinson, 1998;Bonifacino and Dell'Angelica, 1999;Kirchhausen, 1999;Marsh and McMahon, 1999). Most internalized proteins contain sorting signals such as the tyrosine-based motif, Yxx , where x is any amino acid and is a bulky hydrophobic side chain (Lobel et al, 1989;Collawn et al, 1990;Boll et al, 1996) or the di-leucine-based motif, LL (Letourneur and Klausner, 1992). For the tyrosine-based motif, the interaction is mediated by the medium chains of adaptor protein (AP) complexes (Ohno et al, 1995).…”