Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4

Area of Science:

• Cell Biology
• Molecular Biology
• Protein Trafficking

Background:

• Adaptor protein (AP) complexes mediate intracellular trafficking by recognizing sorting signals on membrane proteins.
• The medium (mu) chains of AP-1, AP-2, and AP-3 bind distinct tyrosine-based (YXXphi) sorting signals.
• The adaptor protein complex AP-4 and its mu4 subunit's function in signal recognition remain largely uncharacterized.

Purpose of the Study:

• To determine the signal-binding specificity and affinity of the mu4 subunit of AP-4.
• To elucidate the structural determinants governing mu4's recognition of YXXphi signals.
• To investigate the role of AP-4 in intracellular protein trafficking.

Main Methods:

• Two-hybrid combinatorial peptide library screening using mu4 as bait.
• In vitro binding assays to analyze mu4-YXXphi interactions.
• Surface plasmon resonance to quantify binding affinity.
• Construction and analysis of a Tac chimera with a mu4-specific signal.

Main Results:

• Mu4 exhibits specificity for YXXphi signals, preferring aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3.
• Mu4 specifically recognizes the YXXphi signal from human lysosomal protein LAMP-2 (HTGYEQF).
• The apparent dissociation constant for the mu4-YXXphi interaction is in the micromolar range.
• A Tac chimera with a mu4-specific signal was targeted to the endosomal-lysosomal system, bypassing plasma membrane internalization.

Conclusions:

• Mu4 possesses defined signal-binding preferences, contributing to the specificity of AP-4 mediated trafficking.
• The identified YXXphi signal motif is crucial for targeting proteins to the endosomal-lysosomal pathway via AP-4.
• AP-4 plays a role in directing integral membrane proteins to the endosomal-lysosomal system.