Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding

Area of Science:

• Cell biology
• Molecular biology
• Biochemistry

Background:

• Clathrin-coated vesicles mediate essential cellular transport processes.
• The precise choreography of coat protein assembly during endocytosis remains incompletely understood.
• Transient protein-protein interactions involving short motifs are critical for dynamic protein complex formation.

Purpose of the Study:

• To elucidate the role of Asp-Pro-Phe (DPF) motifs in Eps15/R in orchestrating endocytic coat assembly.
• To investigate the differential decoding of DPF motifs by endocytic pioneers Fcho1/2 and AP-2.
• To determine the necessity of Fcho1/2 and Eps15/R for stable AP-2 complex formation and endocytosis.

Main Methods:

• Structural analysis of Eps15/R⋅Fcho1 micro-homology domain complex.
• Biochemical assays to characterize DPF motif binding to Fcho1/2 and AP-2.
• Cellular experiments using gene-deficient and protein-sequestered cell models to assess endocytic function.

Main Results:

• Arrayed DPF motifs in Eps15/R are differentially recognized by Fcho1/2 and AP-2.
• A spacing-dependent DPF triad in Eps15/R binds Fcho1/2 distinctively compared to single DPF binding to AP-2.
• Loss of FCHO1/2 or Eps15/R leads to unstable AP-2 assemblies and impaired endocytosis.

Conclusions:

• Distinct DPF-based codes within Eps15/R direct the formation of Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes.
• Fcho1/2 facilitates AP-2 conformational activation, promoting cargo engagement.
• This mechanism highlights a crucial role for endocytic pioneers in regulating AP-2 function and cargo selection.