A phosphotyrosine switch for cargo sequestration at clathrin-coated buds

Area of Science:

• Cell biology
• Molecular and structural biology

Background:

• The AP-2 complex is crucial for endocytic cargo selection.
• AP-2 mediates cargo capture both directly and indirectly through clathrin-associated sorting proteins (CLASPs).

Purpose of the Study:

• To investigate the role of Tyr-888 on the AP-2 β2 subunit appendage in CLASP binding and cargo selection.
• To determine if phosphorylation of Tyr-888 affects AP-2 complex assembly and localization.

Main Methods:

• Site-directed mutagenesis to create Y888E and Y888F substitutions in the AP-2 β2 subunit.
• Analysis of AP-2 complex formation and localization using β2-YFP.
• Assessment of interactions between the mutated β2 appendage and CLASPs (ARH and β-arrestin).

Main Results:

• A phosphomimetic Y888E mutation did not disrupt AP-2 assembly or localization.
• The Y888E mutation selectively impaired interactions with ARH and β-arrestin, while Y888F did not.
• The mutated appendage remained structurally sound, indicating functional folding.

Conclusions:

• Tyr-888 on the AP-2 β2 appendage is a critical phosphorylation site regulating interactions with specific CLASPs.
• Reversible phosphorylation of Tyr-888 likely controls cargo sorting by modulating CLASP binding affinity.
• This mechanism provides a dynamic way for AP-2 to manage endocytic traffic.