An intermediate polymer in the assembly of clathrin baskets

Area of Science:

• Biochemistry
• Cell Biology
• Structural Biology

Background:

• Clathrin (8S) typically polymerizes into 150S or 300S basket structures under standard buffer conditions (100 mM Mes, pH 5.9-6.7).
• These structures are crucial for the isolation of coated vesicles.

Purpose of the Study:

• To investigate the self-assembly behavior of clathrin under very low salt conditions.
• To characterize the resulting clathrin species and its relationship to known basket structures.

Main Methods:

• Sedimentation analysis (sedimentation coefficient, sedimentation equilibrium) to determine species size and molecular weight.
• Light-scattering studies to assess the stability of different clathrin species.
• Fluorescent labeling and trypsin digestion to compare protein conformation.

Main Results:

• Under very low salt conditions (2 mM Mes, pH 5.9), clathrin forms a homogeneous 27S species.
• Increasing salt concentration to 50 mM Mes fully converts the 27S species into 150S baskets.
• The 27S species, with a molecular weight six times that of the protomer, represents a highly cooperative, reversible self-association.
• Conformation of clathrin in 27S species and baskets is similar, but differs from the 8S protomer.

Conclusions:

• Clathrin self-assembly is highly sensitive to salt concentration, enabling the formation of distinct oligomeric states.
• A novel 27S clathrin species exists, representing an intermediate in the assembly pathway towards basket formation.
• These findings provide new perspectives on the dynamic assembly of clathrin cages.