Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Sorting and export of transmembrane and lysosomal cargoes at the trans-Golgi network (TGN) are understood.
• Mechanisms for segregating secretory cargoes for extracellular release from the TGN are challenging.
• The soluble TGN-resident protein Cab45, requiring Ca(2+), is necessary for secretory cargo sorting at the TGN.

Purpose of the Study:

• To elucidate the mechanism of secretory cargo sorting at the TGN mediated by Cab45.
• To investigate the role of Ca(2+) in Cab45-dependent cargo selection.
• To characterize the interaction between Cab45 and secretory proteins.

Main Methods:

• In vitro biochemical assays to study Cab45 oligomerization and protein binding.
• Site-directed mutagenesis to disrupt Ca(2+)-binding sites in Cab45.
• Cell-based assays to assess cargo sorting in intact cells.
• Superresolution microscopy to visualize the localization of Cab45, secretory proteins, and SPCA1 within the TGN.

Main Results:

• Cab45 reversibly assembles into oligomers in a Ca(2+)-dependent manner.
• Cab45 oligomers specifically bind secretory proteins (e.g., COMP, LyzC) in vitro.
• Mutating Ca(2+)-binding sites in Cab45 impairs its oligomerization and the sorting of COMP and LyzC.
• Cab45 colocalizes with secretory proteins and SPCA1 in TGN microdomains.

Conclusions:

• Ca(2+)-dependent oligomerization of Cab45 is a key mechanism for sorting specific secretory cargoes at the TGN.
• Cab45 acts as a Ca(2+)-regulated sorting platform for secretory proteins.
• This process involves specific TGN microdomains and interaction with the SPCA1 Ca(2+) pump.