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Assembly and function of AP-3 complexes in cells expressing mutant subunits.
Andrew A Peden1, Rachel E Rudge, Winnie W Y Lui
1Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.
The Journal of Cell Biology
|January 25, 2002
Summary
Mouse mutants mocha and pearl reveal AP-3 complex assembly and function. Specific subunits are crucial for AP-3 complex assembly and lysosome-related organelle trafficking, with beta3
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The adaptor protein 3 (AP-3) complex is essential for intracellular trafficking.
- AP-3 deficiency leads to defects in lysosome-related organelle biogenesis, as observed in mocha and pearl mouse mutants.
Purpose of the Study:
- To investigate AP-3 complex assembly and function using mocha and pearl mouse mutant cells.
- To identify the specific domains and subunits of AP-3 critical for its function.
Main Methods:
- Analysis of AP-3 subunit assembly in mocha (AP-3 delta deficient) and pearl (AP-3 beta3A deficient) mouse cells.
- Yeast two-hybrid system to confirm subunit interactions.
- Functional rescue assays using various beta3A constructs in pearl cell lines, assessing LAMP-1 sorting.
Main Results:
- In mocha cells, beta3 and mu3 form heterodimers, while sigma3 remains monomeric. In pearl cells, delta and sigma3 form heterodimers, and mu3 is degraded.
- All tested beta3A constructs assembled into complexes and localized to membranes.
- Full functional rescue of LAMP-1 sorting was achieved with beta3A, beta3B, and a point mutant lacking a clathrin binding site. Partial rescue was observed with a chimera and a short deletion mutant, while a truncation mutant showed no rescue.
Conclusions:
- The hinge and/or ear domains of the beta3 subunit are critical for AP-3 complex function.
- The clathrin binding site on the beta3 subunit is not essential for AP-3 mediated trafficking.