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Published 21 January 2002. doi:10.1083/jcb.200107140
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© The Rockefeller University Press, 0021-9525/2002/1/327 $5.00
The Journal of Cell Biology, Volume 156, Number 2, January 21, 2002 327-336

Article

Assembly and function of AP-3 complexes in cells expressing mutant subunits



Andrew A. Peden, Rachel E. Rudge, Winnie W.Y. Lui and Margaret S. Robinson

Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK

Address correspondence to Margaret S. Robinson, University of Cambridge, CIMR, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK. Tel.: (44) 1223-330163. Fax: (44) 1223-762640. E-mail: msr12{at}mole.bio.cam.ac.uk

The mouse mutants mocha and pearl are deficient in the AP-3 {delta} and ß3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the ß3 and µ3 subunits coassemble into a heterodimer, whereas the {sigma}3 subunit remains monomeric. In pearl cells, the {delta} and {sigma}3 subunits coassemble into a heterodimer, whereas µ3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of ß3 and µ3 can interact with each other. Pearl cell lines were generated that express ß3A, ß3B, a ß3Aß2 chimera, two ß3A deletion mutants, and a ß3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only ß3A, ß3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The ß3Aß2 chimera and the ß3A short deletion mutant gave partial functional rescue, whereas the ß3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of ß3 are important for function, but the clathrin binding site is not needed.

Key Words: coated vesicles; adaptors; clathrin; mocha; pearl


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