The mouse mutants mocha and pearl are deficient in the AP-3 δ and β3A 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 β3 and μ3 subunits coassemble into a heterodimer, whereas the σ3 subunit remains monomeric. In pearl cells, the δ and σ3 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 β3 and μ3 can interact with each other. Pearl cell lines were generated that express β3A, β3B, a β3Aβ2 chimera, two β3A deletion mutants, and a β3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only β3A, β3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The β3Aβ2 chimera and the β3A short deletion mutant gave partial functional rescue, whereas the β3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of β3 are important for function, but the clathrin binding site is not needed.
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21 January 2002
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January 21 2002
Assembly and function of AP-3 complexes in cells expressing mutant subunits
Andrew A. Peden,
Andrew A. Peden
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
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Rachel E. Rudge,
Rachel E. Rudge
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
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Winnie W.Y. Lui,
Winnie W.Y. Lui
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
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Margaret S. Robinson
Margaret S. Robinson
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
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Andrew A. Peden
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
Rachel E. Rudge
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
Winnie W.Y. Lui
Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK
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: [email protected]
Andrew A. Peden and Rachel E. Rudge contributed equally to this paper.
Andrew A. Peden's present address is Howard Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5426.
*
Abbreviations used in this paper: AP, adaptor proteins; HPS, Hermansky Pudlak syndrome.
Received:
July 31 2001
Revision Received:
December 04 2001
Accepted:
December 04 2001
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2002
J Cell Biol (2002) 156 (2): 327–336.
Article history
Received:
July 31 2001
Revision Received:
December 04 2001
Accepted:
December 04 2001
Citation
Andrew A. Peden, Rachel E. Rudge, Winnie W.Y. Lui, Margaret S. Robinson; Assembly and function of AP-3 complexes in cells expressing mutant subunits . J Cell Biol 21 January 2002; 156 (2): 327–336. doi: https://doi.org/10.1083/jcb.200107140
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