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Originally published as MBC in Press, 10.1091/mbc.01-08-0409 on February 4, 2002
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Vol. 13, Issue 3, 1071-1082, March 2002

Role of Adaptor Complex AP-3 in Targeting Wild-Type and Mutated CD63 to Lysosomes

Brian A. Rous,* Barbara J. Reaves,dagger Gudrun Ihrke,* John A.G. Briggs,* Sally R. Gray,* David J. Stephens,Dagger George Banting,Dagger and J. Paul Luzio§

 *University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge, CB2 2XY, United Kingdom;  dagger Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom; and  Dagger Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

CD63 is a lysosomal membrane protein that belongs to the tetraspanin family. Its carboxyterminal cytoplasmic tail sequence contains the lysosomal targeting motif GYEVM. Strong, tyrosine-dependent interaction of the wild-type carboxyterminal tail of CD63 with the AP-3 adaptor subunit µ3 was observed using a yeast two-hybrid system. The strength of interaction of mutated tail sequences with µ3 correlated with the degree of lysosomal localization of similarly mutated human CD63 molecules in stably transfected normal rat kidney cells. Mutated CD63 containing the cytosolic tail sequence GYEVI, which interacted strongly with µ3 but not at all with µ2 in the yeast two-hybrid system, localized to lysosomes in transfected normal rat kidney and NIH-3T3 cells. In contrast, it localized to the cell surface in transfected cells of pearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the delta  subunit of AP-3. Thus, AP-3 is absolutely required for the delivery of this mutated CD63 to lysosomes. Using this AP-3-dependent mutant of CD63, we have shown that AP-3 functions in membrane traffic from the trans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes.

§ Corresponding author. E-mail address: jpl10{at}cam.ac.uk.

Molecular Biology of the Cell
Vol. 13, 1071-1082, March 2002
Copyright © 2002 by The American Society for Cell Biology


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