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

Abstract

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 ofpearl and mocha mice, which have genetic defects in genes encoding subunits of AP-3, but to lysosomes in functionally rescued mocha cells expressing the δ 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 thetrans-Golgi network to lysosomes via an intracellular route that appears to bypass early endosomes.

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

“…These results suggest that a significant pool of PI4KII␣ is controlled by the function of the adaptor complex AP-3. We note that PI4KII␣ was reported to interact with CD63 (Berditchevski et al, 1997;Yauch and Hemler, 2000), a membrane protein of lysosomes proposed to be sorted by AP-3-dependent mechanisms (Rous et al, 2002). Thus, it is possible that a direct or indirect interaction with CD63 may account, at least in part, for the localization of PI4KII␣ in AP-3-derived vesicles.…”

Clathrin-dependent Association of CVAK104 with Endosomes and theTrans-Golgi Network

“…Previous work had shown that OCRL localizes to endosomes and the TGN (Ungewickell et al, 2004;Lowe, 2005). CVAK104 overlapped also significantly with the adaptor protein complex AP3, which is involved in the sorting of lysosomal membrane proteins (Le Borgne et al, 1998;Rous et al, 2002) (Figure 4C). In contrast, very little colocalization of CVAK104 with early endosomal markers such as early endosomal antigen (EEA)1 ( Figure 4C) and rab5 (our unpublished data) was seen.…”

The tetraspanin CD63 is involved in granule targeting of neutrophil elastase

“…20 Therefore, we hypothesized that NE precursor (proNE) is routed to granules through the protein-delivery pathway of CD63, which recruits AP-3 adaptor complex through the C-terminal lysosomal targeting motif GYEVM. 21 Thus, cooperation with CD63 was thought to facilitate the secretory lysosome targeting of proNE.To corroborate our hypothesis, we examined whether CD63 and proNE interacted upon coexpression in COS cells. Furthermore, we examined how CD63 depletion affected proNE trafficking.…”

“…The CD63AEVM mutant has a single amino acid mutation in the C-terminal lysosomal sorting sequence that decreases intracellular CD63 and plasma membrane sorting. 21 Although not formally proved, CD63AEVM might produce nonfunctional complexes that are likely to be degraded or become transferred to the plasma membrane.In the 5 clones investigated, CD63 siRNA expression showed a mean of 6.5-fold (the range was from 16-fold to 3.7-fold) suppression of CD63 mRNA compared with wild-type cells, CD63 siRNA mock, or a reverted CD63siRNA clone ( Figure 5A). The CD63 mRNA levels of 5 cell clones with scrambled siRNA control vector did not differ from wild-type cells ( Figure S2A).…”