In this study, we have shown that SMAP1 is a novel Arf6GAP. We also revealed that it is not involved in actin reorganization; rather, it regulates clathrin-dependent endocytosis. Furthermore, we showed that SMAP1 associates directly with clathrin through its clathrin box. To our knowledge, the association of an ArfGAP with clathrin has not been described previously.
An in vitro GAP assay revealed that SMAP1 prefers Arf6 to Arf1 as a substrate. Supporting this is that the overexpression of SMAP1 had no effect on the distribution of the Golgi, whose membrane traffic is dependent on the function of Arf1. Arf6, on the other hand, regulates actin reorganization. This was confirmed when we transfected HeLa cells with Arf6 and treated them with AlF, because these cells reorganized their peripheral actin and induced membrane protrusions. Molecules such as ACAP1, ACAP2, and GIT2 are all considered to possess Arf6GAP activity, and cotransfection of one of these inhibits the protrusion of the actin (Jackson et al., 2000
; Mazaki et al., 2001
). Unlike these molecules, however, coexpression of SMAP1 did not perturb the formation of the Arf6-induced membrane protrusion (). Thus, SMAP1 appears to be distinct from the other Arf6GAP members that have been identified to date.
Arf6 also regulates the endocytosis of both transferrin and IL-2R, which are transported via clathrin-dependent and -independent pathways, respectively. It has been shown that overexpression of Arf6GAP such as GIT1 does not affect the transferrin internalization. On the other hand, when one kind of Arf6 mutant is introduced into cells, it affects only the actin reorganization but not the vesicle trafficking (Al-Awar et al., 2000
). Overexpression of SMAP1 impaired clathrin-dependent endocytosis but did not perturb clathrin-independent endocytosis or actin reorganization. Therefore, it is plausible to hypothesize that Arf6 separately regulates actin rearrangement and the clathrin-dependent and -independent pathways of vesicle trafficking by interacting with different sets of Arf6GAPs. This notion is supported by observations that suggest that the Arf6GEFs EFA6 and ARNO are separately involved in the distinct functions exerted by Arf6. For instance, overexpression of EFA6 inhibits transferrin endocytosis and simultaneously induces membrane ruffles (Franco et al., 1999
), whereas overexpression of ARNO enhances the endocytosis of the beta2-adrenergic receptor (Claing et al., 2001
The recruitment of AP-2 to the plasma membrane was impaired in SMAP1-transfected cells, whereas it was rather enhanced in the cells transfected by the GAP-negative mutant of SMAP1. It is thus evident that the GAP activity of SMAP1 is one factor that determines the subcellular localization of AP-2. A GAP-negative mutant of SMAP1 probably leads Arf6 to its active, GTP-bound form. In accordance, we have observed previously that the transfection of HeLa cells with the constitutively active Arf6Q67L protein does not impair the membrane recruitment of AP-2 (unpublished data). Curiously, however, transfection of Arf6Q67L does block transferrin internalization (unpublished data). These apparent discrepancies may be due to the possibility that Arf6Q67L cannot be involved in the triggering of vesicle formation because it cannot cycle between the GTP/active and GDP/inactive forms. Alternatively, Arf6Q67L may affect the actin reorganization that is associated with and is necessary for vesicle formation. In contrast, SMAP1 (and its mutant) may regulate the activity of Arf6 molecules, but only those Arf6 molecules that are engaged in the clathrin-dependent endocytic pathway.
SMAP1 harbors a clathrin-box in its central region. This motif is found in many proteins that are known to be essential components of clathrin-mediated membrane traffic (; ter Haar et al., 2000
), including β-adaptin (Shih et al., 1995
; Dell'Angelica et al., 1998
), β-arrestin (Krupnick et al., 1997
), AP-180 (Morris et al., 1993
), amphiphysin (Ramjaun and McPherson, 1998
), epsin 1 (Rosenthal et al., 1999
), auxilin (Smith et al., 2004
), and phosphatidylinositol 3-kinase (Gaidarov et al., 2001
). These proteins function in the assembly/disassembly of clathrin and the membrane-association of clathrin. The AP-180 molecule, which facilitates clathrin assembly, is particularly notable because its overexpression inhibits the internalization of transferrin, whereas its mutant that lacks a clathrin-box does not have this inhibitory effect on endocytosis (Ford et al., 2001
). We observed that SMAP1 also needs its clathrin-box to inhibit transferrin internalization. This suggests that SMAP1, probably as in the case of AP-180, regulates endocytosis by interacting with the clathrin molecule.
At the Golgi membrane, Arf1 recruits both coat proteins (COPI) and Arf1GAP (GAP1), and these three components together form a ternary complex. Indeed, GAP1 is able to directly interact with β-COP and γ-COP, which are components of COPI-coated vesicles (Eugster et al., 2000
). This interaction with COPI further potentiates the ability of GAP1 to activate the Arf1-GTPase (Goldberg, 1999
). Furthermore, cargo proteins, if present, suppress the GAP1- and COPI-guided hydrolysis of Arf1-bound GTP, thereby facilitating vesicle formation. On the other hand, noncargo proteins have no effect on GTPase activity (Lanoix et al., 1999
; Goldberg, 2000
). Thus, at the Golgi, GAP1 plays a critical role in decoding the signals that allow cargo and noncargo proteins to be sorted from each other. Similarly, another member of Arf1GAP, AGAP1, can bind to the AP-3 molecule and regulate AP-3–dependent vesicle transport from the endosome to the lysosome (Nie et al., 2003
). A somewhat analogous but distinct system may be functioning in the constitutive and clathrin-dependent endocytosis at the plasma membrane. We showed that SMAP1 is an Arf6GAP and can directly interact with the clathrin molecule. Although the interaction between Arf6 and coat proteins has not been reported, one of the effector molecules of Arf6 is PIP2. The enhanced production of PIP2 probably favors the recruitment of AP-2 to the membrane (Krauss et al., 2003
). Therefore, it is possible that Arf6, Arf6GAP (SMAP1), AP-2, and clathrin form a quaternary complex and that clathrin and/or its adaptor proteins modulate the activity of SMAP1. SMAP1 is the first example of a GAP that is capable of interacting with clathrin. As such, it may be a valuable tool in elucidating the mechanism underlying Arf6- and clathrin-dependent endocytosis.