RESUMO
We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Clatrina/metabolismo , Endossomos/fisiologia , Fator de Transcrição AP-1/metabolismo , Rede trans-Golgi/fisiologia , Animais , Células COS , Chlorocebus aethiops , Células HeLa , HumanosRESUMO
ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Clatrina/metabolismo , Endocitose , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Membrana/metabolismo , Fator 6 de Ribosilação do ADP , Actinas/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Alinhamento de SequênciaRESUMO
The endocytosis of cell membrane proteins is initiated by the binding of activated Arf6, a member of Ras-related GTPases, to the PM. A GAP specific for Arf6 triggers the budding of endocytotic vesicles from the PM by inactivating GTP-bound Arf6. We recently identified the SMAP gene that encodes an ArfGAP and is involved in the endocytosis of TfnR and possibly E-cadherin. In this review, we summarize the process of intracellular membrane trafficking, highlighting the roles played by the SMAP gene. Progression of cancer to malignancy occurs in parallel with the disappearance of E-cadherin, a central component of the adherens junction in epithelial cells. Therefore, elucidation of the molecular mechanism of E-cadherin endocytosis should be one of the key elements in tumor cell biology.