Arf GTPase-activating proteins (Arf GAP) play important roles in the formation of the membrane vesicles that traffic between subcellular membranous organelles. The small Arf GTPase-activating protein (SMAP) subfamily of Arf GAPs has two members, SMAP1 and SMAP2, in mammals. The present study investigated whether these two proteins may have an overlapping function in addition to their previously reported distinct functions. Results showed that the presence of either SMAP1 or SMAP2 was sufficient for endocytosis of the transferrin receptor, and that transferrin incorporation was impaired only by the absence of both SMAP1 and SMAP2. This suggests the involvement of both SMAP1 and SMAP2 in transferrin endocytosis. Results also demonstrated a physical association between SMAP1 and SMAP2, which might serve as a basis for a functional interaction, and identified the intramolecular domains responsible for this association.
The sorting machinery in early endosomes is crucial for intracellular homeostasis and signal transduction and its disruption leads to the development of various diseases. In spite of its significance, the molecular mechanism underlying this machinery remains largely unknown. Actin filaments are implicated in intracellular trafficking, including membrane fission at endocytosis, membrane stretching at the Golgi complex, and maturation of endosomes. We have recently found that actin is required for receptor sorting in early endosomes and identified cortactin as a candidate for actin regulation in early endosomes. Inhibition of actin dynamics leads to enlargement of early endosomes and impairment of the sorting; the latter is also observed in cortactin-depleted cells. The endosomal localization of cortactin was enhanced by dynasore, a dynamin inhibitor that effectively inhibits endosomal sorting, indicating that cortactin is involved in the sorting machinery in early endosomes. Here we discuss the role of actin filaments in early endosomes and other molecules implicated in endosomal trafficking.
A large GTPase dynamin, which is required for endocytic vesicle formation, regulates the actin cytoskeleton through its interaction with cortactin. Dynamin2 mutants impair the formation of actin comets, which are induced by Listeria monocytogenes or phosphatidylinositol-4-phosphate 5-kinase. However, the role of dynamin2 in the regulation of the actin comet is still unclear. Here we show that aberrant actin comets in dynamin2-depleted cells were rescued by disrupting of microtubule networks. Depletion of dynamin2, but not cortactin, significantly reduced the length and the speed of actin comets induced by Listeria. This implies that dynamin2 may regulate the actin comet in a cortactin-independent manner. As dynamin regulates microtubules, we investigated whether perturbation of microtubules would rescue actin comet formation in dynamin2-depleted cells. Treatment with taxol or colchicine created a microtubule-free space in the cytoplasm, and made no difference between control and dynamin2 siRNA cells. This suggests that the alteration of microtubules by dynamin2 depletion reduced the length and the speed of the actin comet.
SMAP2 is an Arf GTPase-activating protein that is located and functions on early endosome membranes. In the present study, the trans-Golgi network (TGN) was verified as an additional site of SMAP2 localization based on its co-localization with various TGN-marker proteins. Mutation of specific stretches of basic amino acid residues abolished the TGN-localization of SMAP2. Over-expression of wild-type SMAP2, but not of the mutated SMAP2, inhibited the transport of vesicular stomatitis virus-G protein from the TGN to the plasma membrane. In contrast, this transport was enhanced in SMAP2 (-/-) cells characterized by increased levels of the activated form of Arf. SMAP2 therefore belongs to an ArfGAP subtype that resides on the TGN and functions as a negative regulator of vesicle budding from the organelle.
Early endosomes (EEs) are known to be a sorting station for internalized molecules destined for degradation, recycling, or other intracellular organelles. Segregation is an essential step in such sorting, but the molecular mechanism of this process remains to be elucidated. Here, we show that actin is required for efficient recycling and endosomal maturation by producing a motile force. Perturbation of actin dynamics by drugs induced a few enlarged EEs containing several degradative vacuoles and also interfered with their transporting ability. Actin repolymerization induced by washout of the drug caused the vacuoles to dissociate and individually translocate toward the perinuclear region. We further elucidated that cortactin, an actin-nucleating factor, was required for transporting contents from within EEs. Actin filaments regulated by cortactin may provide a motile force for efficient sorting within early endosomes. These data suggest that actin filaments coordinate with microtubules to mediate segregation in EEs.
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