engleski

Requirements for IqgC protein recruitment to macropinosomes in amoeba Dictyostelium discoideum

Large-scale endocytosis is an evolutionary conserved, Ras/PI3K (phosphoinositide 3-kinases) regulated pathway that encompasses nonselective uptake of extracellular fluid or macropinocytosis and particle uptake or phagocytosis. Dictyostelium discoideum IQGAP-related protein IqgC is a RasGAP (Ras GTPase activating protein) specific for the small GTPase RasG and negatively regulates large- scale endocytosis. IqgC strongly localizes to nascent macropinosome where it colocalizes with active Ras during macropinosome formation. However, IqgC remains on the internalized macropinosome after Ras dissociated from the vesicle. Such dynamics suggests that IqgC has a role in early macropinosome maturation, independent of its interaction with RasG. Our goal is to elucidate the mechanism of IqgC recruitment to macropinosomes. We will clarify: 1) which IqgC domains are required for its recruitment to the membrane during macropinosome formation and early maturation ; 2) whether RasG is required for membrane recruitment of IqgC and 3) which phospholipid interactors mediate strong localization of IqgC to maturing macropinosomes. To investigate which IqgC domains are important for its localization, we constructed four different fluorescent fusion proteins containing: 1) IqgC RasGAP domain ; 2) RasGAP domain and N- terminal region of IqgC ; 3) IqgC RasGAP_Cterminal (RGCT) domain and 4) RGCT and C-terminal end of IqgC. Localizations of these truncated proteins during macropinocytosis were assessed in the iqgC null cells using confocal microscopy. To investigate the role of RasG in membrane recruitment of IqgC, we examined the localization of IqgC fused with fluorescent protein in the rasG null cells. We also mutated the conserved arginine finger in IqgC and examined the localization of this catalitically inactive RasGAP in the iqgC null cells. To analyze the lipid-binding specificity of IqgC, we performed a protein-lipid overlay assay using purified IqgC protein and cell lysates containing full-length or truncated versions of IqgC. Confocal microscopy results showed that RasGAP and RGCT domains alone do not localise to macropinosomes. Furthermore, we showed loss of localization of IqgC to macropinosomes in rasG null strain. The same was found for the mutant that has no GAP activity for Ras. Protein- lipid overlay assay showed that IqgC interacts with phosphoinositides, with strongest binding affinity to phosphatidylinositol 3-phosphate (PI(3)P) and PI-bisphosphates PI(4, 5)P2, PI(3, 5)P2 and PI(3, 4)P2. Neither the RasGAP and RGCT domains alone, nor with adjacent N- and C- terminal end respectively, are sufficient for the recruitment of IqgC to macropinosomes, which suggests that full-length IqgC or a combination of regions are required for its recruitment to macropinosomes. RasG is required for the recruitment of IqgC to the membrane during macropinosome formation, but it is dispensable for its retention during early macropinosome maturation. The latter is probably mediated by phosphoinositides to which IqgC preferentially binds and which are known to be involved in macropinosome formation and early maturation.

macropinocytosis, large-scale endocytosis, IqgC, RasGAP, IQGAP

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