engleski

IQGAP-related protein IqgC interacts with RasG and Rab5A on Dictyostelium macropinosomes

Dictyostelium IqgC is a GAP (GTPase activating protein) specific for the small GTPase RasG, one of the main positive regulators of macropinocytosis (bulk fluid uptake). By deactivating RasG, IqgC negatively regulates macropinocytosis in amoeba Dictyostelium discoideum. IqgC strongly localizes to macropinosomes, where it colocalizes with active Ras. However, it remains on the internalized vesicle even after Ras has dissociated from the macropinosme (Previously published in: Marinović M et al. (2019) Proc Natl Acad Sci U S A 116, 1289- 1298). This finding suggests that IqgC functions independently of RasG, probably in early macropinosome maturation. First, we examined the role of RasG in the recruitment of IqgC to forming macropinocytic cups. We observed the loss of IqgC localization to macropinosomes in rasG null cells. Mutant IqgC proteins unable to bind Ras and IqgC lacking its RasG-binding RasGAP domain also failed to localize correctly. These results show that interaction with RasG is indispensable for IqgC recruitment to forming macropinosomes. Next, we explored novel IqgC interactor(s) that could bind IqgC on the nascent macropinosome after Ras detached. Potential protein candidates were selected from the previously determined interactome, according to their known function in the early endosome maturation. Using Co-IP, and afterwards GST-pull-down assay with purified IqgC, we identified the early endosome marker, GTPase Rab5A, as a direct binding partner of IqgC. Furthermore, using confocal microscopy of live cells, we showed that both proteins colocalize on the primary macropinocytic vesicle. Consistently, a lipid dot blot using cell lysates identified phosphoinositides (PIs) involved in macropinosome formation and early maturation as IqgC membrane phospholipid interactors. Altogether, these results suggest that RasG is essential for IqgC loading to the forming macropinosome, but Rab5A and PIs likely mediate its retention on the RasG- vacated vesicle.

IqgC ; RasGAP ; RasG ; Rab5A ; macropinocytosis

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