engleski

Small GTPases interacting with IqgC on Dictyostelium macropinosomes

The Dictyostelium IQGAP-related protein IqgC is a GAP (GTPase-activating protein) specific for the small GTPase RasG [1]. It deactivates RasG and thus negatively regulates macropinocytosis in the amoeba Dictyostelium discoideum. Macropinocytosis is a Ras-regulated endocytic process for non- selective uptake of extracellular fluid [2]. Previous study has shown that IqgC strongly localizes to macropinosomes [1]. It colocalizes with active Ras during the formation of the macropinocytic cup and on the nascent macropinosome. However, IqgC remains on the internalized macropinosome even after Ras has dissociated from the vesicle. This dynamics suggests that IqgC has a role independent of RasG, probably in early macropinosome maturation. We aim to (I) clarify the role of RasG in the recruitment of IqgC to the forming macropinosomes, and (II) identify novel protein interactor(s) of IqgC that could retain it after RasG disappears. To examine the role of RasG in IqgC recruitment, we analyzed the localization of IqgC in rasG null cells by confocal microscopy. We observed the loss of IqgC localization to macropinosomes in the absence of RasG. Next, we monitored the localization of a truncated version of IqgC, which lacked the RasG- binding RasGAP domain, in iqgC null cells and again observed mislocalized IqgC. Finally, IqgC proteins mutated in amino acids important for its GAP activity and/or interaction with RasG again did not localize properly. Altogether, these data demonstrate that interaction with RasG is indispensable for the recruitment of IqgC to the macropinosome during its formation. To identify novel protein interactor(s) that could explain IqgC functions during early macropinosome maturation, we selected several protein candidates from the previously published IqgC interactome [1], based on their known involvement in early endocytic pathways. Using this approach, we found another small GTPase from the Ras superfamily, Rab5A, to co-immunoprecipitate with endogenous IqgC. By performing GST-Rab5A pull-down assay with purified IqgC, we have demonstrated a direct interaction between Rab5A and IqgC. Confocal microscopy of wild-type cells co-expressing fluorescently labelled IqgC and Rab5A showed their colocalization on internalised macropinosomes. More specifically, Rab5A associates with the macropinocytic vesicle when IqgC signal starts to diminish. By the time Rab5A, whose presence increases gradually, reaches maximal intensity, IqgC has dissociated from the vesicle. In summary, interaction with RasG is necessary for the correct localization of IqgC to forming macropinosomes. After macropinosome closure, RasG dissociates from the macropinocytic vesicle. However, IqgC remains on the vesicle where it interacts with the early endosome marker Rab5A [3]. Deciphering the physiological significance of this interaction requires further investigation.

IqgC ; RasGAP ; RasG ; Rab5A ; macropinocytosis

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