Naslov
IqgC protein interacts with RasG and Rab5A GTPases during macropinocytosis in amoeba Dictyostelium discoideum

Autori
Putar, Darija ; Ćutić, Tamara ; Mijanović, Lucija ; Weber, Igor ; Filić, Vedrana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts of the Congress of the Croatian Society of Biochemistry and Molecular Biology "HDBMB22: From Science to Knowledge" / Dulić, Morana ; Sinčić, Nino ; Vrhovac Madunić, Ivana - Zagreb : Hrvatsko Društvo za Biotehnologiju, 2022, 125-125

Skup
Congress of the Croatian Society of Biochemistry and Molecular Biology: From Science to Knowledge (HDBMB22)

Mjesto i datum
Brela, Hrvatska, 28.09.2022. - 01.10.2022

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
IqgC ; RasGAP ; RasG ; Rab5A ; macropinocytosis

Sažetak
Dictyostelium discoideum protein IqgC is a GAP (GTPase activating protein) specific for the small GTPase RasG. GTPases from the Ras superfamily regulate macropinocytosis, the endocytic process for nonselective uptake of extracellular fluid. By deactivating RasG, IqgC negatively regulates macropinocytosis in amoeba D. discoideum. IqgC strongly localizes to forming macropinosome, where it colocalizes with active Ras. However, Ras dissociates from the internalized macropinosome prior to IqgC1, which suggests RasG-independent roles of IqgC during macropinosme maturation. First, we examined the role of RasG in IqgC recruitment to macropinocytic cups. By monitoring the localization of IqgC in rasG null cells, or localization of IqgC mutants unable to bind Ras in iqgC null cells, we observed the loss of IqgC localization to macropinosomes. These results demonstrate that RasG is indispensable for the recruitment of IqgC to forming macropinosomes. Next, we searched for novel IqgC interactor(s) that could mediate IqgC functions in early macropinosome maturation, after detachment of Ras. Potential protein candidates, previously identified by mass spectroscopy, were selected according to their known function in the early endosome maturation. From those, early endosome marker, GTPase Rab5A was shown to colocalize with IqgC on the primary macropinocytic vesicle in live cells that co-express both fluorescently labelled proteins. Furthermore, by performing GST-Rab5A pull-down assay with purified IqgC, we identified Rab5A as a direct interactor of IqgC. Using lipid dot blot assay with lysates of cells expressing full length or truncated IqgC variants, we demonstrated that IqgC binds to various phosphoinositides (PIs). Although its binding was generally of low specificity, IqgC bound with the highest affinity to PI(4, 5)P2 via its N-terminus and RasGAP domain. Nevertheless, the binding was abrogated for RasG-binding mutants, confirming the necessity of RasG for IqgC loading to macropinosome membrane. Altogether, these results show that RasG is necessary for IqgC recruitment to the forming macropinosome, but PIs and Rab5A likely mediate its retention on the RasG-vacated vesicle. The biological significance of Rab5-IqgC interaction is under investigation.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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