engleski

Recruitment mechanisms of RasGAP protein IqgC to macropinosomes in amoeba Dictyostelium discoideum

Large-scale endocytosis encompasses nonselective uptake of extracellular fluid or macropinocytosis and particle uptake or phagocytosis. This evolutionary conserved, Ras-regulated process is a primary way of feeding for amoeba Dictyostelium discoideum. Wild-type D. discoideum isolates predominantly use phagocytosis to feed on bacteria, while their macropinocytosis is not adequate to sustain growth. On the other hand, laboratory strains have strongly upregulated macropinocytosis that enables them to thrive in liquid medium. This is due to a deletion of a gene encoding RasGAP (Ras GTPase activating protein) protein NF1 in axenic strains, which supresses formation of large macropinosomes and phagosomes in wild-type strains. In our previous study, we have identified a RasG-specific GAP IqgC that negatively regulates large-scale endocytosis in axenic strain. Nevertheless, deletion of iqgC induced only modest increase in fluid uptake, possibly because axenic strains already have almost maximally upregulated macropinocytosis. To investigate this, we have generated iqgC knock-out in wild-type DdB strain and preliminary results demonstrated increased growth rates in axenic medium. IqgC strongly localizes to nascent macropinosome where it colocalizes with active Ras. However, IqgC remains on the internalized macropinosome after Ras dissociated from the vesicle. Such dynamics suggests that IqgC also functions in early macropinosome maturation, probably in RasG- independent way. To elucidate the mechanism of IqgC recruitment to macropinosomes, we have analysed its localization in rasG- cells. The loss of localization demonstrated essential role of RasG in IqgC recruitment to nascent macropinosome. Next, we analysed IqgC domains required for its recruitment to the membrane during macropinosome formation and early maturation. Examination of several fluorescent fusion proteins by confocal microscopy showed that RasGAP and RGCT domains alone, or with adjacent N- and C- terminal ends, are not sufficient for IqgC recruitment to macropinosomes. Since we were unable to demonstrate direct interactions between IqgC and binding partners previously identified in a pull- down assay, we performed a protein-lipid overlay assay to see if there are any lipid interactors. We showed that IqgC preferentially binds to phosphatidylinositol 3-phosphate (PI(3)P) and PI- bisphosphates PI(4, 5)P2, PI(3, 5)P2 and PI(3, 4)P2, which are known to be involved in macropinosome formation and early maturation. In summary, IqgC is recruited to nascent macropinosome via interaction with RasG. However, RasG is dispensable for its retention on maturing vesicle, which is probably mediated by phosphoinositides.

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

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