Abstract
Endocytosis of glycosylphosphatidyl inositol (GPI)-anchored proteins (GPI-APs) and the fluid phase takes place primarily through a dynamin- and clathrin-independent, Cdc42-regulated pinocytic mechanism. This mechanism is mediated by primary carriers called clathrin-independent carriers (CLICs), which fuse to form tubular early endocytic compartments called GPI-AP enriched endosomal compartments (GEECs). Here, we show that reduction in activity or levels of ARF1 specifically inhibits GPI-AP and fluid-phase endocytosis without affecting other clathrin-dependent or independent endocytic pathways. ARF1 is activated at distinct sites on the plasma membrane, and by the recruitment of RhoGAP domain-containing protein, ARHGAP10, to the plasma membrane, modulates cell-surface Cdc42 dynamics. This results in the coupling of ARF1 and Cdc42 activity to regulate endocytosis at the plasma membrane. These findings provide a molecular basis for a crosstalk of endocytosis with secretion by the sharing of a key regulator of secretory traffic, ARF1.
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Acknowledgements
We thank M. Kalia and B. Srinag for help with biochemistry, H. Krishnamurthy and Wellcome Trust-aided imaging and flow-cytometry facility at NCBS for help with confocal imaging and sorting transfected cells. We are indebted to: G. D. Gupta for making ARF6 shRNA; R. Alexander for ARF1 RNAi resistant and mRFP–ABD vectors; N, Sabu for ARHGAP10 shRNA; J. Gruenberg for GFP-tagged wild-type ARF1 and ARF1T31N plasmids; S.G. Ferguson for HA-tagged wild-type ARF1, ARF1T31N and ARF1Q71L constructs; P. Chavrier for GFP–ARHGAP10 domains; R. A. Kahn for ARF1, ARF3, ARF4 and ARF5 shRNA plasmids; R. Vishwakarma for fluorescent folate analogues; S. Bourgoin for ARF6-specific antibodies; and R. E. for C6-LacCer. We thank other members of the Mayor Laboratory and NCBS for generous support and encouragement. S.K. is supported by a pre-doctoral fellowship from Council of Scientific and Industrial Research (Government of India). Work in S.M.'s laboratory is supported by intramural funds from NCBS, and a J. C. Bose fellowship.
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S.K. executed and analysed all experiments. S.K. and S.M. planned all experiments and wrote the manuscript.
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Kumari, S., Mayor, S. ARF1 is directly involved in dynamin-independent endocytosis. Nat Cell Biol 10, 30–41 (2008). https://doi.org/10.1038/ncb1666
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DOI: https://doi.org/10.1038/ncb1666
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