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Regulation of a Golgi flippase by phosphoinositides and an ArfGEF

Nature Cell Biology volume 11, pages 1421–1426 (2009)Cite this article

Abstract

The essential role for phosphatidylinositol-4-phosphate (PtdIns(4)P) in vesicle-mediated protein transport from the trans-Golgi network (TGN) was first described in the budding yeast Saccharomyces cerevisiae1,2,3. However, the identity of downstream effectors of PtdIns(4)P in this system has been elusive. Here, we show that Drs2p, a type IV P-type ATPase required for phospholipid translocase (flippase) activity and transport vesicle budding from the TGN4,5,6,7,8, is an effector of PtdIns(4)P. Drs2p-dependent flip of a fluorescent phosphatidylserine analogue across purified TGN membranes requires synthesis of PtdIns(4)P by the phosphatidylinositol-4-kinase (PI(4)K) Pik1p. PtdIns(4)P binds to a regulatory domain in the C-terminal tail of Drs2p that has homology to a split PH domain and is required for Drs2p activity. In addition, basic residues required for phosphoinositide binding overlap a previously mapped binding site for the ArfGEF Gea2p9. ArfGEF binding to this C-terminal domain also stimulates flippase activity in TGN membrane preparations. These interactions suggest the presence of a coincidence detection system used to activate phospholipid translocation at sites of vesicle formation.

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Figure 1: Loss of Drs2p does not perturb phosphoinositide metabolism.
Figure 2: NBD-PS flippase activity in TGN membranes requires Drs2p and PI(4)K activity catalysed by Pik1p.
Figure 3: Stimulation of NBD-PS flippase activity by phosphoinositides.
Figure 4: Functional requirement for a phosphoinositide binding motif in the Drs2p C-terminal tail.
Figure 5: Synergistic activation of NBD-PS flippase activity by phosphoinositide and ArfGEF.

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Acknowledgements

We thank Scott D. Emr and Anjon Audhya for yeast strains, John York for providing the GST-Inp53-Sac1 plasmid, Jonathan Goldberg for the His-Gea2p-Sec7 plasmid and our lab colleagues for comments on this manuscript. These studies were supported by NIH Grant GM62367 (to T.R.G.)

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Authors and Affiliations

  1. Department of Biological Sciences, Vanderbilt University, Nashville, 37235-1634, TN, USA

    Paramasivam Natarajan, Ke Liu, Dustin V. Patil & Todd R. Graham

  2. Department of Molecular & Cell Biology, University of California, Berkeley, 94720-3200, CA, USA

    Vicki A. Sciorra

  3. Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, 91198, France

    Catherine L. Jackson

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  1. Paramasivam Natarajan
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Contributions

P.N. performed most of the project planning, experimental work, data analysis and manuscript preparation. Experimental work was also contributed by K.L. (Fig. 1b), D.V.P (Fig. 4c) V.A.S. (Fig. 1a), and C.L.J. (Fig, 5c). TRG contributed to project planning, data analysis and manuscript preparation.

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Correspondence to Todd R. Graham.

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Natarajan, P., Liu, K., Patil, D. et al. Regulation of a Golgi flippase by phosphoinositides and an ArfGEF. Nat Cell Biol 11, 1421–1426 (2009). https://doi.org/10.1038/ncb1989

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