Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide

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Abstract

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

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Figure 1: FAPP2 is a GlcCer-transfer protein.
Figure 2: FAPP2 is required for complex GSL synthesis.
Figure 3: GSL synthesis depends on PtdIns4P, and not on membrane trafficking at the GC.
Figure 4: Model of FAPP2 mechanism of action.

Change history

  • 06 September 2007

    In the AOP version of this Article, the y-axes of Fig. 2c was incorrectly labelled. This was corrected on 6 September 2007.

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Acknowledgements

We thank A. Luini, R. Ghidoni, P. Viani, L. Riboni for discussions; A. Luini, D. Corda and M. Gimona for critical reading of the manuscript; T. Scanu for sharing the data on Bet3 knockdown; C. Iurisci for technical assistance; A. Spaar for the bioinformatic analysis; G. Perinetti for the statistic analysis of the data; J. P. Slotte for the fluorescence facility; J. Saus for anti-CERT antibody; C. P. Berrie for editorial assistance; and E. Fontana for the artwork. This work was supported by the Telethon Electron Microscopy Core Facility, by a fellowship from FIRC to G.D.A., and by Telethon, AIRC, the Academy of Finland, Sigrid Jusélius, Magnus Ehrnrooth, K. Albin Johansson Foundations, Medicinska Understödsföreningen Liv och Hälsa, ISB Graduate School and Åbo Akademi University.

Author Contributions M.A.D.M. supervised the entire project and wrote the manuscript with G.D.A. and with comments from all coauthors; G.D.A designed and conducted the experiments of sphingolipid labelling; G.D.A. designed the experiments of membrane trafficking, which were conducted by A.D.C. and G.D.T.; G.D.A. designed the cloning of FAPP2, which was conducted by G.D.T. and M.S.; G.D.T. and M.S. prepared all the constructs, recombinant proteins and anti-FAPP1, FAPP2, PI(4)KIIIβ, PI(4)KIIα, BET3 and GM130 antibodies; E.P. and R.P. designed and conducted the experiments for electron microscopy; A.G. performed the trafficking experiment in FAPP1-knockdown cells; P.M. designed the experiments of intervesicular lipid transfer, which were conducted by G.W.; A.S. and C.C.C. conducted the HPLC measurements of GSLs under the supervision of F.M.P.; J.B. conducted the sphingolipid analysis by LC–MS under the supervision of Y.A.H.

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Correspondence to Maria Antonietta De Matteis.

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Supplementary information

Supplementary Information

This file contains Supplementary Data, Supplementary Figures S1-S7 with Legends, Supplementary Methods, Supplementary Discussion and Supplementary Notes with additional references. (PDF 1073 kb)

Supplementary Video 1

This file contains Supplementary Video 1 which shows an electron microscopy tomography of the Golgi complex in mock-treated HeLa cells. (MOV 1309 kb)

Supplementary Video 2

This file contains Supplementary Video 2 which shows an electron microscopy tomography of the Golgi complex in FAPP2-KD HeLa cells. (MOV 828 kb)

Supplementary Video 3

This file contains Supplementary Video 3 which shows an electron microscopy tomography of the Golgi complex in GCS-KD HeLa cells. (MOV 1181 kb)

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D’Angelo, G., Polishchuk, E., Tullio, G. et al. Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide. Nature 449, 62–67 (2007) doi:10.1038/nature06097

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