Abstract
The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of the cytidine 5′-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis.
Similar content being viewed by others
Accession codes
References
Farquhar, M.G. & Palade, G.E. The Golgi apparatus: 100 years of progress and controversy. Trends Cell Biol. 8, 2–10 (1998).
Donaldson, J.G. & Lippincott-Schwartz, J. Sorting and signaling at the Golgi complex. Cell 101, 693–696 (2000).
Mollenhauer, H.H. & Morre, D.J. Perspectives on Golgi apparatus form and function. J. Electron Microsc. Tech. 17, 2–14 (1991).
Rothman, J.E. The protein machinery of vesicle budding and fusion. Protein Sci. 5, 185–194 (1996).
Warren, G. & Malhotra, V. The organisation of the Golgi apparatus. Curr. Opin. Cell Biol. 10, 493–498 (1998).
Bankaitis, V.A., Malehorn, D.E., Emr, S.D. & Greene, R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J. Cell Biol. 108, 1271–1281 (1989).
Cockcroft, S. Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic. Bioessays 20, 423–432 (1998).
Wirtz, K.W. Phospholipid transfer proteins revisited. Biochem. J. 324, 353–560 (1997).
McMaster, C.R. Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery. Biochem. Cell Biol. 79, 681–692 (2001).
Roth, M.G. Lipid regulators of membrane traffic through the Golgi complex. Trends Cell Biol. 9, 174–179 (1999).
Huijbregts, R.P., Topalof, L. & Bankaitis, V.A. Lipid metabolism and regulation of membrane trafficking. Traffic 1, 195–202 (2000).
Wang, Y.J. et al. Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi. Cell 114, 299–310 (2003).
Weigert, R. et al. CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature 402, 429–433 (1999).
Godi, A. et al. FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P. Nature Cell Biol. 6, 393–404 (2004).
Simon, J.P. et al. An essential role for the phosphatidylinositol transfer protein in the scission of coatomer-coated vesicles from the trans-Golgi network. Proc. Natl Acad. Sci. USA 95, 11181–11186 (1998).
Corda, D., Hidalgo Carcedo, C., Bonazzi, M., Luini, A. & Spano, S. Molecular aspects of membrane fission in the secretory pathway. Cell Mol. Life Sci. 59, 1819–1832 (2002).
Baron, C.L. & Malhotra, V. Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the plasma membrane. Science 295, 325–328 (2002).
Chernomordik, L., Kozlov, M.M. & Zimmerberg, J. Lipids in biological membrane fusion. J. Membr. Biol. 146, 1–14 (1995).
Shemesh, T., Luini, A., Malhotra, V., Burger, K.N. & Kozlov, M.M. Prefission constriction of Golgi tubular carriers driven by local lipid metabolism: a theoretical model. Biophys. J. 85, 3813–3827 (2003).
Bankaitis, V.A. Cell biology. Slick recruitment to the Golgi. Science 295, 290–291 (2002).
Pagano, R.E. What is the fate of diacylglycerol produced at the Golgi apparatus? Trends Biochem. Sci. 13, 202–205 (1988).
Lev, S. The role of the Nir/rdgB protein family in membrane transport and cytoskeleton remodeling. Exp. Cell Res. 297, 1–10 (2004).
Vihtelic, T.S., Goebl, M., Milligan, S., O'Tousa, J.E. & Hyde, D.R. Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein. J. Cell Biol. 122, 1013–1022 (1993).
Lev, S. et al. Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein. Mol. Cell. Biol. 19, 2278–2288 (1999).
Litvak, V. et al. Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain. Curr. Biol. 12, 1513–1518 (2002).
Litvak, V. et al. Mitotic phosphorylation of the peripheral Golgi protein Nir2 by Cdk1 provides a docking mechanism for Plk1 and affects cytokinesis completion. Mol. Cell 14, 319–330 (2004).
Litvak, V., Tian, D., Carmon, S. & Lev, S. Nir2, a human homolog of Drosophila melanogaster retinal degeneration B protein, is essential for cytokinesis. Mol. Cell. Biol. 22, 5064–5075 (2002).
Griffiths, G., Pfeiffer, S., Simons, K. & Matlin, K. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J. Cell Biol. 101, 949–964 (1985).
Balch, W.E. & Keller, D.S. ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments. J. Biol. Chem. 261, 14690–14696 (1986).
Liljedahl, M. et al. Protein kinase D regulates the fission of cell surface destined transport carriers from the trans-Golgi network. Cell 104, 409–420 (2001).
Randazzo, P.A. & Kahn, R.A. Myristoylation and ADP-ribosylation factor function. Methods Enzymol. 250, 394–405 (1995).
Jamora, C. et al. Gbetagamma-mediated regulation of Golgi organization is through the direct activation of protein kinase D. Cell 98, 59–68 (1999).
Wang, E., Norred, W.P., Bacon, C.W., Riley, R.T. & Merrill, A.H. Jr. Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme. J. Biol. Chem. 266, 14486–14490 (1991).
Merrill, A.H. Jr., van Echten, G., Wang, E. & Sandhoff, K. Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J. Biol. Chem. 268, 27299–27306 (1993).
Levine, T.P. & Munro, S. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components. Curr. Biol. 12, 695–704 (2002).
Riebeling, C., Allegood, J.C., Wang, E., Merrill, A.H. Jr. & Futerman, A.H. Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors. J. Biol. Chem. 278, 43452–43459 (2003).
Ichikawa, S. & Hirabayashi, Y. Glucosylceramide synthase and glycosphingolipid synthesis. Trends Cell Biol. 8, 198–202 (1998).
Lipsky, N.G. & Pagano, R.E. Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc. Natl Acad. Sci. USA 80, 2608–2612 (1983).
Henneberry, A.L., Lagace, T.A., Ridgway, N.D. & McMaster, C.R. Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth. Mol. Biol. Cell 12, 511–520 (2001).
Henneberry, A.L., Wright, M.M. & McMaster, C.R. The major sites of cellular phospholipid synthesis and molecular determinants of fatty acid and lipid head group specificity. Mol. Biol. Cell 13, 3148–3161 (2002).
Boggs, K.P., Rock, C.O. & Jackowski, S. Lysophosphatidylcholine and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine inhibit the CDP-choline pathway of phosphatidylcholine synthesis at the CTP:phosphocholine cytidylyltransferase step. J. Biol. Chem. 270, 7757–7764 (1995).
McGee, T.P., Skinner, H.B., Whitters, E.A., Henry, S.A. & Bankaitis, V.A. A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes. J. Cell Biol. 124, 273–287 (1994).
Skinner, H.B. et al. The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity. Proc. Natl Acad. Sci. USA 92, 112–116 (1995).
Kearns, B.G., Alb, J.G. Jr. & Bankaitis, V. Phosphatidylinositol transfer proteins: the long and winding road to physiological function. Trends Cell Biol. 8, 276–282 (1998).
Cleves, A.E. et al. Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell 64, 789–800 (1991).
Kearns, B.G. et al. Essential role for diacylglycerol in protein transport from the yeast Golgi complex. Nature 387, 101–105 (1997).
Allen-Baume, V., Segui, B. & Cockcroft, S. Current thoughts on the phosphatidylinositol transfer protein family. FEBS Lett. 531, 74–80 (2002).
van Tiel, C.M., Schouten, A., Snoek, G.T., Gros, P. & Wirtz, K.W. The structure of phosphatidylinositol transfer protein alpha reveals sites for phospholipid binding and membrane association with major implications for its function. FEBS Lett. 531, 69–73 (2002).
Buccione, R. et al. Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study. J. Biol. Chem. 271, 3523–3533 (1996).
Bligh, E.G. & Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Med. Sci. 37, 911–917 (1959).
Acknowledgements
We thank Z. Elazar, M. Liscovitch and A. Futerman for stimulating discussions. We also thank members of the Futerman laboratory (S. Boldin, Y. Kacher and M. Jmoudiak) for assistance in lipid analysis. Finally, we thank C. Brodie for the GFP–PKD construct. S. L. is an incumbent of the Helena Rubinstein Career Development Chair. This work was supported by the Israel Science Foundation (No. 1073/03), the Israel Cancer Research Foundation, and by the Harry and Jeanette Weinberg Fund for the Molecular Genetics of Cancer.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary information
Supplementary figures S1, S2, S3, S4 and supplementary methods plus movie legends (PDF 301 kb)
Supplementary information
Movie S1 (MOV 2702 kb)
Supplementary information
Movie S2 (MOV 2350 kb)
Rights and permissions
About this article
Cite this article
Litvak, V., Dahan, N., Ramachandran, S. et al. Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function. Nat Cell Biol 7, 225–234 (2005). https://doi.org/10.1038/ncb1221
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb1221
- Springer Nature Limited
This article is cited by
-
PITPNC1 promotes the thermogenesis of brown adipose tissue under acute cold exposure
Science China Life Sciences (2022)
-
Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy
BMC Biology (2020)
-
Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes
Cell Death & Differentiation (2016)
-
The phosphatidylinositol‐transfer protein Nir2 binds phosphatidic acid and positively regulates phosphoinositide signalling
EMBO reports (2013)
-
Links between lipid homeostasis, organelle morphodynamics and protein trafficking in eukaryotic and plant secretory pathways
Plant Cell Reports (2011)