Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission

Abstract

Endophilins have been proposed to have an enzymatic activity (a lysophosphatidic acid acyl transferase or LPAAT activity) that can make phosphatidic acid in membranes1,2,3. This activity is thought to change the bilayer asymmetry in such a way that negative membrane curvature at the neck of a budding vesicle will be stabilized. An LPAAT activity has also been proposed for CtBP/BARS (carboxy-terminal binding protein/brefeldin A-ribosylated substrate), a transcription co-repressor that is implicated in dynamin-independent endocytosis and fission of the Golgi in mitosis4,5,6. Here we show that the LPAAT activity associated with endophilin is a contaminant of the purification procedure and can be also found associated with the pleckstrin homology domain of dynamin. Likewise, the LPAAT activity associated with CtBP/BARS is also a co-purification artefact. The proposed locus of activity in endophilins includes the BAR domain, which has no catalytic site but instead senses positive membrane curvature. These data will prompt a re-evaluation of the molecular details of membrane budding.

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Figure 1: LPAAT activity may be a co-purification artefact.
Figure 2: Endophilin and CtBP/BARS do not have LPAAT activity and endophilin effects positive, rather than negative membrane curvature.

References

  1. 1

    Schmidt, A. et al. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401, 133–141 (1999)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Modregger, J., Schmidt, A. A., Ritter, B., Huttner, W. B. & Plomann, M. Characterization of Endophilin B1b, a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1. J. Biol. Chem. 278, 4160–4167 (2003)

    CAS  Article  Google Scholar 

  3. 3

    Guichet, A. et al. Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction. EMBO J. 21, 1661–1672 (2002)

    CAS  Article  Google Scholar 

  4. 4

    Weigert, R. et al. CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature 402, 429–433 (1999)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Hidalgo Carcedo, C. et al. Mitotic Golgi partitioning is driven by the membrane-fissioning protein CtBP3/BARS. Science 305, 93–96 (2004)

    ADS  Article  Google Scholar 

  6. 6

    Bonazzi, M. et al. CtBP3/BARS drives membrane fission in dynamin-independent transport pathways. Nature Cell Biol. 7, 570–580 (2005)

    CAS  Article  Google Scholar 

  7. 7

    Leung, D. W. The structure and functions of human lysophosphatidic acid acyltransferases. Front. Biosci. 6, D944–D953 (2001)

    CAS  Article  Google Scholar 

  8. 8

    Umezu-Goto, M. et al. Lysophosphatidic acid production and action: validated targets in cancer? J. Cell. Biochem. 92, 1115–1140 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Schuske, K. R. et al. Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin. Neuron 40, 749–762 (2003)

    CAS  Article  Google Scholar 

  10. 10

    Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001)

    CAS  Article  Google Scholar 

  11. 11

    Huttner, W. B. & Schmidt, A. Lipids, lipid modification and lipid-protein interaction in membrane budding and fission—insights from the roles of endophilin A1 and synaptophysin in synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10, 543–551 (2000)

    CAS  Article  Google Scholar 

  12. 12

    Karbowski, M., Jeong, S. Y. & Youle, R. J. Endophilin B1 is required for the maintenance of mitochondrial morphology. J. Cell Biol. 166, 1027–1039 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Kumar, V. et al. Transcription corepressor CtBP is an NAD+-regulated dehydrogenase. Mol. Cell 10, 857–869 (2002)

    CAS  Article  Google Scholar 

  14. 14

    Nardini, M. et al. CtBP/BARS: a dual-function protein involved in transcription co-repression and Golgi membrane fission. EMBO J. 22, 3122–3130 (2003)

    CAS  Article  Google Scholar 

  15. 15

    Chinnadurai, G. CtBP family proteins: more than transcriptional corepressors. BioEssays 25, 9–12 (2003)

    CAS  Article  Google Scholar 

  16. 16

    Zhang, Q., Piston, D. W. & Goodman, R. H. Regulation of corepressor function by nuclear NADH. Science 295, 1895–1897 (2002)

    ADS  CAS  PubMed  Google Scholar 

  17. 17

    Hamada, F. & Bienz, M. The APC tumour suppressor binds to C-terminal binding protein to divert nuclear β-catenin from TCF. Dev. Cell 7, 677–685 (2004)

    CAS  Article  Google Scholar 

  18. 18

    Hildebrand, J. D. & Soriano, P. Overlapping and unique roles for C-terminal binding protein 1 (CtBP1) and CtBP2 during mouse development. Mol. Cell. Biol. 22, 5296–5307 (2002)

    CAS  Article  Google Scholar 

  19. 19

    tom Dieck, S. et al. Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex. J. Cell Biol. 168, 825–836 (2005)

    Article  Google Scholar 

  20. 20

    Schmitz, F., Konigstorfer, A. & Sudhof, T. C. RIBEYE, a component of synaptic ribbons: a protein's journey through evolution provides insight into synaptic ribbon function. Neuron 28, 857–872 (2000)

    CAS  Article  Google Scholar 

  21. 21

    Kooijman, E. E. et al. Spontaneous curvature of phosphatidic acid and lysophosphatidic acid. Biochemistry 44, 2097–2102 (2005)

    CAS  Article  Google Scholar 

  22. 22

    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)

    CAS  Article  Google Scholar 

  23. 23

    Brown, A. P., Coleman, J., Tommey, A. M., Watson, M. D. & Slabas, A. R. Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases. Plant Mol. Biol. 26, 211–223 (1994)

    CAS  Article  Google Scholar 

  24. 24

    Coleman, J. Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. J. Biol. Chem. 265, 17215–17221 (1990)

    CAS  PubMed  Google Scholar 

  25. 25

    Coleman, J. Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC). Mol. Gen. Genet. 232, 295–303 (1992)

    CAS  PubMed  Google Scholar 

  26. 26

    Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank members of the laboratory for support and encouragement; A Brown for JC201 and pPlsC; F. Hamada and M. Bienz for GST–APC(two-motif repeats). J.L.G. was the recipient of a Medical Research Council Pre-doctoral Fellowship.

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Correspondence to Harvey T. McMahon.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Lysophosphatidic acid acyl transferase (LPAAT) activities. Full TLC plates of LPAAT activities of proteins purified from BL21 and JC201 bacteria and Coomassie gels of protein purifications. (PDF 235 kb)

Supplementary Figure 2

Growth kinetics for JC201 bacteria expressing various endophilins and controls (PDF 113 kb)

Supplementary Figure 3

Binding of endophilin to C16-CoA agarose and ultracentrifugation in the presence of palmitoyl-CoA (PDF 438 kb)

Supplementary Figure 4

Endophilin A1 does not bind CoA, shown by ultracentrifugation and Coomassie gels of liposome binding. (PDF 826 kb)

Supplementary Figure 5

NADH binds to CtBP but does not compete with LPAAT activity and binding of APC protein fragment to CtBP (PDF 176 kb)

Supplementary Figure 6

Oligomerisation of CtBP is unaffected by Oleoyl-CoA (PDF 542 kb)

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Gallop, J., Butler, P. & McMahon, H. Endophilin and CtBP/BARS are not acyl transferases in endocytosis or Golgi fission. Nature 438, 675–678 (2005). https://doi.org/10.1038/nature04136

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