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

Nature volume 438, pages 675–678 (2005)Cite this article

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.

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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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  1. MRC Laboratory of Molecular Biology, Hills Road, CB2 2QH, Cambridge, UK

    Jennifer L. Gallop, P. Jonathan G. Butler & Harvey T. McMahon

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Correspondence to Harvey T. McMahon.

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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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