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Characterization of the self-palmitoylation activity of the transport protein particle component Bet3

Authors

  • D. Kuemmel
  • J. Walter
  • M. Heck
  • U. Heinemann
  • M. Veit

Journal

  • Cellular and Molecular Life Sciences

Citation

  • Cell Mol Life Sci 67 (15): 2653-2664

Abstract

  • Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.


DOI

doi:10.1007/s00018-010-0358-y