Analysis of DHHC Acyltransferases Implies Overlapping Substrate Specificity and a Two-Step Reaction Mechanism

Authors

  • Haitong Hou,

    1. University of Osnabrück, Department of Biology, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany
    Search for more papers by this author
  • Arun T. John Peter,

    1. University of Osnabrück, Department of Biology, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany
    Search for more papers by this author
  • Christoph Meiringer,

    1. University of Osnabrück, Department of Biology, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany
    Search for more papers by this author
    • 2

      Present address: Roche Diagnostics GmbH, 82377 Penzberg, Germany

  • Kanagaraj Subramanian,

    1. University of Osnabrück, Department of Biology, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany
    Search for more papers by this author
    • 3

      Present address: The Scripps Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

  • Christian Ungermann

    Corresponding author
    1. University of Osnabrück, Department of Biology, Biochemistry section, Barbarastrasse 13, 49076 Osnabrück, Germany
      Christian Ungermann, Christian.Ungermann@biologie.uni-osnabrueck.de
    Search for more papers by this author

Christian Ungermann, Christian.Ungermann@biologie.uni-osnabrueck.de

Abstract

Asp-His-His-Cys (DHHC) cysteine-rich domain (CRD) acyltransferases are polytopic transmembrane proteins that are found along the endomembrane system of eukaryotic cells and mediate palmitoylation of peripheral and integral membrane proteins. Here, we address the in vivo substrate specificity of five of the seven DHHC acyltransferases for peripheral membrane proteins by an overexpression approach. For all analysed DHHC proteins we detect strongly overlapping substrate specificity. In addition, we now show acyltransferase activity for Pfa5. More importantly, the DHHC protein Pfa3 is able to trap several substrates at the vacuole. For Pfa3 and its substrate Vac8, we can distinguish two consecutive steps in the acylation reaction: an initial binding that occurs independently of its central cysteine in the DHHC box, but requires myristoylation of its substrate Vac8, and a DHHC-motif dependent acylation. Our data also suggest that proteins can be palmitoylated on several organelles. Thus, the intracellular distribution of DHHC proteins provides an acyltransferase network, which may promote dynamic membrane association of substrate proteins.

Ancillary