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J Biol Chem. 2015 Dec 4;290(49):29259-69. doi: 10.1074/jbc.M115.691147. Epub 2015 Oct 20.

The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc.

Author information

1
From the Department of Molecular Medicine and.
2
the Core Proteomics and Mass Spectrometry Facility, Cornell University, Ithaca, New York 14853.
3
From the Department of Molecular Medicine and mel237@cornell.edu.

Abstract

DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.

KEYWORDS:

S-acylation; acyltransferase; fatty acylation; mass spectrometry (MS); post-translational modification (PTM); protein palmitoylation; zinc

PMID:
26487721
PMCID:
PMC4705932
DOI:
10.1074/jbc.M115.691147
[Indexed for MEDLINE]
Free PMC Article

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