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

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Zhang, Sheng Linder, Maurine E. Gottlieb, Colin D.
Description	Author Affiliation: Gottlieb CD ( From the Department of Molecular Medicine and.); Zhang S ( the Core Proteomics and Mass Spectrometry Facility, Cornell University, Ithaca, New York 14853.); Linder ME ( 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.
ISSN	00219258
e-ISSN	1083351X
Journal	Journal of Biological Chemistry
Issue Number	49
Volume Number	290
Language	English
Publisher	American Society for Biochemistry and Molecular Biology (United States)
Publisher Date	2015-12-04
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Membrane Proteins Metabolism Zinc Chemistry Amino Acid Motifs Animals Biotin Catalytic Domain Chelating Agents Cysteine Iodoacetamide Ions Lipoylation Lysine Mass Spectrometry Mice Mutation Protein Binding Protein Processing, Post-Translational Sf9 Cells Spodoptera Biochemistry Molecular Biology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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