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J. Biol. Chem., Vol. 283, Issue 36, 25014-25026, September 5, 2008

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Palmitoylation and Membrane Interactions of the Neuroprotective Chaperone Cysteine-string Protein^*

Jennifer Greaves, Christine Salaun, Yuko Fukata^¶||, Masaki Fukata^¶||, and Luke H. Chamberlain¹

From the Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom, the Faculte de Medecine Paris Descartes, Site Necker, INSERM U845, 156 Rue de Vaugirard, Paris 75730 cedex 15, France, the ^¶Division of Membrane Physiology, National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan, and ^||Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 3-5 Chiyoda, Tokyo 102-0075, Japan

Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.

Received for publication, March 18, 2008 , and in revised form, July 1, 2008.

^* This work was funded by a Wellcome Trust Research Career Development fellowship (to L. H. C.), by an Medical Research Council Senior Research fellowship (to L. H. C.), and by Tenovus Scotland (Grant S06/1 to L. H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ To whom correspondence should be addressed: Tel.: 44-131-650-8313; Fax: 44-131-650-8313; E-mail: Luke.Chamberlain{at}ed.ac.uk.

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