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

Jennifer Greaves,Christine Salaun,Yuko Fukata,Masaki Fukata,Luke H Chamberlain

doi:10.1074/jbc.m802140200

Abstract

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.

Highlights

Protein sorting and the micro-localization of proteins within membranes [5,6,7,8,9]
Cysteine-string protein (CSP) Palmitoylation Is Inefficient in HEK293 Cells and Is Enhanced by Co-expression of Specific Golgi-localized DHHC Proteins—As previously shown [24], when EGFP-CSP is expressed in PC12 cells, two bands are detected by immunoblotting (Fig. 1A)
To ensure that the observed enhancement of CSP palmitoylation was directly related to DHHC protein expression, we examined the effects of increasing amounts of DHHC3 plasmid on palmitoylation of co-transfected EGFP-CSP

Summary

Introduction

Protein sorting and the micro-localization of proteins within membranes [5,6,7,8,9]. Despite several attempts to identify palmitoyl transferases over many years, it was only relatively recently that palmitoylating enzymes containing a conserved DHHCCRD (cysteine-rich domain)2 were first identified in yeast [10, 11], where they mediate the majority of palmitoylation reactions [12]. DHHC Proteins Regulate Stable Membrane Binding of CSP— In PC12 cells, palmitoylated EGFP-CSP associates tightly with membranes (Fig. 3A) [24]. Because specific DHHC proteins enhance CSP palmitoylation in HEK293 cells (Fig. 1B), we tested whether these enzymes were sufficient to catalyze stable membrane binding of CSP.

Results

Conclusion