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Kittler JT, Moss SJ, editors. The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology. Boca Raton (FL): CRC Press/Taylor & Francis; 2006.

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The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology.

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Chapter 5Protein Palmitoylation by DHHC Protein Family

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5.1. INTRODUCTION

Palmitoylation is the post-translational modification of proteins with palmitic acid (16-carbon saturated fatty acid) and regulates the membrane targeting, subcellular trafficking and function of proteins [1]. Palmitoylation occurs either through amide-linkage (N-palmitoylation) or thioester linkages (S-palmitoylation). S-palmitoylation occurs on cysteine residues in diverse sequence contexts and is more commonly found in most palmitoylated proteins. Here, the term of protein palmitoylation will mean S-palmitoylation. Protein palmitoylation is the frequent lipid modification of neuronal proteins and modifies many important proteins, including synaptic vesicle proteins, ion channels, guanosine triposphate (GTP)-binding proteins, neurotransmitter receptors and synaptic scaffolding proteins [2]. Examples include PSD-95, a protein that scaffolds receptors and signaling enzymes at the post-synapse; NCAM140, a neural cell adhesion molecule that localizes at the growth cone and regulates neurite outgrowth; and SNAP-25, a t-SNARE protein that regulates neurotransmitter release [2]. PSD-95 palmitoylation is necessary for sorting of PSD-95 to dendrites and participates in the post-synaptic clustering of PSD-95 in dendritic spines, thereby regulating the clustering of α-amino-3-hydroxy-5-methyl-4-isox-azole propionic acid (AMPA)-type glutamate receptors [3]. Unlike other irreversible lipid modifications such as myristoylation and prenylation, palmitoylation is relatively labile and palmitate on proteins turns over rapidly. Importantly, the specific extracellular signal regulates protein-palmitoylation levels [4]. At post-synaptic sites, palmitate continuously turns over on PSD-95. Depalmitoylation of PSD-95 is enhanced by glutamate receptor-mediated synaptic activity, and this process dissociates PSD-95 and AMPA receptors from the postsynaptic sites [3].

Although the actions of enzymes that add or remove protein palmitate might mediate the dynamic regulation of palmitoylation, the enzymes have been elusive. Recent genetic studies in yeast have identified proteins that mediate palmitoylation. Erf2p [5–7] and Akr1p [8] are palmitoyl-transferases (PATs) for yeast Ras2p and yeast casein kinase2 (Yck2p), respectively (Figure 5.1). Erf2p and Akr1p are integral membrane proteins harboring a cysteine-rich domain containing a conserved DHHC (Asp-His-His-Cys) motif. In genomes of human and mouse, 23 kinds of DHHC-containing proteins are predicted (Figure 5.2). To identify the physiological PATs for substrates, systemically evaluating functions of the family of 23 DHHC-containing proteins is necessary. For this purpose, we isolated all mouse DHHC proteins and established a screening method that allows us to identify the candidate PAT for specific substrates [9]. We found that a subset of DHHC proteins specifically palmitoylates PSD-95 (P-PATs), and that DHHC proteins have substrate specificity (Figure 5.2) [9]. P-PAT activity regulates synaptic clustering of PSD-95 and AMPA receptors, as well as modulating AMPA receptor function in hippocampal neurons. Thus, the DHHC protein-mediated reaction is a potential general mechanism of protein palmitoylation in cells. In this chapter, we describe procedures to screen the DHHC protein family to identify the specific PAT. The procedures include three steps.

FIGURE 5.1. Domain structures of yeast DHHC proteins, Erf2p and Akr1p.

FIGURE 5.1

Domain structures of yeast DHHC proteins, Erf2p and Akr1p. Each has several transmembrane domains (black boxes) and a conserved cysteine-rich domain containing DHHC (Asp-His-His-Cys) motif (DHHC-CRD, gray boxes; DHYC in Akr1p). The DHHC sequence is thought (more...)

FIGURE 5.2. Phylogenetic tree of the mouse DHHC protein family and summary of screening.

FIGURE 5.2

Phylogenetic tree of the mouse DHHC protein family and summary of screening. DHHC proteins apparently have substrate specificity. Among them, DHHC-3 and DHHC-7 palmitoylate PSD-95, GAP43, Gs, and SNAP-25, suggesting that DHHC-3 and DHHC-7 show broad substrate (more...)

5.2. OVERVIEW OF PAT SCREENING

Transfection of the target substrate cDNA together with individual DHHC clone into cultured cells (e.g., HEK293T cells and COS-7 cells) Metabolic labeling with [3H]palmitic acid SDS-PAGE and fluorography

5.3. PROTOCOLS

5.3.1. Transfection

Seed HEK293T (or COS-7) cells (3 × 105 to 6 × 105 cells/well, six-well format) and incubate at 37°C for 16 to 20 hours in the growth medium without antibiotics.

Transfect about 80% confluent cells using lipofectamine plus according to the manufacturer’s instructions (Invitrogen).

Dilute the DNA mixtures (2 μg) in 100 μl of DMEM (or Opti-MEM from Invitrogen). The DNA mixture includes 1 μg each of plasmids for the substrate and individual DHHC clone.

Add 6 μl of Plus reagent from Invitrogen and mix gently. Incubate for 15 min at room temperature.

Combine the diluted DNA mixture with diluted lipofectamine (4 μl in 100 μl of DMEM or Opti-MEM). Total volume will be 200 μl. Incubate for 15 min at room temperature.

Add 800 μl of DMEM or Opti-MEM to the tube containing the complexes (total volume will be 1 ml).

Remove the cultured medium from cells and add 1 ml of the diluted complexes.

Incubate cells at 37°C for 4 hours and then add 1 ml of growth medium including 20% serum (twice the normal concentration of serum). Incubate the cells at 37°C for 20 to 24 hours.

5.3.2. Metabolic Labeling

Dry [3H]palmitic acid (PerkinElmer, 45 Ci/mM, 5 mCi/ml in ethanol) under reduced pressure in a concentrator such as SpeedVac and reconstitute in small amount of 100% of ethanol (e.g., 1 μCi in 10 μl ethanol).

Preincubate transfected cells for 30 min in serum-free DMEM supplemented with fatty-acid free bovine serum albumin (10 mg/ml, Sigma).

Label cells for 4 hours with 0.5 mCi [3H]palmitate diluted in 1ml of preincubation medium (for each well). Note that final concentration of ethanol in medium must be less than 1%.

5.3.3. SDS-PAGE and Fluorography

Wash labeled cells with PBS, collect cells in 1 ml of SDS-PAGE sampling buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.001% bromophenol blue) with 10 mM DTT. Pipette up and down cell suspension on the dish before transferring it to a tube to help cell lysis. Heat the suspension at 95°C for no more than 2 min.

For fluorography, separate 60 μl of cell suspensions with SDS-PAGE (13.5 cm × 8.5 cm separating gel). After fixing the gels for 30 min in a fixing solution (isopropanol:water:acetic acid = 25:65:10), treat the gel with Amplify (Amersham) for 30 min, dry under vacuum and expose to (Kodak Biomax MS) at 80°C for 24 hours. Signals for autopalmitoylation of most DHHC clones and enhanced palmitoylation of substrates by some DHHC clones are usually detected within 24 hours, 72 hours at the most (Figure 5.3).

FIGURE 5.3. Screening of SNAP-25 palmitoylating enzymes.

FIGURE 5.3

Screening of SNAP-25 palmitoylating enzymes. Individual DHHC clones (DHHC-1 through DHHC-12 among 23 DHHCs are shown) were transfected together with SNAP-25 wild-type (WT) or cysteine-deleted SNAP-25 (ΔCys) [10] into HEK293 cells. After metabolic (more...)

Scan the radio-labeled bands in the autoradiograph and analyze with NIH software. To quantitate the relative PAT activity of each DHHC clone, we quantitate the ratio of the palmitoylated protein to total amount of protein (measured by Coomassie staining or quantitative immunoblot).

5.3.4. Notes

A construct of substrate with mutations in palmitoylation sites (e.g., deletion or replacement of cysteine residues) is a good negative control (Figure 5.3; note that no palmitoylation signals around 28 kDa in SNAP-25 ΔCys). For another negative control to see the basal level of palmitoylation, transfect the substrate with mock vector but not with DHHC clones. As a positive control, PSD-95 and PSD-95-PATs P-PATs) such as DHHC-3 and DHHC-15 might be useful [9].

Incubation time of transfection is critical. Longer transfection (over 24 hours) increases the basal level of palmitoylation in the mock cells (in the absence of DHHC clones) and more-or-less hinders DHHC protein-enhanced palmitoylation. Most cultured cells express endogenous DHHC proteins.

Efficiency of transfection (especially for co-transfection) is an important factor for screening. HEK293T cells usually give a better efficiency (about 90% co-transfection) than COS-7 cells (about 60% co-transfection).

We usually do not immunoprecipitate the substrate to detect the DHHC clone-induced palmitoylation. The immunoprecipitation method does not necessarily reflect the total palmitoylated proteins because usual extraction conditions using Triton X-100 or radio-immunoprecipitation assay (RIPA) buffer cannot extract all the palmitoylated proteins, which strongly associate with membrane and go to the insoluble fraction. If immunoprecipitation of the substrate proteins is necessary, 1% SDS (followed by Triton X-100 neutralization) might be suitable for the extraction of palmitoylated proteins [9].

Treat the cell suspension carefully because thioester linkage is labile and sensitive to heat and reducing condition.

Some palmitoylated proteins (such as PSD-95, SNAP-25 and so on) shift their apparent molecular weights on SDS-PAGE (Figure 5.3).

To identify the physiological enzymes, the following experiments are suggested after this screening:

Confirm that the palmitoylation signal for the substrate is not overlapped with the autopalmitoylation signal of the DHHC protein (Figure 5.3; note the autopalmitoylated signals indicated by open arrowheads). Transfection of the DHHC clone without the substrate might be necessary.

Examine whether the candidate DHHC clone palmitoylates the substrate in other cell lines. If viral vectors (e.g., Sindbis or Semliki Forest virus) of DHHC clones are available, seeing the DHHC clone-enhanced palmitoylation of endogenous proteins in primary hippocampal neurons is possible [9].

Examine whether the candidate DHHC clone expresses in tissues where the substrate express.

Examine whether palmitoylation of the endogenous substrate is affected when the candidate DHHC clone is knocked-down by RNAi or inhibited by dominant-negative mutants.

5.4 ACKNOWLEDGMENTS

We thank Dr. Roger A. Nicoll and Dr. Hillel Adesnik of the University of California, San Francisco, for helpful suggestions and discussion, and Dr. Paul A Roche of the National Institute of Health for a kind gift of SNAP-25 cDNA. We also thank Dr. Josef Kittler of University College London for giving us this opportunity. Dr. Yuko Fukata is supported by a long-term fellowship of The International Human Frontier Science Program Organization. Our study is supported by grants from the National Institutes of Health, Christopher Reeves Paralysis Foundation, the Human Frontier Science Program and the American Heart Association.

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Copyright © 2006, Taylor & Francis Group, LLC.
Bookshelf ID: NBK2551PMID: 21204476

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