Palmitoylation motif

Berzat, A.C., Buss, J.E., Chenette, E.J., Weinbaum, C.A., Shutes, A., Der, C.J., Minden, A., and Cox, A.D. (2005). Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif. J Biol Chem 280 33055-33065. 

Varner AS, Ducker CE, Xia Z, Zhuang Y, De Vos ML, Smith CD. Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs. Biochem. J. 2003 373 91-99. 

The use of fluorescent protein that is fused with a peptide motif for targeting proteins to plasma membrane such as the palmitoylation motif of GAP-43 [27] and the farnesylation motif (CAAX box) of K-ras [28] facilitates visualization of axons. However, it should be careful not to extremely overexpress the membrane-targeted version of fluorescent protein, because we have noticed that it sometimes causes morphological abnormality of axons. 

The (3 subunit of the Py complex of transducin is a seven-bladed (3 propeller (Fig.ll-7C). It is composed of seven GH-WD repeat units - [GH - Xn - WD]4 8-where GH = Gly - His, WD = Trp-Asp, and Xn is a core repeating sequence, usually 32 -42 residues in length. This motif is also found in at least 40 other eukaryotic proteins.188/237 -240 In the Py complex (Fig. 11-7B) the y subunit assumes an elongated, largely a-helical structure. It is often anchored at its C terminus by a famesyl or geranylgeranyl chain, while Ga may be myristoylated or palmitoylated.195/230/243... 

Synaptic exocytosis involves three SNARE proteins the R-SNARE synaptobrevin/VAMP (isoforms 1 and 2) on the vesicle, and the Q-SNAREs syntaxin (isoforms 1 and 2) and SNAP-25 on the plasma membrane (Figure 4). Since SNAP-25 has two SNARE-motifs, synaptobrevin, syntaxin, and SNAP-25 together have four SNARE-motifs. Synaptobrevins and SNAP-25 are relatively simple SNARE proteins that are composed of little else besides SNARE motifs and membrane-attachment sequences (a transmembrane region for synaptobrevin, and a cysteine-rich palmitoylated sequence for SNAP-25). Syntaxins, in contrast, are complex proteins. The N-terminal two-thirds of syntaxins include a separate, autonomously folded domain (the so-called Habc-domain), while the C-terminal third is composed of a SNARE motif and transmembrane region just like synaptobrevin. 

Synaptobrevin 2 is a small protein composed of 118 amino acids. It contains a SNARE motif with a short N-terminal proline-rich extension but lacks an independently folded N-terminal domain. Like syntaxin 1, the protein possesses a C-terminal transmembrane domain that is connected to the SNARE motif by a short linker (Figure 1). Synaptobrevin is palmitoylated at cysteine residues close to its transmembrane domain. Synaptobrevin 2 is highly expressed in neurons and neuroendocrine cells, but unlike syntaxin 1 it is also present in many non-neuronal tissues albeit at low levels. 

SNAP-25, a protein of 208 amino acids, deviates from the typical SNARE structure in that it has two SNARE motifs, joined by a flexible linker region, but lacks a transmembrane domain (Figure 1). The linker contains a cluster of four palmitoylated cysteine residues by which the protein is anchored at the plasma membrane. SNAP-25 can be phosphorylated at positions Thrl38 and Seri 87 by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. SNAP-25 represents a small subgroup of SNAREs with a similar structure, including SNAP-23, SNAP-29, and SNAP-47. In contrast to the neuron-specific SNAP-25 these SNAREs are ubiquitously expressed. 

Fig. 5. Topology and common structural features of GPCRs from class A. The characteristic seven transmembrane domains, which are presumably a-helical, are shown as numbered cylinders TMI-VII. These are connected by intracellular loops ICLI-III and extracellular loops, ECLI-III. A conserved disulfide links ECLI and ECLII. The residues that define common sequence motifs, as discussed in the text, are denoted by white circles. Post-translational glycosylation and palmitoylation modifications are depicted at the N- and C-termini, respectively (see text for additional discussion and references).

Wrch-l/RhoU is an atypical Rho family GTPase of the Cdc42 branch (57% sequence identity to Cdc42) but with additional N- and C-terminal extensions [95]. Wrch-1 terminates in a CCFV motif, which upon first inspection appears to be a permanently prenylated CAAX motif but is not rather, it specifies reversible palmitoylation of the second cysteine... 

CXX motif) [96], This palmitoylation step is necessary for membrane localization of Wrch-1, thereby serving a function similar to prenylation of other small GTPases. Since Wrch-1 is not prenylated, it does not interact with RhoGDI. However, we have shown that phosphorylation of Wrch-1 within its HVD near the lipidated cysteine does drive changes in both protein localization and function [97,98]. This indicates that hypervariable domain phosphorylation has both prenylation-dependent and -independent effects on Rho family activity. 

GAP-43 lacks any hydrophobic region found in usual membrane proteins and the palmitoylation which has been implicated in the membrane anchoring is not present in the purified protein. However, the effector domain of basic amphiphilic nature has the ability to bind acidic phospholipids. The domain adopts an a helical conformation when put into hydrophobic environments as shown by the CD and NMR analyses. A growing body of evidence suggests that the basic amphiphilic a-helical domain, which has been initially found as a calmodulin binding motif, serves as a reversible membrane-association signal. 

Hancock, J. F., Paterson, H., Marshall, C. J. (1990). A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21 to the plasma membrane. Cell 63,133-139. 

Particularly common are the iV myristoyl- and S-palmitoyl anchoring motifs shown here. AAmyristoylation al ys occurs at an iV-terminal glycine residue, whereas thioester linkages occur at cysteine residues within the polypeptide chain. G-protein—coupled receptors, with seven transmembrane segments, may contain one (and sometimes two) palmitoyl anchors in thioester linkage to cysteine residues in the C-terminal segment of the protein. 

Antisera developed against recombinant RP2 showed that in cultured cells the protein appeared to localize to the plasma membrane (Chappie et al., 2000). Bioinformatic analyses revealed that this could be a consequence of a dnal acylation motif at the N-terminus of the protein. Therefore we designed a series of experiments to test the significance of this potential motif for myristoylation and palmitoylation. 

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