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First published online 22 January 2008
doi: 10.1242/jcs.020107

Journal of Cell Science 121, 421-427 (2008)
Published by The Company of Biologists 2008
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Palmitoylation and localisation of RAS isoforms are modulated by the hypervariable linker domain

Alex J. Laude and Ian A. Prior*

Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, UK


Figure 1
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  		Fig. 1. Palmitoylated RAS hypervariable regions (HVRs). Alignment of palmitoylated RAS HVR sequences reveals regions of strong homology between isoforms that are conserved between species (A). Patches B1 and B2 containing basic (+) and hydrophobic ({phi}) residues are conserved together with a precisely located central pair of acidic (–) residues. K(B)-RAS is shown for reference (B). (C) GFP-tagged HVR mutants used for the analysis of membrane targeting.

 

Figure 2
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  		Fig. 2. The linker domain influences the targeting of palmitoylated RAS. Singly palmitoylated N- and K(A)-RAS show stronger cell-surface localisation when the linker domain is present (HVR-N/HVR-KA versus tN/tKA). Dually palmitoylated H-RAS does not require an upstream linker sequence for stable cell-surface localisation (tH versus HVR-H). Bars, 10 µm.

 

Figure 3
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  		Fig. 3. The targeting domain basic patch facilitates localisation to the plasma membrane. In the absence of adjacent basic residues, mono-palmitoylated N-RAS and K(A)-RAS targeting domains are confined to the ER/Golgi (tN and tKA-Q) Complementary addition of basic lysine residues to tN is necessary for cell-surface labelling that is also seen with tKA, which has intrinsic basic residues. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. N-RAS linker motifs cooperate to promote cell-surface localisation. Substitution of the linker domain for alanine residues in a minimal GFP-tagged HVR (HVR-N-ala) or full-length N-RAS (N-RAS-ala) causes a complete redistribution to the ER/Golgi. Loss of the basic/hydrophobic patch (HVR-N-{Delta}1, HVR-N-Q), conserved serine (HVR-N-SA) or acidic aspartate residues (HVR-N-DA) results in an increase in endomembrane labelling compared with that of the control (HVR-N). Semi-quantitative analysis of perinuclear and plasma membrane staining is displayed together with a representative example of cells matching each criterion for perinuclear localisation. Plasma membrane labelling was scored positive if even minimal labelling was present on surface areas where cells were not directly apposed. Bars, 10 µm. Golgi labelling: –, no; +, intermediate; ++, strong.

 

Figure 5
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  		Fig. 5. Stable N-RAS palmitoylation requires acidic linker residues. N-RAS constructs exhibit equivalent de-palmitoylation kinetics, with t1/2=12-15 minutes (A). Steady-state [3H]-palmitate labelling is equivalent for N-RAS constructs (B) but significantly reduced when acidic residues are lost compared with control (C; HVR-N-DA, HVR-N-Q-{Delta}2-ala and HVR-N-ala versus HVR-N). Representative blots of [3H]-palmitate labelling are displayed, with re-probed GFP blots used for normalisation; the graphs incorporate the means of the [3H]-palmitate signal corrected for GFP expression and normalised to N-RAS labelling (B,C). The percentage total protein in the P100 fraction reveals equivalent levels of strong membrane binding among the constructs (D). The graphs display means from 3-5 independent experiments.

 

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