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-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.