Fig. 4. The N-terminus of Arl8b is required for its lysosomal localisation. (A) Mutant forms of Arl8b, with the corresponding N-terminal peptides obtained after trypsin digestions of HA-tagged forms as in Fig. 3C. The sequence of the N-terminal peptides identified by MALDI were determined by tandem mass spectrometry (MS/MS). The mass of the peptide stated is for the protonated form, with that from Arl8b(L2F) being the form with an oxidised methionine that was more abundant than the native version and hence used for MS/MS sequencing. (B-D) Confocal micrographs of COS cells transfected with plasmids expressing the indicated forms of Arl8b fused to GFP and then, after fixation and permeabilisation, labelled with an anti-CD63 antibody to label lysosomes. (E-F) Confocal micrographs of COS cells transfected with plasmids expressing GFP (-G) fused to the indicated chimeras of Arf1 and Arl8b, and labelled with the indicated antibodies. Replacement of the first 18 residues of Arl8b with the first 15 of Arf1 does not prevent localisation to lysosomes (labelled with CD63), whereas the first 19 residues of Arl8b do not relocalise Arf1 (17-181) from the Golgi (labelled with golgin-245). Bars, 10 µm.