Fig. 5.

Localisation of PBF deletion mutants. (A) Putative functional domains of WT PBF, as well as three deletion mutants M1, M2 and M3. NLS, nuclear localisation signal. Site-directed mutagenesis was used to delete the C-terminal 30 amino acids (149-180) for M1. M2 had a deletion of amino acids 29-93 and M3 lacked amino acids 94-149, containing the potential transmembrane domain. (B) (i) Confocal images demonstrating the localisation of each of the mutant PBF proteins compared with wild-type PBF in COS-7 cells. M1 is predominantly in the cell membrane whereas M2 and M3 appear to be in the endoplasmic reticulum. (ii) Coexpression of CD8 confirmed the presence of M1 in the plasma membrane. (iii) Endogenous PDI staining verified endoplasmic reticulum expression of M2 and M3. (iv) Intracellular expression of WT and mutated PBF with CD63 in COS-7 cells. Unlike the WT, none of the mutants show significant colocalisation with CD63. Scale bars: 20 μM.