Fig. 5. hNMD3 possesses a conserved, C-terminal NES sequence. (A) Scheme illustrating the position of sequence motifs in NMD3 from different species and constructs used for transient expression. NMD3 proteins from all species possess an N-terminal conserved domain containing 4 putative zinc fingers (gray boxes). NMD3s in eukaryotes have acquired a C-terminal domain harboring the NLS (white box) and two potential C-terminal NES sequences, according to Ho et al. and Gadal et al. (Ho et al., 2000b; Gadal et al., 2001). Deletion of NES1 (black box) in S.c.Nmd3p dramatically affects cell growth and leads to a nuclear localization of the protein, whereas the potential NES2 (striped box) does show some deviation from the NES consensus sequence but in conjunction with NES2 is needed for viability of yeast (Gadal et al., 2001). The NES1 sequence is conserved throughout higher eukaryotes. Conserved residues are highlighted in bold. For comparison, the PKI NES is shown. Accession numbers of the different NMD3 proteins are the following: S.c. P38861, S.p. Q09817, H.s. NP_057022, M.m. NP_598548, A.g. EAA11292, D.m. CAB42049, C.e. CAA96689, A.t. AAL07089 and O.s. AAK00432. Full-length wild-type hNMD3, C-terminal deletion mutants lacking NES1 (hNMD3C27), or both NESs (hNMD3C71), and an NES mutant (hNMD3-NESmut), in which L487 and I489 were changed to alanines, are depicted. (B) hNMD3 contains an NES sequence in the last 20 amino acids. GFP fusions of the different constructs as presented in A were expressed in transiently transfected HeLa cells. Eighteen hours post-transfection, the cells were transferred into fresh medium without or with leptomycin B (LMB) and fixed after 4 hours. The intracellular distribution of the individual proteins was determined by fluorescence microscopy.