Influence of the nuclear export signal on cytoplasmic localization of 16.4.1. (A) Sequences identified in 16.4.1 and in proteins contained in a database of experimentally verified NES by a common weight matrix. Protein sequences in NESbase version 1  were analysed with a collection of 7 weight matrices. These identified 48 of 75 NES in their native protein context at default threshold (0.84). Analysis of the 16.4.1 sequence with reduced threshold settings (0.74) yielded a single match with matrix M5 but not with any other matrix. This match identified the 16.4.1 core NES (shown at top). Sequences in proteins of the NES database identified by M5 at default threshold are shown below. All sequence matches map to experimentally verified NES. (B) To assess functionality of the novel 16.4.1 NES in the context of the full length protein, a 16.4.1 mutant was generated in which L92, I97 and I99 were replaced by alanine residues. HeLa cells were transfected with plasmids for expression of GFP fusion proteins containing wild type (16.4.1-GFP) or mutated (16.4.1(NESmut)-GFP) 16.4.1 and subcellular distribution of GFP fusion proteins were evaluated by quantitative fluorescence microscopy. Cells expressing 16.4.1(NESmut)-GFP showed a significantly higher nuclear proportion of fluorescence (34%) than cells expressing wild type 16.4.1-GFP (27%). This effect was significant (p < 0.005). Scale bars: 10 μm.
Kramer-Hämmerle et al. BMC Cell Biology 2005 6:20 doi:10.1186/1471-2121-6-20