Figure 2. The putative NLS in OCR-2 is sufficient to drive nuclear localization of a GFP::LacZ fusion protein, and requires basic residues within the NLS.

The osm-10 promoter [30] was used to express GFP::LacZ fusion proteins in the ASH neurons. (A) The GFP::LacZ fusion (osm-10p::gfp::lacZ) alone is excluded from the nucleus of the ASH neurons. GFP fluorescence was restricted to the cytoplasm in 78/78 animals assessed. (B) Inclusion of the putative NLS, upstream of and in frame with GFP::LacZ (osm-10p::ocr-2_841–856::gfp::lacZ), resulted in robust nuclear translocation of GFP. The ASH neurons of 97/103 worms examined displayed pronounced nuclear accumulation of GFP. (C) Six of the basic lysine and arginine residues (indicated in bold) were replaced with the nonpolar amino acid alanine (indicated with an asterisk and in bold) and the mutated NLS was fused upstream of and in frame with GFP::LacZ (osm-10p::ocr-2_{841–856(NLSmut)}::gfp::lacZ). (D) Mutating these basic clusters abolished nuclear localization. The OCR-2_{841–856(NLSmut)}::GFP::LacZ fusion was restricted to the cytoplasm of ASH in 44/44 animals examined. Numbers represent the combined data of ≥3 independent transgenic lines for each construct.