Comparative spatial localization of protein-A-tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy

The nuclear pore complex (NPC) mediates protein and RNP import in and RNA a nd RNP export out of the nucleus of eukaryotic cells. Due to its genetic tr actability, yeast offers a versatile system for investigating the chemical composition and molecular architecture of the NPC. In this context, protein A tagging is a commonly used tool for characterizing and localizing yeast NPC proteins (nucleoporins). By preembedding antiprotein A immunogold elect ron microscopy (immunogold EM), we have localized two yeast nucleoporins, N sp1p and Nic96p, in mutant yeast strains recombinantly expressing these nuc leoporins tagged with four (Nsp1p) or two (Nic96p) IgG binding domains of p rotein A (i.e., ProA-Nsp1p and ProtA-Nic96p). We have compared the location of the recombinant fusion proteins ProtA-Nsp1p and ProtA-Nic96p (i.e., as specified by their protein A tag) to the location of authentic Nsp1p and Ni c96p (i.e., as defined by the epitopes recognized by corresponding nucleopo rin antibodies) and found all of them to reside at the same three NPC sites . Hence, recombinant expression and protein A tagging of the nucleoporins N sp1p and Nic96p have not caused any significant mislocation of the fusion p roteins and thus enabled mapping of these two yeast nucleoporins at the ult rastructural level in a faithful manner. (C) 2000 Academic Press.