Figure 2.

In the presence of wild-type Nsp1p, Nsp1-ala6p no longer interacts with Nup82p and Nic96p and is not targeted to the NPC. (A) Yeast cells with a disrupted chromosomal copy of NSP1 and complemented by ProtA-C-Nsp1p (wt) or ProtA-C-nsp1-ala6p (ala6) were grown at 24°C. Whole-cell lysates from these two strains were affinity purified by IgG-Sepharose chromatography. The affinity-purified fractions were analyzed by Western blotting for the presence of ProtA-C-Nsp1p (asterisks) or ProtA-C-nsp1-ala6p (open circles), Nup159p (using the rat7#5 antibody), and Nic96p, or by Coomassie blue staining. In this nsp1⁻ background, both ProtA-C-Nsp1p and ProtA-C-nsp1-ala6p were able to interact with Nup159p and Nic96p. Nic96p (arrow) and Nup82p (arrowhead) could also be detected by Coomassie blue staining of the two eluates. Note that in this strain background, Nup159p was degraded during the purification procedure and was therefore hardly detectable by Coomassie blue staining. (B) Whole-cell lysates from NUP82-HA cells carrying a wild-type chromosomal copy of NSP1 and expressing ProtA-C-Nsp1p (wt) or ProtA-C-nsp1-ala6p (ala6) were affinity purified byIgG-Sepharose chromatography as described in MATERIALS AND METHODS. The eluates were analyzed by silver staining (Silver) or by Western blot using an anti-IgG coupled to HRP to detect ProtA-C-Nsp1p (asterisks) or ProtA-C-nsp1-ala6p (open circles), an anti-HA antibody to detect Nup82-HAp (arrrowhead), or an antiNic96p (arrow). In the presence of wild-type Nsp1p, the interaction between ProtA-C-nsp1-ala6p and both Nup82-HAp and Nic96p was inhibited (a very faint band was detectable only when a fivefold excess of the ProtA-C-nsp1-ala6 eluate was loaded). (C) The GFP-C-NSP1 and GFP-C-nsp1-ala6 fusion genes were expressed in an NSP1-disrupted strain (nsp1⁻) and in a wild-type strain (NSP1). The GFP-C-Nsp1p and GFP-C-nsp1-ala6p signals were observed by fluorescence microscopy in living cells grown at 24°C as described in MATERIALS AND METHODS.