Pom33 interacts with the Rtn protein Rtn1. (A) Affinity purification by IgG chromatography of Pom33-ProtA in Rtn1-GFP–, rtn1-K48I-GFP–, or Yop1-GFP–expressing strains. Total soluble extracts (inputs) and affinity-purified fractions (eluates, 2.5-fold equivalent for the anti–[α]-ProtA and 2,500-fold equivalent for the other antibodies) from strains expressing (+) or not expressing (−) Pom33-ProtA were analyzed by Western blotting using the indicated antibodies. Dpm1 and the nucleolar protein Nop1 were used as controls. (B) Spinning disk confocal images of rtn1Δ nup133Δ cells expressing Rtn1-GFP or rtn1K48I-GFP and Nup49-mCherry. Note that unlike wt Rtn1, the rtn1-K48I mutant is not enriched at the clustered pores labeled with Nup49-mCherry. Bar, 5 µm. (C) Affinity purification by IgG chromatography of Pom33-ProtA or Pom34-ProtA in Rtn1-GFP–expressing cells. Total soluble extracts (inputs) and affinity-purified fractions (eluates, fivefold equivalent for the anti-ProtA and 2,500-fold equivalent for the other antibodies) were analyzed by Western blotting using the indicated antibodies. Black lines indicate that intervening lanes have been spliced out. Size markers on the sides of the gel blots indicate kD.