Brr6 and Brl1 localization and the role of disulfide bonds. (A) Localization of yeGFP-tagged Brr6 and Brl1 expressed under control of P_ADH1. Plot profiles along the yellow lines indicate the distribution of yeGFP signals on the NE and the cortical ER. Bar, 5 µm. (B) Strains expressing NUP188-tdTomato in combination with BRR6-yeGFP, BRL1-yeGFP, or NUP85-yeGFP under the endogenous promoter. The enlargements (top left) show NEs that were used for the plot profiles (right). Black arrowheads in the graph indicate colocalization of Brr6 or Brl1 with Nup188 on the NE. Bars: (overview) 5 µm; (enlargement) 1 µm. (C) Immuno-EM analysis of yeGFP-Pom34, Asi3-yeGFP, yeGFP-Brr6, and yeGFP-Brl1 cells with anti-GFP antibody. Blue arrowheads, 10-nm gold particles reflecting yeGFP localization. Orange stars indicate NPCs. Red squares represent the red marked enlargements on the right. Green boxed pictures on the right show additional examples with Brr6 and Brl1 signals at NPCs taken from other EM micrographs. N, nucleus; C, cytoplasm. Bars: (overviews) 500 nm; (enlargements) 50 nm. (D) Plot of gold particles of the NE at NPCs. n is given in E. NPC occupancy is described in Materials and methods. (E) Quantification of gold-labeled particles on different sides of the NE. (F) Topology of Brl1 by BiFC. Bar, 5 µm. (G) In vivo biotinylation of HBH-Brl1, HBH-Brr6, and Brr6-HBH. (H) Scheme of Brl1 and Brr6. In vivo redox analysis of Brr6 and Brl1. Immunoblot with antibodies against Brl1 or His-tag. (I) Serial dilutions of brl1Δ CEN-URA3-BRL1 cells with the indicated LEU2-based CEN plasmids. 5-FOA removes the URA3-based plasmid. (J) Model for the topology of the disulfide bonds in Brr6 and Brl1.