Fig. 1. Mislocalization of Arn1p in a vps4-ts strain and localization to the endosome in END+ and end4-1 strains. (A–C) Strains SEY6210 (VPS+) and MBY3 (vps4-ts) were transformed with pArn1-HA and grown in iron-poor medium at 25°C. Aliquots of culture were shifted to 37°C (A and C) for 1 h or grown at 25°C (B) prior to fixation and preparation for indirect immunofluorescence. Mouse monoclonal HA.11 was the primary antibody and Cy3-conjugated donkey anti-mouse was the secondary antibody. Images are in sets of three: fluorescence on the left, differential interference contrast (DIC) in the center and the merged image on the right. (D and E) Congenic RH144–3D (END+; D) and RH268–1C (end4-1; E) strains were transformed with pMetArn1-HA and grown in iron-poor medium at 22°C. Cells were shifted to methionine-free, iron-poor medium and cultured at 37°C for 2 h prior to fixation and preparation for indirect immunofluorescence microscopy. A wild-type strain that did not carry an HA-tagged allele was treated identically, as a control (F). Images are in pairs with fluorescence on the left and DIC on the right.