The Δgef1 strain (RGY84) was transformed with pKF31 (optCLC2) and pKF56 (MNT1-3 × myc) plasmids and additionally with a plasmid encoding full-length Kha1p (pKF68) or Kha1pΔC382 (pKF67). Cells were grown in SD medium without methionine. Subcellular fractions pelleted at 4000, 12000, 20000, 35000, 105000 g and the 105000 g supernatant were subjected to SDS/PAGE (10% polyacrylamide) and further analysed by immunoblotting. Equal amounts of protein of each fraction were loaded as indicated (H, homogenate; P, Pellet; and S, supernatant). The Kha1p variants were detected using anti-GFP antibodies. The same samples were additionally probed with anti-S. cerevisiae ferrochelatase antibodies and antibodies specific to the c-Myc epitope of Mnt1p–Myc. The positions of molecular-mass markers are shown on the right.