FIGURE 8: Mitochondrial fraction of Ydj1 is tightly associated with the outer membrane. (A) A fraction of Ydj1 is localised to mitochondria. WCE, PMFs and mitochondria isolated from 2×FLAG-YDJ1/ydj1Δ yeast cultures grown at 30^oC and 37^oC were analyzed by SDS-PAGE and immunoblotting with antibodies against the FLAG epitope, cytosolic protein Hog1 and the ADP/ATP mitochondrial carrier Aac2. Anti-FLAG antibody staining is present in all fractions suggesting pancellular localisation of Ydj1, which is expected. Ydj1 is also present in the gradient-purified mitochondrial fraction pointing at associations with mitochondria under normal (30^oC) and elevated (37^oC) temperatures. (B and C) Mitochondrial fraction of Ydj1 is associated with the outer membrane. Gradient-purified mitochondria iso-lated from 2×FLAG-YDJ1/ydj1Δ yeast cultures were subfractionated by osmotic disruption of the outer membrane (Swelling) com-bined with Proteinase K (Prot. K) treatment. Western blotting using anti-FLAG demonstrated Ydj1 localised to the outer mitochondrial mem-brane upon normal (30^oC) and higher (37^oC) temperature growth conditions. (D and F) Salt and (E and G) alkaline extractions of Ydj1 suggest tight peripheral association with the outer mitochondrial membrane. Western blotting analysis of salt extracts demonstrates presence of Ydj1 only in pelleted fractions isolated from gradient-purified mitochondria from the cells grown at both 30°C and 37°C. Positive antibody staining for Ydj1 appeared in Western blots which analysed the supernatants obtained from protein extracts under indicated alkaline conditions. Anti-serum against transmembrane pro-tein Aac2 was used as a marker of integral membrane association. Ponceau S staining of the blots was used to visualise protein loads.