Figure 6
Ssa1 breaks the interaction of Aco1-C with Aco1-N terminal domains. (A) Aco1 polypeptides originating from separate lysates form complexes in vitro. Separate cell lysates prepared from yeast cells expressing the aconitase C-terminus fused to GST (GST-C) and either Aco1 or Aco1-N fused to HA (HA-Aco1 or HA-N respectively) were mixed and incubated with glutathione sepharose beads. The mixed lysates were pulled down with glutathione sepharose beads and analyzed by Western blotting using GST and HA antisera. Arrows indicate the bands of GST-C (top panel), HA-Aco1 (bottom panel, top arrow) and HA-N (bottom panel, bottom arrow). (B) Ssa1 reduces the amount of HA-Aco1 pulled down by GST-C. Separate cell lysates prepared from yeast cells expressing GST-C and HA-Aco1 were mixed in the presence of different amounts of purified Ssa1 (GenScript, Lot number- U9459BB190S01/P20011602). Glutathione sepharose beads were used to pull down the GST fusion proteins and associated proteins which was analysed by Western blotting using GST and HA antisera. (C) Ssa1 reduces the amount of HA-N pulled down by GST-C. Separate cell lysates prepared from yeast cells expressing GST-C and HA-N were analyzed as described in (B). (D) Aco1-N and endogenously co-expressed aconitase import efficiency in the absence or presence of over-expressed Ssa1. Yeast strains harboring plasmids encoding the indicated proteins were induced in galactose medium and pulse labeled with [35S] methionine–cysteine and CCCP. An aliquot of these cultures was further incubated with DTT in order to restore post-translational import of proteins into mitochondria in the presence of cold methionine–cysteine. Total cell extracts were immunoprecipitated with Aco1, antisera and SDS-PAGE followed by autoradiography was performed. Precursor, p; Mature, m. Asterisk indicates nonspecific bands. This experiment was repeated three times. A significant difference in the standard error was found between the ratios of mature (m) versus total [precursor (p) + mature (m)] forms of chromosomally encoded wild type aconitase with and without Ssa1 overexpression (lanes 3 and 6).
