Fig 6.

Mdy2 effects on the interaction of Sgt2 and Ydj1. (A) Purified proteins: GST-Sgt2 (lane 1), GST-sgt2(ΔN) (lane 2), glutathione S-transferase (GST) (lane 3), and Mdy2 (lane 4) were expressed in bacteria and were purified as described in “Materials and Methods.” Five micrograms each were displayed by sodium dodecyl sulfate (SDS) gel electrophoresis, and a Coomassie Brilliant Blue–stained gel is given here. Molecular mass markers (kDa) are phosphorylase b (97 000), bovine serum albumin, ovalbumin (66 000), carbonic anhydrase (30 000), and soybean trypsin inhibitor (22 000). The apparent molecular mass of the recombinant Mdy2 is larger than that predicted, and the lower band (marked by a circle) is a degradation product of his-tagged Mdy2 (lane 4). (B) Interaction between Sgt2 and Mdy2. GST pull-down assays were carried out as described and were used to determine whether the region containing the N-terminal 58 amino acids of Sgt2 was essential for interacting with Mdy2. The proteins pull-down by glutathione (GSH)-Sepharose were analyzed by SDS gel, and a Coomassie Blue–stained gel is shown here. Lanes 1, 2, and 3 are GST-Sgt2, GST-sgt2(ΔN), and GST with Mdy2, respectively. The location of Mdy2 on the gel is indicated with an asterisk. (C) Association of Ydj1 with Sgt2 is elevated by Mdy2. GST-Sgt2, GST-sgt2(ΔN), and GST were incubated with Ydj1 purified from yeast in the presence and absence of Mdy2. The association of Ydj1 with GST fusion proteins was determined by Western blot analysis with anti-Ydj1 antibodies. Lane 1 contains ¹/₁₀ of the Ydj1 used for the assays. Lanes 2 and 5 contain GST-Sgt2 without and with Mdy2, respectively. Lanes 3 and 6 contain GST-sgt2(ΔN) without and with Mdy2, respectively. Lanes 4 and 7 contain GST without and with Mdy2, respectively.