Figure 3.

Ydj1 and Sis1 enhance TA transfer from Ssa1 to Sgt2.A, scheme of the assay to measure Bos1^Bpa transfer from Ssa1 to Sgt2. 0.1 μM Bos1^Bpa was preincubated with 3 μM Ssa1 and/or JDP, followed by addition of 0.3 μM Sgt2 to initiate the transfer reaction. At specified times after Sgt2 addition (t), aliquots of the reaction were frozen and analyzed by UV crosslinking at –20 °C. B, representative Western blot analysis of the time courses of Bos1^Bpa transfer from Ssa1 to Sgt2 in the absence and presence of Ydj1. C, quantification of the Bos1-Sgt2 crosslink from the data in (B) and their replicates. D, representative Western blot image of Bos1^Bpa transfer from Ydj1 alone or Ssa1·Ydj1 to Sgt2 and Sgt2-TPRmt. Two crosslinked bands were observed between Bos1 and Sgt2-TPRmt, possibly due to different conformations of the Bos1·Sgt2-TPRmt complex. E, quantification of the efficiency of Bos1-Sgt2 crosslinking from the data in (D) and their replicates. F, representative Western blot analysis of the time courses of Bos1^Bpa transfer from Ssa1 to Sgt2 in the absence and presence of Sis1. G, quantification of the Bos1-Sgt2 crosslink from the data in (F) and their replicates. H, representative Western blot image of Bos1^Bpa transfer from Ssa1 and Sis1 to Sgt2 and Sgt2-TPRmt. All values in (C) and (E) represent mean ± SD, with n ≥ 3; “∗” and “∗∗” denote p < 0.005 and p < 0.001, respectively, from Student's t test. Error bars are shown but may not be visible in some cases. Values from two independent experiments are shown in (G) as black (Ssa1 alone) and red (Ssa1+Sis1) circles. TA, tail-anchored protein.