Fig. 1.

Vps46p interacts with Vta1p and Vps4p. (A and B) Protein extracts were prepared from WT, vps4Δ, vps32Δ, and vta1Δ (KEBY88, KEBY92, KEBY112, and JLY03) yeast expressing Vps46p–HA (pJL11). GST pull-downs were performed as described in Materials and Methods. Approximately 10% of input (solubilized P15 fraction) and samples from the GST pull-downs were run on the gel. GST alone was used as a negative control. Blots were probed with anti-HA mAb. GST pull-downs were performed by using GST–Vta1p (A) and GST–Vps4p (B). (C) Vps46p was cloned into a bacterial expression vector to contain a C-terminal 6XHIS tag. Vps46p–6XHIS was recombinantly expressed, purified, and used for in vitro binding analysis. Vps46p–6XHIS was incubated with glutathione Sepharose and GST alone (negative control), GST–Vta1p, GST–Vps4p, or GST–Vma13p (negative control). Unbound protein was recovered from the supernatant and saved. The beads were washed, and bound proteins were eluted by heating in sample buffer. Blots were probed with an anti-6XHIS mAb.