FIGURE 3.

Ypk2 directly binds to Avo1. A, Tor2-Ypk2 interaction in the WT or avo2Δbit61Δ background. Lysates from HA-Tor2-expressing yeast cells transformed with a plasmid expressing GST or GST-Ypk2 were subjected to GST pulldown procedures. Pulled down proteins were separated and detected by Western analysis using anti-HA antibodies for HA-Tor2 and by Coomassie Blue staining for GST and GST-Ypk2. Expression levels of HA-Tor2 are shown in the bottom panel. B, in vitro binding assay. ³⁵S-Labeled Avo1, Avo3, and Lst8 were prepared using an in vitro transcription/translation kit and incubated with glutathione-Sepharose bead-bound recombinant GST or GST-Ypk2 produced in E. coli. After washes, the bound ³⁵S-labeled proteins were analyzed by SDS-PAGE and autoradiography; GST fusion proteins were stained with Coomassie Blue. Shown in the bottom panels are input ³⁵S-labeled proteins (one-twentieth of the amount used in pulldown) analyzed by SDS-PAGE and autoradiography. prot., protein.