Figure 7.

The N-terminal PIN domain in Rrp44 mediates protein–protein interactions. (A) Sedimentation behavior of WT and Rrp44-ΔPIN on glycerol gradients. Gal::rrp44 strains transformed with plasmids encoding protA-tagged Rrp44 [full-length (WT) or ΔPIN] were grown on glucose for 8.5 h to deplete endogenous Rrp44. Protein extracts from 27.5 ODs of cells were then separated on linear 10–30% glycerol gradients and analyzed by western blotting. (B) Recombinant, GST-tagged WT Rrp44 (WT), Rrp44-endo (endo), Rrp44-ΔPIN (ΔPIN), the PIN domain alone (PIN) and free GST were expressed in E. coli and purified on a glutathione sepharose column. The proteins (indicated by arrowheads) were separated on an 8% polyacrylamide/SDS gel and stained with Coomassie blue. Asterisk: E. coli GroEL. (C) Western blot analysis of TAP-purified exosomes from Csl4-TAP strains grown at 25°C. Exosomes were purified on IgG sepharose at 150 mM NaCl (lane 1) or treated with 800 mM MgCl₂ to dissociate endogenous Rrp44 (lane 2). TEV eluates (2.5 μl) were separated on an 8% polyacrylamide/SDS gel and analyzed by immunoblotting with anti-Rrp44, anti-Rrp6 and anti-Rrp43 antibodies. (D) GST-pull down experiment. The GST-tagged Rrp44 constructs or free GST (B) were coupled to glutathione sepharose beads and incubated with purified yeast exosomes (C). Bound material was eluted under denaturing conditions, separated on an 8% polyacrylamide/SDS gel and analyzed by immunoblotting with an anti-Rrp43 antibody.