Figure 8.

Interaction of PGM48 with Other PG Proteins.

(A) Yeast two-hybrid assays for interactions between PGM48 and selected proteins. PGM48 was used as bait, and selected candidate proteins were used as prey. Bait plasmid contains Cub-PLV and prey plasmid contains NubG. NubG moiety was fused to the N terminus of prey proteins. The resulting plasmids were transferred into the different yeast strains for bait and prey. The transformed yeasts harboring bait and prey constructs were mated, and resulting transformants were analyzed on selective medium lacking Ade, His, Try, Leu, Ura, and Met (upper lane) and for β-galactosidase (β-Gal) activity (lower lane). Soluble NubG and Nub-WT were used as negative and positive controls, respectively.

(B) Ni-NTA resin-mediated pull-down of PGM48-His with GST-CCD4. Purified GST alone (10 µg) or GST-fused CCD4 (2 µg) was incubated with purified PGM48 fused with His6 (2.0 µg), together with Ni-NTA resin. Input (50% of the reaction) and eluted samples (80%) were loaded into a SDS-PAGE gel and analyzed by silver staining. i, input; pd, pull-down eluate.

(C) Ni-NTA resin-mediated pull-down of PGM48-His with GST-PES1. Purified GST alone (10 µg) or GST-fused PES1 (3 µg) was incubated with purified PGM48 fused with His6 (2.0 µg), together with Ni-NTA resin. Input (50%) and eluted samples (80%) were loaded into a SDS-PAGE gel and analyzed by silver staining.

(D) Glutathione resin-mediated pull-down of GST-PES1 and GST-CCD4 with PGM48-His. Purified GST-PES1 (2 µg), GST-CCD4 (2 µg), GST-PGSAG (2 µg), and GST alone (10 µg) were incubated with purified PGM48-His (3 µg) recombinant protein, together with glutathione resin. Input (50% of the reaction) and eluted samples (80%) were loaded into a SDS-PAGE gel and analyzed by silver staining.