FIG. 1.

Mutation of the glycogen-binding domains of Sip2 and Gal83. (A) Conservation of the glycogen-binding domain among β subunits. Amino acid sequences of rat AMPK β1, Gal83, and Sip2 were aligned with CLUSTAL W (40) and formatted by an Excel macro as previously described (34). Residues that are conserved between the rat and yeast sequences are shaded black, and conservative substitutions are shaded gray. Numbers indicate amino acid positions. Asterisks mark sites of mutations. (B) Glycogen-binding assay. Wild-type and mutant glycogen-binding domains of Gal83 and Sip2, expressed as His₆-tagged proteins in bacteria, were incubated with glycogen. Glycogen and bound protein were separated from unbound protein by centrifugation. Protein in the input (I), supernatant (S; glycogen unbound) and pellet (P; glycogen bound) were detected by Western blot analysis of each fraction (20 μl) with anti-His₆ tag antibody.