FIG. 8.

Changes in biophysical characteristics of Ycf1p determined by immunoprecipitation (IP), sucrose density gradient fractionation, and limited trypsin digestion. WT and glr1Δ cells were transformed with an expression construct of YCF1 or YCF1(C436A) tagged with an HA epitope at the C terminus. Cells were cultured with and without CdCl₂ supplementation (15 μM, 9 h) in the media. Cells lysates were prepared by vortexing cells with glass beads in a degassed buffer (50 mM HEPES, pH 7.4) containing iodoacetic acid (5 mM) to alkylate –SH groups (29). The samples were subjected to IP (A, B), limited trypsin digestion (C), and western blotting using anti-HA antibodies (A–C). Anti-Pgk1p immunoblotting and staining of the membranes determined equal loading. Representative figures of a minimum of two independent experiments are presented. Asterisks indicate the fragments displaying difference in glr1Δ cells.