FIGURE 3.

Grain setting defect 1 association with PM through S-acylation. (A) Alignment of the GSD1 C-terminal sequences with its homologous proteins. The C-terminal sequences contain a coiled-coil domain but vary at the end amino acid residues. Asterisk indicates possibly S-acylated cysteine residues. (B) Biotin switch assays of S-acylation state of GSD1 in rice membrane fractions. S-acylated GSD1 is detected by Western blotting in the elution fraction of the hydroxylamine-treated sample but not detected in the untreated sample. Hyd+, with hydroxylamine; Hyd-, without hydroxylamine. (C) Comparison of the GSD1 S-acylation with or without 2-bromopalmitate (2-BP) (an S-acylation inhibitor) treatment. 2-bromopalmitate treatment substantially reduced the amount of S-acylated GSD1. (D) Western blotting of GSD1 in sucrose density gradient fractions of membrane proteins from rice with GSD1 specific antibodies. GSD1 is predominantly detected in the upper fractions (fractions: 4–6), whereas 2-BP treatment substantially reduces GSD1 in these fractions. (E) Confocal microscopic observation of the subcellular localization of GFP-GSD1 in tobacco leaves under 2-BP treatment or mock solution. Arrowhead denotes GFP fluorescent in nucleus. Bars = 50 μm.