FIG. 4.

Distribution of nuclear and cytoplasmic RPD3, RbAp48, SIN3, and histone deacetylation activity on sucrose gradients. (A) Fractionation of nuclear and cytoplasmic endogenous RPD3, SIN3, and RbAp48 and of exogenous RPD3 on sucrose gradients. Extracts from nuclei and cytoplasm of uninjected oocytes or oocytes which had been microinjected with RPD3 mRNA were fractionated on sucrose gradients (200 oocyte equivalents per gradient). One-third of every second fraction was analyzed by Western blotting with the indicated antibodies. SIN3, RPD3, and RbAp48 were detected on the same blot for uninjected oocytes. Size markers indicated that fractions 8, 12, and 16 corresponded to 669, 443, and 150 kDa, respectively (25). (B) Expressed RPD3 cofractionates with increased histone deacetylase activity in nuclei and cytoplasm. Histone deacetylation activity of 7 μl of every second fraction was determined in uninjected and RPD3-injected nuclear and cytoplasmic fractions. Released acetate as counts per minute of tritium is indicated by numbers on the y axis. The error of the deacetylase assays is ±5%.