RNA polymerase II regulation in α-amanitin-resistant rat myoblast mutants. Changes in wild-type and mutant enzyme levels during growth in α-amanitin

Abstract

A series of independent α-amanitin-resistant (Ama R ) rat myoblast mutants, possessing both wild-type α-amanitin-sensitive and mutant α-amanitin-resistant forms of RNA polymerase II, have been examined with respect to the levels of these two forms of the enzyme when these mutant cells are grown in the presence of α-amanitin. Under these conditions, there is no change in the growth rates of the Ama R mutants. Upon exposure of the mutant cells to α-amanitin, the wild-type enzyme is inactivated by α-amanitin and this decline in wild-type enzyme activity is compensated by a subsequent rise in the specific activity of the mutant RNA polymerase II, maintaining the total RNA polymerase II activity level roughly constant. This regulation of RNA polymerase II activity occurs rapidly, within one generation time, is completely reversible on removal of α-amanitin from the culture medium, and occurs without any apparent change in the specific activity of RNA polymerase I. This regulatory phenotype is exhibited by both diploid and tetraploid Ama R mutants. γ-[ 3 H]amanitin† equilibrium binding measurements to RNA polymerase II in cell lysates of independent mutants grown in the presence and absence of α-amanitin indicate that wild-type RNA polymerase II enzyme levels are reduced dramatically at short times after the shift into α-amanitin (50% reduction at 3 to 6 hours in 1 μ m -α-amanitin). These results suggest that wild-type RNA polymerase II or its α-amanitin binding subunit is rapidly degraded in vivo when complexed with α-amanitin. Ama416, a tetraploid Ama R mutant appears to contain 1·7 to 1·8-fold more RNA polymerase II enzyme activity and γ-[ 3 H]amanitin binding activity than the wild-type parental cell line when grown in 1 μ m -α-amanitin. This result is consistent with the notion that this mutant is defective in membrane transport for α-amanitin.