Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI(+)], a prion form of the S. cerevisiae Sup35 protein (Sup35([PSI+])), and the three N(alpha)-acetyltransferases, NatA, NatB, and NatC, which collectively modify approximately 50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI(+)] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI(+)] NatA mutants continue to propagate heritable Sup35([PSI+]). This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI(+)] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35([PSI+]) complexes and their phenotypic effects.