CAC3 (MSI1) suppression of RAS2(G19V) is independent of chromatin assemblyfactor I and mediated by NPR1

Cac3p/Msilp, the Saccharomyces cerevisiae homolog of retinoblastoma-associa ted protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF -I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transductio n pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of e ither CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating th at Cac3p has at least two distinct, separable functions, one in chromatin a ssembly and one in regulating RAS function. Unlike Cac1p, which localizes p rimarily to the nucleus, Cac3p localizes to both the nucleus and the cytopl asm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that st ablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p , suppressed the RAS/cAMP pathway. Furthermore, NPRI overexpression interfe red with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating t hat extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Dele tion of NPR1 did not affect the quantity, phosphorylation state, or localiz ation of Ras2p. Consistent with the idea that Npr1p exerts its effect on th e RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubi quitin suppressed both the heat shock sensitivity and the sporulation defec ts caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MS I1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism tha t involves the sequestration of Npr1p and may be due to the increased ubiqu itination of an Npr1p substrate.