Studies of the Ras /cAMP pathway in the budding yeast Saccharomyces cerevisiae

Abstract

The Ras/cAMP pathway plays an important role in regulating cell growth, metabolism, stress responses and differentiation in the budding yeast Saccharomyces cerevisiae. Many of the components of the Ras pathway have been identified. In this study, two approaches were employed to identify new components of the Ras signaling pathway. One involves isolating extragenic suppressors of hyperactivated Ras signaling induced by inactivation of Ras GTPase activating proteins. More than one hundred mutants were characterized genetically. Some of the known components of the Ras pathway were identified, demonstrating the effectiveness of the screen. In addition, a novel component of the Ras pathway was found. In the second approach we identified GNP1, which we demonstrated to encode a high affinity glutamine permease. The involvement of Grip1 in Ras signaling remains to be established. In addition, we found that unlike other amino acid permeases, GNP1 is expressed on both rich and poor nitrogen sources by transcription factors of the GATA family.;The second half of this study is focused on the function of Msi1, a negative regulator of Ras signaling in yeast and a homolog of mammalian retinoblastoma (Rb) binding protein RbAp48 and RbAp46. In a two-hybrid screen, Cac1 was identified as the major Msi1 interacting protein. Msi1 and Cac1 are two subunits of the yeast chromatin assembly factor (yCAF-I). However, MSI1 inhibits Ras signaling even in the absence of CAC1, demonstrating that MSI1's effects on Ras signaling are not mediated via yCAF-I and suggesting that Msi1 is a bifunctional protein. We demonstrated that in multiple genetic backgrounds, MSI1 overexpression suppresses the heat shock sensitivity induced by increased cAMP content. However, MSI1 does not inhibit cAMP synthesis or total cellular PKA activity, nor does MSI1 stimulate expression of cAMP repressible genes. Moreover, MSI1 suppresses hyperactivated protein kinase A (PKA) in the presence of the regulatory subunit Bcy1 and Msi1l is physically associated with Bcy1. Taken together, these observations suggest that Msi1 may misregulate either the localization or the activity of a subpopulation of PKA and thereby negatively regulates Ras signaling.