The Ras/cAMP-dependent Protein Kinase Signaling Pathway Regulates an Early Step of the Autophagy Process in Saccharomyces cerevisiae

Abstract

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMPdependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.

Functional Analysis of the ATG8 Homologue Ao atg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

“…This result agrees with the localization of AoAtg8 and DsRed2 during conidial germination. In A. nidulans and Aspergillus fumigatus, the Ras signaling pathway, which plays an important role in the control of cell growth and response to nutrients, is involved in germination and autophagy (2,4,24). Furthermore, in S. cerevisiae, a relationship has been reported between the Ras signaling pathway and the Tor kinase (central negative regulator of autophagy) (29), which supports our data.…”

“…The NSR13AA strain, NSR13 transformed with adeA (6), was used as a control for the phenotypic assay. Czapek Dox (CD) medium (0.3% NaNO 3 2 O, and 2% glucose (pH 5.5)] supplemented with 0.15% methionine was used as a selective medium for disrupting the Aoatg8 gene. CD medium lacking sodium nitrate (CDϪN) or CD medium containing 200 ng/ml rapamycin (CDϩR) was used for inducing autophagy.…”

Cell growth control: little eukaryotes make big contributions

“…At present, no bona fide targets of ATG1 are known and it is still a matter of debate whether ATG1 primarily controls maroautophagy via a phosphorylation event(s) or via a proposed structural role in autophagic complex formation Kamada et al, 2000;Abeliovich et al, 2003). Notably, macroautophagy is also regulated by additional protein kinases, such as PKA (Budovskaya et al, 2004), SNF1 (Wang et al, 2001) and GCN2 (Talloczy et al, 2002).…”

Autophagic Processes in Yeast: Mechanism, Machinery and Regulation

“…High levels of glucose result in the production of cAMP, which binds to, and inactivates, Bcy1, the regulatory subunit of PKA. As a consequence, PKA is activated and inhibits macroautophagy (Budovskaya et al 2004). PKA directly phosphorylates Atg1 and Atg13, at sites that are distinct from those targeted by TOR, and this posttranslational modification regulates the association of these proteins with the PAS (Budovskaya et al 2005;Stephan et al 2009).…”