An Evolutionary Proteomics Approach For The Identification Of Pka Targets In Saccharomyces Cerevisiae Identifies Atg1 And Atg13, Two Proteins That Play A Central Role In The Regulation Of Autophagy By The Ras/Pka Pathway And The Tor Pathway

A cell in its natural environment spends its time in a quiescent state known as G₀. A challenge the cell faces is to determine when the conditions are favorable for it to grow. The cell accomplishes this task through the combined actions of signaling pathways. These pathways link cells to the extracellular environment and act as molecular switches that engage the cell into a growth program when the conditions are favorable. In the budding yeast Saccharomyces cerevisiae, two signaling pathways that regulate growth in response to the availability of nutrients are the Tor kinase pathway and the Ras/cAMP dependent protein kinase (PKA) pathway.

In Chapter 2, we describe an evolutionary proteomics approach for the identification of PKA targets in S. Cerevisiae. In this method, we use a PKA target consensus site and mine the yeast proteome for occurrences of this consensus. Next, we ask if these sites have been conserved through evolution in related yeast species. We found that conservation of a PKA site correlated with the likelihood of that site being recognized by PKA. This approach identified 44 substrates of PKA in yeast. One subset of targets, the AuTophaGy-related proteins Atg1, Atg13 and Atg18, was involved in the process of autophagy. Autophagy has been implicated in a number of cellular processes including aging and cancer. In yeast, this process is fully induced following nutrient limitation and allows the cell to survive prolonged periods of starvation. Previous work in yeast has shown that both the Tor and the Ras/pathways negatively regulate autophagy during the normal phase of growth. We focus on one particular PKA target, Atg1, and show that Atg1 is regulated by PKA.

In chapter 3, we extend our analysis to Atg13. We find that this protein is a critical target of the Ras/PKA pathway. Atg13 is also regulated by the Tor pathway, and our data suggest that it might be a nexus of signal integration within the autophagy machinery. Tor and PKA appear to respond to different nutritional cues to provide separate inputs in the regulation of Atg13, thus regulating different aspects of the autophagy process.