Autophagy Stimulus-Dependent Role of the Small GTPase Ras2 in Peroxisome Degradation

Cells adjust their metabolism in response to nutrient availability. mTOR and PKA are key regulators of growth and degradation, primarily activated by nitrogen and glucose, respectively. While their roles in bulk autophagy are well understood, their influence on selective autophagy remains unclear. Selective autophagy pathways, such as peroxisome degradation (pexophagy), involve more specific regulatory mechanisms. Prior research has shown that mTOR and PKA promote cell growth while suppressing degradation. However, the role of the Ras2 GTPase, a key component of the PKA pathway, in selective autophagy has remained unexplored. This gap motivated the study of Ras2's function in peroxisome degradation. No prior work had resolved how Ras2 activity interacts with mTOR inhibition and glucose sensing. The study aimed to clarify this context-dependent role, particularly in relation to pexophagy. The Ras2 protein is homologous to human Ras, which is linked to cancer, making its function in autophagy biologically relevant. Understanding Ras2’s role could provide insights into both yeast biology and human disease mechanisms.

The study aimed to determine how Ras2 activity influences peroxisome degradation under different nutrient conditions. Specifically, it focused on the interaction between Ras2 and mTOR inhibition, as well as glucose sensing. The researchers sought to clarify whether Ras2 promotes or suppresses pexophagy depending on the type of mTOR inhibition. They wanted to test if direct mTOR inhibition via rapamycin and indirect inhibition via nutrient shift affect peroxisome degradation differently. The study also aimed to assess how glucose availability modulates Ras2’s role in pexophagy. By comparing two distinct mTOR inhibition methods, the researchers hoped to identify context-specific functions of Ras2. Their goal was to determine if Ras2 acts as an inhibitor of peroxisome degradation in glucose-rich conditions or as a facilitator in nitrogen-limited environments. This would help clarify the signaling pathways involved in selective autophagy.

The researchers used the yeast model system to study peroxisome degradation. They employed two types of mTOR inhibition: direct inhibition with rapamycin and indirect inhibition via nutrient shift. Cells were grown in oleate medium, which lacks glucose, and then transferred to pexophagy medium containing glucose and limited nitrogen. The study monitored peroxisome degradation using fluorescent markers and microscopic analysis. Ras2 activity was manipulated through genetic inactivation or overexpression to assess its impact on pexophagy. The researchers compared peroxisome degradation levels in wild-type cells and Ras2-mutant cells under both types of mTOR inhibition. They also evaluated how glucose availability influenced Ras2’s role in pexophagy. The study combined genetic, biochemical, and imaging techniques to track autophagy dynamics. This approach allowed them to determine whether Ras2 acts as an inhibitor or a facilitator of peroxisome degradation under different conditions.

When mTOR was inhibited directly with rapamycin, peroxisome degradation was partially suppressed by active Ras2. In contrast, inactivating Ras2 led to increased peroxisome degradation, suggesting a role in suppressing pexophagy. This result implies that Ras2 may act as an inhibitor of peroxisome degradation in glucose-rich conditions. However, when mTOR inhibition occurred via nutrient shift from oleate to pexophagy medium, Ras2 activity was required for efficient peroxisome degradation. In this context, inactivating Ras2 reduced pexophagy, indicating a facilitatory role. The study found that the role of Ras2 depends on the type of mTOR inhibition and glucose availability. In glucose-grown cells, Ras2 suppresses peroxisome degradation, while in nitrogen-limited conditions, it promotes it. These findings suggest a context-dependent function of Ras2 in pexophagy. The results highlight the importance of glucose sensing in modulating Ras2’s activity during selective autophagy.

The study concludes that Ras2 has a stimulus-dependent role in peroxisome degradation. In glucose-rich conditions, Ras2 suppresses pexophagy when mTOR is inhibited directly. However, when mTOR inhibition occurs via nutrient shift to a glucose-containing, nitrogen-limited medium, Ras2 activity is required for efficient degradation. These findings suggest that Ras2 functions differently depending on the type of mTOR inhibition and glucose availability. The authors propose that the role of Ras2 in glucose sensing-associated signaling is more important in the nutrient shift context than in mTOR-related autophagy inhibition. The study highlights the importance of context in determining Ras2’s function in selective autophagy. The results support the idea that Ras2 acts as a modulator of peroxisome degradation rather than a universal inhibitor or activator. The findings may have broader implications for understanding how GTPase signaling regulates selective autophagy in response to environmental cues.