Phosphorylation of the C-terminus of the α-Arrestin Aly3 Regulates Glucose Transporter Ght5 Localization and Cell Proliferation in Fission Yeast under Low Glucose Conditions

Core Concepts

Phosphorylation of the C-terminal serine residues of the α-arrestin Aly3 by the TORC2 pathway inhibits Aly3-mediated ubiquitination and vacuolar transport of the glucose transporter Ght5, thereby ensuring cell-surface localization of Ght5 and enabling cell proliferation under low glucose conditions in fission yeast.

Abstract

The study investigates the molecular mechanisms by which fission yeast cells adapt to low glucose conditions. The key findings are: Phosphorylation of the C-terminal serine residues of the α-arrestin Aly3, but not the glucose transporter Ght5, is required for cell proliferation under low glucose conditions. Cells expressing a phosphorylation-defective Aly3 mutant exhibit increased ubiquitination and vacuolar localization of the glucose transporter Ght5, leading to defective cell proliferation in low glucose. Phosphorylation of at least one of the three C-terminal serine residues (S582, S584, S585) of Aly3 is sufficient to maintain Ght5 at the cell surface and enable cell proliferation under low glucose. Aly3 physically interacts with the HECT-type ubiquitin ligases Pub1 and Pub3, and these interactions are required for Ght5 surface localization and cell proliferation in low glucose. Deletion of the pub3 gene partially suppresses the proliferation defect of the TORC2 pathway mutant gad8ts under low glucose conditions, suggesting that Pub3-mediated ubiquitination of Ght5 acts downstream of the TORC2 pathway. The study reveals how the TORC2 pathway regulates the α-arrestin Aly3 via phosphorylation to control glucose transporter localization and enable fission yeast cells to adapt to low glucose conditions.

Stats

Cells expressing the phosphorylation-defective ght5(ST11A) mutant proliferated in low glucose conditions, similar to cells expressing the wild-type ght5 gene. Cells expressing the phosphorylation-defective aly3(ST18A) or aly3(4th A) mutants failed to proliferate in low glucose conditions. Reverting one of the alanine residues in the aly3(4th A) mutant back to serine (aly3(4th A;A584S)) restored cell proliferation in low glucose. Ght5 exhibited increased ubiquitination and vacuolar localization in cells expressing the aly3(ST18A) or aly3(4th A) mutants compared to cells expressing wild-type Aly3. Deletion of the pub3 gene partially suppressed the proliferation defect of the gad8ts mutant under low glucose conditions.

Quotes

"Phosphorylation of Aly3 at the C-terminus attenuates ubiquitination and subsequent vacuolar transport of the high-affinity glucose transporter, Ght5, ensuring cell proliferation in low glucose." "Cells expressing the mutant Aly3 protein in which potentially phosphorylatable serine residues are replaced with alanine, i.e., Aly3(ST18A) and Aly3(4th A), exhibited massive accumulation of Ght5 in vacuoles as well as defective cell proliferation in low glucose, suggesting that dephosphorylation activates Aly3 as an adaptor of a ubiquitin ligase for Ght5, which in turn promotes transport of Ght5 from the cell surface to vacuoles." "Essentially the same defective phenotypes caused by expression of Aly3(4th A) were restored by reverting one of the mutated residues back to serine, i.e., Aly3(4th A;A584S). These two analogous results of phenotype restoration suggest that the TORC2 pathway mediates phosphorylation of Aly3 at the C-terminus in low glucose."

Key Insights Distilled From

Phosphorylation of Aly3 C-terminus impedes aberrant endocytosis of S. pombe hexose transporter Ght5

by Toyoda,Y., M... at www.biorxiv.org 03-27-2024

https://www.biorxiv.org/content/10.1101/2024.03.24.586504v1

Deeper Inquiries

How do changes in nitrogen availability affect the phosphorylation status of Aly3 and the localization of the glucose transporter Ght5?

In response to changes in nitrogen availability, the phosphorylation status of Aly3 and the localization of the glucose transporter Ght5 are intricately regulated. Under conditions of nitrogen starvation, TORC1 is inactivated, leading to decreased phosphorylation of Aly3 at the C-terminus. This dephosphorylation likely occurs due to increased phosphatase activity, such as that of PP2A, which is negatively regulated by TORC1. The dephosphorylated state of Aly3 promotes its interaction with the glucose transporter Ght5, facilitating ubiquitination and subsequent vacuolar transport of Ght5. This process ensures efficient nitrogen acquisition through the degradation of biomolecules in vacuoles. Conversely, in the presence of nitrogen, TORC2 is active and phosphorylates Aly3 at specific serine residues, such as S584, inhibiting its interaction with Ght5. This phosphorylation prevents ubiquitination of Ght5, maintaining its localization on the cell surface and enabling cell proliferation in low glucose.

How do changes in nitrogen availability affect the phosphorylation status of Aly3 and the localization of the glucose transporter Ght5?

The phosphorylation-dependent activity of the α-arrestin Aly3 likely regulates various cellular processes and signaling pathways beyond the localization of the glucose transporter Ght5. α-arrestins, including Aly3, are known to act as ubiquitin ligase adaptors for specific membrane proteins, promoting their ubiquitination and subsequent endocytosis. Therefore, the phosphorylation status of Aly3 may impact the turnover and localization of other membrane proteins involved in nutrient uptake, signaling, or cellular homeostasis. Additionally, α-arrestins have been implicated in regulating endocytosis of G-protein-coupled receptors (GPCRs) and other signaling receptors, suggesting a broader role in modulating signal transduction pathways. The phosphorylation-dependent regulation of Aly3 could potentially influence the dynamics of these signaling cascades, affecting cellular responses to environmental cues and stress conditions.

Could the insights from this study on the regulation of nutrient transporter localization be applied to understand adaptations in other eukaryotic cell types or organisms?

The findings from this study on the regulation of nutrient transporter localization through the phosphorylation-dependent activity of Aly3 provide valuable insights that could be extrapolated to understand adaptations in other eukaryotic cell types or organisms. The mechanisms uncovered, such as the role of TORC2 signaling in phosphorylating Aly3 to inhibit ubiquitination of nutrient transporters, are likely to be conserved across different species. By studying the orthologs of Aly3 and related signaling pathways in other organisms, researchers can gain a deeper understanding of how cells adapt to changing nutrient conditions. This knowledge could be particularly relevant in the fields of cell biology, metabolism, and physiology, shedding light on the molecular mechanisms underlying nutrient sensing and utilization in diverse biological systems.