Novel TORC1 inhibitor Ecl1 is regulated by phosphorylation in fission yeast

Area of Science:

• Cellular Biology
• Molecular Biology
• Yeast Genetics

Background:

• Extender of chronological lifespan 1 (Ecl1) is crucial for cellular stress responses, including starvation, in fission yeast.
• Ecl1's inhibition of the target of rapamycin complex 1 (TORC1) pathway is known, but its posttranslational modifications remain largely uncharacterized.

Purpose of the Study:

• To investigate the impact of posttranslational modifications, specifically phosphorylation, on Ecl1 regulation and function.
• To elucidate the relationship between Ecl1 phosphorylation, stress response, and TORC1 inhibition.

Main Methods:

• Mass spectrometry was employed to analyze Ecl1 phosphorylation levels in yeast under sulfur or metal starvation.
• Phosphorylation-mimetic mutations were created to assess the functional consequences of Ecl1 phosphorylation at Thr7.
• Co-immunoprecipitation or similar techniques were used to investigate physical interactions between Ecl1 and TORC1 components.

Main Results:

• Mass spectrometry identified Thr7 as a phosphorylation site on Ecl1, with levels decreasing under starvation conditions.
• A phosphorylation-mimetic mutation at Thr7 impaired Ecl1's role in starvation response, indicating suppression of function by phosphorylation.
• Ecl1 inhibited TORC1 independently of Thr7 phosphorylation status, even upon overexpression of the phosphorylation-mimetic mutant.
• A physical interaction between Ecl1 and the TORC1 subunit RAPTOR (Mip1) was newly identified.

Conclusions:

• Ecl1 exhibits dual functional modes: quantity-dependent inhibition of TORC1 and Thr7 phosphorylation-dependent control over cellular functions.
• Thr7 phosphorylation acts as a negative regulator of Ecl1's contribution to cellular stress adaptation.
• Ecl1's interaction with RAPTOR (Mip1) provides a mechanistic link for its TORC1 inhibition.