Evaluation of Translation Rate Through L-azidohomoalanine (AHA) Incorporation and Subsequent Alkyne Fluorophore-Mediated Click Chemistry in Yeast.

Abstract

Accurate measurement of protein translation rates is crucial for understanding cellular processes and disease mechanisms. However, existing methods for quantifying translation rates in yeast cells are limited. Here, we present a streamlined protocol for measuring protein translation rates in Saccharomyces cerevisiae using the methionine analog L-azidohomoalanine (AHA), which is the L isoform of this synthetic amino acid, and fluorophore-labeled alkyne dye-based Click chemistry. Our method involves incorporating AHA into newly synthesized proteins, followed by detection using confocal microscopy, flow cytometry, and SDS-PAGE. We validated our protocol by measuring translation rates under various stress conditions, including heat stress, endoplasmic reticulum (ER) stress induced by tunicamycin, and translation inhibition by cycloheximide. Confocal microscopy revealed differential AHA incorporation and fluorescence intensity across conditions. Flow cytometry quantitatively confirmed significant increases in translation rates under heat stress and decreases under ER stress compared to unstressed conditions at 6 and 24 h post-treatment. Imaging of gels under fluorescence detectors following SDS-PAGE further visualized newly synthesized proteins, with no detectable translation after cycloheximide treatment. Our protocol offers enhanced precision and selectivity compared to existing methods for mammalian cells and represents the first standardized approach for measuring translation rates in yeast. Despite limitations in required specialized equipment and expertise, this method holds promise for diverse applications in biotechnology and biomedical research, enabling investigations into protein synthesis regulation in yeast systems. Key features • This study presents the first standardized protocol for measuring protein translation in budding yeast using AHA and Click chemistry, addressing yeast-specific challenges effectively. • The study uses microscopy, flow cytometry, and fluorescence gel imaging to robustly validate yeast translation rates, ensuring reliable, reproducible results across cellular and biochemical levels. • The method detects translation changes under stress: increased with heat, decreased with ER stress, and halted by cycloheximide, highlighting its sensitivity for proteostasis research. • Despite requiring specialized equipment and expertise, the method offers valuable applications in biomedical research, metabolic engineering, and drug screening focused on protein homeostasis in yeast.