Jha Mainak Pratim, Mapa Koyeli
Protein Homeostasis Laboratory, Department of Life Sciences, School of Natural Sciences, Shiv Nadar Institution of Eminence, Delhi-NCR, India.
Bio Protoc. 2025 Jul 20;15(14):e5379. doi: 10.21769/BioProtoc.5379.
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 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.
准确测量蛋白质翻译速率对于理解细胞过程和疾病机制至关重要。然而，现有用于量化酵母细胞中翻译速率的方法存在局限性。在此，我们提出了一种简化的方案，用于使用甲硫氨酸类似物L-叠氮高丙氨酸（AHA）（该合成氨基酸的L异构体）和基于荧光团标记炔烃染料的点击化学来测量酵母中的蛋白质翻译速率。我们的方法包括将AHA掺入新合成的蛋白质中，然后使用共聚焦显微镜、流式细胞术和SDS-PAGE进行检测。我们通过在各种应激条件下测量翻译速率来验证我们的方案，这些应激条件包括热应激、衣霉素诱导的内质网（ER）应激以及环己酰亚胺引起的翻译抑制。共聚焦显微镜揭示了不同条件下AHA掺入和荧光强度的差异。流式细胞术定量证实，与未应激条件相比，在处理后6小时和24小时，热应激下翻译速率显著增加，ER应激下翻译速率降低。SDS-PAGE后在荧光检测器下对凝胶进行成像进一步可视化了新合成的蛋白质，环己酰亚胺处理后未检测到翻译。与现有的哺乳动物细胞方法相比，我们的方案具有更高的精度和选择性，并且是测量酵母中翻译速率的首个标准化方法。尽管在所需的专业设备和专业知识方面存在局限性，但该方法在生物技术和生物医学研究中有多种应用前景，能够对酵母系统中的蛋白质合成调控进行研究。关键特性 • 本研究提出了首个使用AHA和点击化学测量芽殖酵母中蛋白质翻译的标准化方案，有效解决了酵母特有的挑战。 • 该研究使用显微镜、流式细胞术和荧光凝胶成像来有力地验证酵母翻译速率，确保在细胞和生化水平上获得可靠、可重复的结果。 • 该方法可检测应激下的翻译变化：热应激时增加，ER应激时降低，环己酰亚胺使其停止，突出了其对蛋白质稳态研究的敏感性。 • 尽管需要专业设备和专业知识，但该方法在专注于酵母蛋白质稳态的生物医学研究、代谢工程和药物筛选中具有重要应用价值。
