确定酿酒酵母菌整体内质网膜蛋白拓扑结构的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-04-03 DOI:10.1016/j.xpro.2025.103727

Ritika Chaudhary, Vineet Choudhary

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引用次数: 0

摘要

在这里，我们提出了一种方案来确定Fld1（少量脂滴）的拓扑结构，Fld1是酿酒酵母中的一种完整的内质网（ER）膜蛋白。我们描述了生成功能性n端GFP和c端mCherry与Fld1融合的步骤。我们详细介绍了进行亚细胞分离以分离er源性微粒体的策略，这些微粒体受到盐洗涤剂提取分析。然后，我们提供了使用蛋白酶K处理确定Fld1拓扑结构的程序。

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Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.

Here, we present a protocol to determine the topology of Fld1 (few lipid droplets), an integral endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae. We describe steps to generate functional N-terminal GFP and C-terminal mCherry fusion with Fld1. We detail the strategy to perform subcellular fractionation to isolate ER-derived microsomes that were subjected to salt detergent extraction analysis. We then provide procedures to determine the topology of Fld1 using proteinase K treatment.

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