CryoAPEX-用于膜蛋白亚细胞定位的电子断层成像工具。
CryoAPEX - an electron tomography tool for subcellular localization of membrane proteins.
机构信息
Department of Biological Sciences, Purdue University, 915 W. State St., LILY G-227, West Lafayette, IN 47907, USA.
Department of Biological Sciences, Purdue University, 915 W. State St., LILY G-227, West Lafayette, IN 47907, USA
出版信息
J Cell Sci. 2019 Mar 18;132(6):jcs222315. doi: 10.1242/jcs.222315.
We describe a method, termed cryoAPEX, which couples chemical fixation and high-pressure freezing of cells with peroxidase tagging (APEX) to allow precise localization of membrane proteins in the context of a well-preserved subcellular membrane architecture. Further, cryoAPEX is compatible with electron tomography. As an example, we apply cryoAPEX to obtain a high-resolution three-dimensional contextual map of the human FIC (filamentation induced by cAMP) protein, HYPE (also known as FICD). HYPE is a single-pass membrane protein that localizes to the endoplasmic reticulum (ER) lumen and regulates the unfolded protein response. Alternate cellular locations for HYPE have been suggested. CryoAPEX analysis shows that, under normal and/or resting conditions, HYPE localizes robustly within the subdomains of the ER and is not detected in the secretory pathway or other organelles. CryoAPEX is broadly applicable for assessing both lumenal and cytosol-facing membrane proteins.
我们描述了一种称为 cryoAPEX 的方法，该方法将细胞的化学固定和高压冷冻与过氧化物酶标记（APEX）相结合，以允许在保存良好的亚细胞膜结构背景下精确定位膜蛋白。此外，cryoAPEX 与电子断层扫描兼容。作为一个例子，我们应用 cryoAPEX 来获得人类 FIC（cAMP 诱导的丝状）蛋白 HYPE（也称为 FICD）的高分辨率三维上下文图谱。HYPE 是一种单次跨膜蛋白，定位于内质网（ER）腔并调节未折叠蛋白反应。已经提出了 HYPE 的替代细胞位置。cryoAPEX 分析表明，在正常和/或休息条件下，HYPE 在 ER 的亚域中强烈定位，并且在分泌途径或其他细胞器中检测不到。cryoAPEX 广泛适用于评估腔和细胞质面向的膜蛋白。
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