Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. The current methods for detecting GTPase activation using fluorescent proteins rely on the interaction between the GTPase and its effector. Consequently, these methods are not applicable to factors, such as Sar1, where the effector also functions as a GTPase-activating protein. Here, we present a novel method, the Small GTPase ActIvitY ANalyzing (SAIYAN) system, for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at the ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion confined the activated Sar1 at the ER exit sites, implying the importance of Sar1 activation through the ERGIC in collagen secretion.

中文翻译：

---

小 GTPase ActIvitY 分析 (SAIYAN) 系统：一种检测活细胞中 GTPase 激活的方法。


小 GTP 酶在各种细胞信号传导途径中至关重要，检测它们在活细胞内的激活对于理解细胞过程至关重要。目前使用荧光蛋白检测 GTP 酶激活的方法依赖于 GTP 酶与其效应子之间的相互作用。因此，这些方法不适用于 Sar1 等因子，其中效应子还充当 GTP 酶激活蛋白。在这里，我们提出了一种新方法，即小 GTPase ActIvitY 分析 (SAIYAN) 系统，用于利用 split mNeonGreen 系统通过荧光信号检测内源性小 GTPase 的激活。我们证明了 Sar1 在内质网 (ER) 出口位点的激活，并成功检测了其在各种细胞条件下的激活状态。利用胶原蛋白分泌细胞中的 SAIYAN 系统，我们发现激活的 Sar1 位于 ER 出口位点和 ER-高尔基中间室 (ERGIC) 区域。此外，胶原蛋白分泌受损将激活的 Sar1 限制在 ER 出口位点，这意味着通过 ERGIC 激活 Sar1 在胶原蛋白分泌中的重要性。