Molekulare Biotechnologie & Systembiologie, RPTU Kaiserslautern-Landau, Paul-Ehrlich Straße 23, D-67663 Kaiserslautern, Germany.
Plant Physiol. 2023 Oct 26;193(3):1772-1796. doi: 10.1093/plphys/kiad335.
In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.
在衣藻（Chlamydomonas reinhardtii）中，质体囊泡诱导蛋白 1 和 2（VIPP1 和 VIPP2）在感知和应对膜应激以及类囊体膜生物发生中发挥作用。为了更深入地了解这些过程，我们旨在鉴定与质体中的 VIPP1/2 相互作用的蛋白质，并为此选择了邻近标记（PL）。我们使用核苷酸交换因子叶绿体 GRPE 同源物 1（CGE1）与基质热休克蛋白 70B（HSP70B）之间的瞬时相互作用作为测试系统。虽然 APEX2 和 BioID 的 PL 被证明效率低下，但 TurboID 导致体内大量生物素化。在常温和 H2O2 应激条件下，以 VIPP1/2 为诱饵的 TurboID 介导的 PL 证实了 VIPP1 与 VIPP2、HSP70B 和叶绿体 DNAJ 同源物 2（CDJ2）的已知相互作用。在 VIPP1/2 邻近组中鉴定的蛋白质可以分为参与类囊体膜复合物生物发生和光合作用电子传递调节的蛋白质，包括质子梯度调节 5 样蛋白 1（PGRL1）。第三组包括 11 种功能未知的蛋白质，它们的基因在叶绿体应激条件下上调。我们将它们命名为 VIPP 邻近标记（VPL）。在相互实验中，我们在 VPL2 和 PGRL1 的邻近组中证实了 VIPP1。我们的结果证明了 TurboID 介导的 PL 在研究衣藻质体中蛋白质相互作用网络方面的稳健性，并为分析 VIPP 在类囊体生物发生和应激反应中的功能铺平了道路。
