TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response

Abstract

In Chlamydomonas (Chlamydomonas reinhardtii) the VESICLE-INDUCING PROTEINS 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 proxiome 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 (VPL1-11). 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.

Chlamydomonas: Fast tracking from genomics

“…Recently, protein-protein interactions have been deduced by proximity labeling, a technique that uses a genetically encoded biotin ligase fused to a protein of interest (interaction bait) that brings the ligase to its partner proteins that become biotinylated (Zhang, Li, et al, 2022). The use of proximity labeling in Chlamydomonas is nascent but has already established a pyrenoid interactome (Lau et al, 2023) and identified interactions of CGE1 and VIPP1/2 (Kreis et al, 2023) under oxidative stress (H 2 O 2 ) conditions with HSP70B, CDJ2, and proteins involved in the regulation of thylakoid biogenesis and photosynthetic electron transport.…”