TurboID reveals the interaction network of VESICLE-INDUCING PROTEIN IN PLASTIDS (VIPPs) in Chlamydomonas reinhardtii
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ABSTRACT: In Chlamydomonas reinhardtii, VIPP1 and VIPP2 play a role 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 under ambient and H2O2 stress conditions and chose proximity labeling (PL) for this purpose. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in significant biotinylation in vivo, which could be enhanced by exogenously added biotin. Using TurboID-mediated PL with VIPP1/2 as baits we could confirm known interactions of VIPP1 with VIPP2, HSP70B and CDJ2. Novel proteins in the VIPP1/2 interaction network can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport. A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named these proteins VIPP PROXIMITY LABELING (VPL1-11) and confirmed the proximity of VIPP1 and VPL2 in a reciprocal experiment. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for targeted analyses of the functions of VIPPs in thylakoid biogenesis and chloroplast stress responses.
INSTRUMENT(S): TripleTOF 6600
ORGANISM(S): Chlamydomonas Reinhardtii
SUBMITTER: Frederik Sommer
LAB HEAD: Michael Schroda
PROVIDER: PXD038515 | Pride | 2024-01-26
REPOSITORIES: Pride
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