Dataset Information

TurboID reveals the interaction network of VESICLE-INDUCING PROTEIN IN PLASTIDS (VIPPs) in Chlamydomonas reinhardtii

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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			Action	DRS
	20201218MS201MSek001.wiff	Wiff
	20201218MS201MSek001.wiff.scan	Wiff
	20201218MS201MSek002.wiff	Wiff
	20201218MS201MSek002.wiff.scan	Wiff
	20201218MS201MSek003.wiff	Wiff

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Publications

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

Kreis Elena E König Katharina K Misir Melissa M Niemeyer Justus J Sommer Frederik F Schroda Michael M

Plant physiology 20231001 3

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 ...[more]

PMID: 37310689

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Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.