TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 inChlamydomonas 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 and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CGE1 and stromal HSP70B as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in significant 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 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 them 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 analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

Proximity labelling allows to study novel factors in chloroplast developmenta

“…Under these conditions we observe that plastid targeted TID mediates the enrichment of the majority of proteins after SA pulldowns compared to a Col0 control (Figure 4 and Figure S3). Done in parallel, two independent studies report functional TID-dependent proximity labelling in plastids of Chlamydomonas reinhardtii (Lau et al, submitted; Kreis et al, submitted). Together, these data suggest that TID-based proximity labelling works in plastids of diverse species.…”

“…At the same time, we also observe important differences between these species. Whereas for Arabidopsis plastids we find addition of 50 μM exogenous biotin enough to obtain sufficient TID dependently enriched proteins for analysis, both studies in Chlamydomonas report the need of supplementing exogenous biotin between 0.5 and 1 mM for efficient labelling inside plastid stroma (Kreis et al, submitted). In case of pyrenoid localised TID labelling even concentrations > 1mM were found to be necessary (Lau et al, submitted).…”

“…In their manuscripts, Kreis and colleagues as well as Lau and colleagues observe that high expression of TID leads to alterations in the endogenous biotinylation pattern. This effect was attributed to competition between TID and the endogenous biotin ligases for endogenous biotin as it could be largely reverted by addition of exogenous biotin (Kreis et al, submitted; Lau et al, submitted). In Arabidopsis we were not able to see such an obvious effect, and both cgl20a-TID and plastid-TID lines did not show any obvious phenotype compared to Col0 after 4 generations of propagation indicating that constitutive presence of TID, at least at moderate levels, does not severely interfere with endogenously required biotinylation.…”

CLPB3 is required for the removal of chloroplast protein aggregates and thermotolerance in Chlamydomonas

“…via DEG1C. In fact, CLPB3 was found in the proxiome of VIPP1 in cells exposed to oxidative stress ( Kreis et al , 2022 ). Moreover, cytosolic HSP101 has been shown to cooperate with the proteasome system, albeit only on a small sub-set of aggregated proteins, while refolding was the preferred path ( McLoughlin et al , 2019 ).…”

RETICULON-LIKE PROTEIN B2 is a proviral factor co-opted for the biogenesis of viral replication organelles in plants

“…Therefore, technically, it is difficult to determine whether the enriched peptides corresponding to p23 actually originated from p23 or from the p82 replicase. In addition, since the p82 protein is produced by read-through of p23, its expression level is very low, and there is often a bias against low abundance proteins during PL analysis ( Kreis et al 2022 ). All these factors may contribute to the low fold change value of p82 in the MS data set even though it interacts with p23.…”