Kreis, E. | Search bioRxiv

bioRxiv2022

TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 in Chlamydomonas reinhardtii

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.

plant biology↗

bioRxiv2022

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

In the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two Chlamydomonas reinhardtii clpb3 mutants. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in protease capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to signals from HSP22E/F, originating largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system. HighlightChloroplast CLPB3 in Chlamydomonas is required for resolving heat-induced protein aggregates and this activity confers thermotolerance under severe heat stress. During heat stress, CLPB3 organizes into stromal foci located next to the thylakoid membrane system, indicating a role for CLPB3 in disentangling protein aggregates from there.

plant biology↗