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

Abstract

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.

TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 inChlamydomonas reinhardtii

“…PL confirms the interaction of VIPP1 with VIPP2 at chloroplast membranes under oxidative stress shown previously by AP-MS (Theis et al, 2020). CLPB3 in the VIPP1 proxiome under oxidative stress conditions supports the idea that CLPB3 may aid in the removal of protein aggregates from thylakoid membranes, as proposed previously based on localization data (Kreis et al, 2022). CDJ2 in the VIPP2 proxiome suggests that the assembly state of VIPP2, like that of VIPP1, is controlled by the HSP70B/CDJ2/CGE1 chaperone system (Liu et al, 2007) (notice that VIPP2 forms rods like VIPP1 (Theis et al, 2020)).…”

“…CLPB3 was enriched with VIPP1-TurboID only in Setup 3 and only under H 2 O 2 stress. It functions as a protein disaggregase in the chloroplast and localizes to punctae situated next to the thylakoid membrane system (Kreis et al, 2022). Of the remaining 13 genes, only two encoded proteins with a clear functional annotation: CHLP1, which was enriched with VIPP1- and VIPP2-TurboID only under ambient conditions, and LPA3 (LOW PS II ACCUMULATION 3), which was enriched only with VIPP1 under H 2 O 2 stress.…”

“…Sample amounts loaded were based on protein determination as described by (Bradford, 1976) or based on chlorophyll concentrations (Porra et al, 1989). Antisera used were against BirA (this work), CDJ1 (Willmund et al, 2008), CGE1 (Schroda et al, 2001), CLPB3 (Kreis et al, 2022), DEG1C (Theis et al, 2019b), the HA epitope (Sigma-Aldrich H3663), HSP22E/F (Rütgers et al, 2017), HSP70B (Schroda et al, 1999), HSP90C (Willmund and Schroda, 2005), mCherry (Crozet et al, 2018), RPL1 (Ries et al, 2017), and VIPP1 (Liu et al, 2005). Anti-rabbit-HRP (Sigma-Aldrich) and anti-mouse-HRP (Santa Cruz Biotechnology sc-2031) were used as secondary antibodies.…”