The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation

Abstract

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Secmediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knockout mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression

“…Immunodetection was performed by enhanced chemiluminescence (ECL) using the FUSION‐FX7 Advance™ imaging system (PeqLab). Antisera were against VIPP1 (Liu et al, ), VIPP2 (this study), HSP70B and CGE1 (Schroda, Vallon, Whitelegge, Beck, & Wollman, ), CF1β (Lemaire & Wollman, ), LHCA2 (Agrisera AS01 006), PsbA (Agrisera AS05 084), SECA (Schroda M., unpublished), HSP22F (Rütgers et al, ), DEG1C (Theis, Lang, et al, ), HA (Abcam ab137838), PsaA (Agrisera AS06 172), cytochrome f (Pierre & Popot, ), HSP90C (Willmund & Schroda, ), and LHCSR3 (Naumann et al, ). Anti‐rabbit‐HRP (Sigma‐Aldrich) was used as secondary antibody.…”

“…Chlamydomonas VIPP1 and VIPP2 have several properties in common. They share that their expression is induced in HL (Figure a; Nordhues et al, ; Perlaza et al, ), after the addition of H 2 O 2 (Figure b), upon depletion of the chloroplast proteases ClpP and DEG1C (Perlaza et al, ; Ramundo et al, ; Theis, Lang, et al, ), and when the translocation or integration of thylakoid membrane proteins is impaired (Figure d,e; Göhre et al, ). VIPP1 and VIPP2 also share the ability to form rod‐like structures in vitro (Figure b,c; Liu et al, ; Theis, Gupta, et al, ).…”

“…Strikingly, it resembled that of VIPP1, with ~43% of HSP22E/F present in the pellet and ~57% in the soluble fraction. We included DEG1C in our analysis, the major chloroplast Deg‐type protease that localizes to the stroma and thylakoid membranes (Theis, Lang, et al, ). Compared with VIPP2, HSP22E/F, and VIPP1, a lower percentage of DEG1C was found in the pellet fraction (~33%; Figure b).…”

“…At chloroplast membranes of HL‐ or H 2 O 2 ‐exposed cells, VIPP1/2 might organize membrane domains that serve as interfaces between membrane and soluble chaperones and proteases for the handling of oxidized, unfolded membrane proteins. DEG1C might be such a protease, as it accumulates under the same stress conditions as VIPP2 and localizes one third of its pool to chloroplast membranes (Figure b; Theis, Lang, et al, ). In fact, Arabidopsis Hsp21 has been shown to maintain the integrity of thylakoid membranes in the gun5 mutant during heat stress, apparently by stabilizing PSII through direct interaction (Chen, He, Chen, & Guo, ).…”

Ascorbate peroxidase 4 plays a role in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress

“…They also find that only one chloroplastic APX protein, that is, CrAPX1, exists in Chlamydomonas and no APX isoforms can be detected in cytosolc and peroxisome. Further, the proteomic investigation of the Chlamydomonas deg1c mutant lacking DEG1C protease activity shows an increase of CrAPX4 protein in the chloroplast stroma 26 . It demonstrates that, in addition to CrAPX1, CrAPX4 also exists in Chlamydomonas chloroplast.…”