Differential Roles of the Thylakoid Lumenal Deg Protease Homologs in Chloroplast Proteostasis

Abstract

ORCID IDs: 0000-0002-7279-0246 (Y.L.); 0000-0001-6202-5826 (Z.A.)Deg proteases are involved in protein quality control in prokaryotes. Of the three Arabidopsis (Arabidopsis thaliana) homologs, Deg1, Deg5, and Deg8, located in the thylakoid lumen, Deg1 forms a homohexamer, whereas Deg5 and Deg8 form a heterocomplex. Both Deg1 and Deg5-Deg8 were shown separately to degrade photosynthetic proteins during photoinhibition. To investigate whether Deg1 and Deg5-Deg8 are redundant, a full set of Arabidopsis Deg knockout mutants were generated and their phenotypes were compared. Under all conditions tested, deg1 mutants were affected more than the wild type and deg5 and deg8 mutants. Moreover, overexpression of Deg5-Deg8 could only partially compensate for the loss of Deg1. Comparative proteomics of deg1 mutants revealed moderate up-regulation of thylakoid proteins involved in photoprotection, assembly, repair, and housekeeping and down-regulation of those that form photosynthetic complexes. Quantification of protein levels in the wild type revealed that Deg1 was 2-fold more abundant than Deg5-Deg8. Moreover, recombinant Deg1 displayed higher in vitro proteolytic activity. Affinity enrichment assays revealed that Deg1 was precipitated with very few interacting proteins, whereas Deg5-Deg8 was associated with a number of thylakoid proteins, including D1, OECs, LHCBs, Cyt b 6 f, and NDH subunits, thus implying that Deg5-Deg8 is capable of binding substrates but is unable to degrade them efficiently. This work suggests that differences in protein abundance and proteolytic activity underlie the differential importance of Deg1 and Deg5-Deg8 protease complexes observed in vivo.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation

“…Impaired protein quality control at thylakoid membranes might be alleviated, but not fully compensated, by the upregulation of the membrane-intrinsic FtsH protease by ;45%. In the Arabidopsis deg1 mutant, FtsH proteases were upregulated as well (Butenko et al, 2018). In the wild type, high light conditions could induce the accumulation of misfolded and unassembled proteins through an increased rate of PSII repair and through protein oxidation.…”

“…Based on this observation and the finding that Deg1 interacted with the D2 protein, the authors also suggested a chaperone role for Deg1 in PSII assembly. In the Arabidopsis deg1 knockout mutant, the abundance of all complexes of the photosynthetic light reactions was reduced by 15% to 30% (Butenko et al, 2018).…”

“…Since most of these upregulated proteins are not localized in the stroma, they cannot be substrates of DEG1C and must be upregulated via signaling pathways responding to decreased DEG1C activity. Note that in the Arabidopsis deg1 mutant, proteins involved in photoprotection were upregulated as well (Butenko et al, 2018).…”

“…There, Deg2 and Deg7 are associated with the stromal side of thylakoid membranes (Haussühl et al, 2001;Sun et al, 2010a), while Deg1, Deg5, and Deg8 are associated with the lumenal side (Itzhaki et al, 1998;Sun et al, 2007). Deg5 lacks a PDZ domain and forms heterooligomeric complexes with Deg8 in a 1:1 stoichiometry (Sun et al, 2007;Butenko et al, 2018). The proteolytic activity of Deg1 and Deg2 was shown to depend on the redox state, with Deg1 being more active under reducing conditions and Deg2 more active under oxidizing conditions (Ströher and Dietz, 2008).…”

Chlorophyll catabolism precedes changes in chloroplast structure and proteome during leaf senescence

“…Mass spectrometry was performed at the De Botton Protein Profiling institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, essentially as recently described (Butenko et al., ). Samples were subjected to in‐solution tryptic digestion using a modified Filter Aided Sample Preparation protocol (FASP).…”