FtsH And CpSRP54 Cooperate Thylakoid Membrane Associated Proteostasis In Arabidopsis Thaliana

The thylakoid membrane-associated proteostasis plays an essential role in the chloroplast development during de-etiolation,including the translocation and assembly of membrane proteins and the degradation of damaged or unassembled proteins.It ensures efficient photosynthesis through protein quality control(PQC)which ensures the correct assembly of photosynthesis proteins with cofactors such as photosynthetic pigments by protein synthesis and degradation.However,the regulation of thylakoid membrane-associated proteostasis is largely unknown and the effective trackable substrates for monitoring this process are still lacked.In this study,LhcB2,a subunit of the Light-harvesting antenna complex in PSII,was selected as the target.And a heterogeneous LhcB2-GFP fusion protein was expressed in Arabidopsis as a substrate and detection target for the thylakoid membrane-associated proteostasis through the generation of WT LhcB2-GFP transgenic strain.The following results were obtained:1)Using western blot and 1-D BN-PAGE followed by 2-D SDS-PAGE,we found that LhcB2-GFP(～55 k Da)was dysfunctional and degraded to a short-form d LhcB2-GFP(～38 k Da)through an N-terminal degradation initiated on thylakoid membranes during de-etiolation.Hence,we decided to use LhcB2-GFP and d LhcB2-GFP as a trackable substrate for monitoring thylakoid membrane-associated proteostasis by analyzing its expression.2)We crossed WT LhcB2-GFP transgenic strain with diverse mutants of chloroplast protease defection to explore the proteases involved in the degradation of LhcB2-GFP to d LhcB2-GFP during the thylakoid membrane-associated proteostasis.The degradation of LhcB2-GFP to form d LhcB2-GFP was disrupted in the AtFtsH2 mutants var2-3 and var2-4.By analyzing the expression of LhcB2-GFP in var2 mutants,we revealed that the activity of the ATPase domain of VAR2/AtFtsH2 could affect the degradation of LhcB2-GFP.The yeast two-hybrid assay showed that the N-terminus degradation peptide of LhcB2-GFP interacts with the M41 protease domain of VAR2/AtFtsH2.Besides,over-accumulation of LhcB2-GFP in var2-3 and var2-4 formed protein aggregates which were insoluble in nonionic detergents.3)We also crossed WT LhcB2-GFP transgenic strain with diverse mutants of defection in chloroplast development to find out other chloroplast factors involved in the LhcB2-GFP homeostasis.And the factors that may be involved were screened by analyzing the expression of LhcB2-GFP in those mutants during de-etiolation.The degradation of LhcB2-GFP to form d LhcB2-GFP was also disrupted in pga4-1 mutant.And over-accumulation of LhcB2-GFP in pga4-1 and pga4-2 also formed protein aggregates which were insoluble in nonionic detergents.Allelism test with pga4-1 confirmed that pga4-2 is an allele mutant of PGA4/cpSRP54.4)Genetic assay showed that pga4-1 suppressed the variegated leaf phenotype of the var2 mutant.The pga4-1 var2-4 mutant was recovered to the var2-4 mutant phenotype using the binary construct ProcpSRP54:cpSRP54-GFP.This suggests the genetic interaction between PGA4/cpSRP54 and VAR2/AtFtsH2,and that pga4-1 is a suppressor mutant of the var2 variegated phenotype.5)Above all,we proposed a model that VAR2/AtFtsH2 and PGA4/cpSRP54 coordinate thylakoid membrane-associated proteostasis during de-etiolation in Arabidopsis.