Coordinated Regulation and Complex Formation of YELLOW VARIEGATED1 and YELLOW VARIEGATED2, Chloroplastic FtsH Metalloproteases Involved in the Repair Cycle of Photosystem II in Arabidopsis Thylakoid Membranes

Abstract

Arabidopsis YELLOW VARIEGATED1 ( VAR1 ) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes ( ftsh1 , ftsh6 , and ftsh8 ) closely related to VAR1 and VAR2 . This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1-and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of ‫ف‬ 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1-and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.

Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases

“…The pea homolog of FtsH3 was detected in mitochondria but not in chloroplasts (Kolodziejczak et al, 2002). Transient expression assays of green fluorescent protein fusions revealed that Arabidopsis FtsH3, 4, and 10 were indeed targeted to mitochondria, whereas all other nine FtsHs were targeted to the chloroplast (Takechi et al, 2000;Sakamoto et al, 2002Sakamoto et al, , 2003.…”

“…Products of the studied genes were previously shown, or predicted, to be targeted to either chloroplasts or mitochondria (Adam et al, 2001;Sokolenko et al, 2002;Sakamoto et al, 2003). We therefore tested whether the effect of environmental conditions on expression could be related to the cellular location of the gene product.…”

“…This is probably due to variations in pin geometry that result in different amount of printed DNA (Schuchhardt et al, 2000). Sokolenko et al (2002), and Sakamoto et al (2003). Number of genes responding to environmental stimuli were taken from the data presented in Figure 1.…”

“…FtsH1, 2, and 5 were found as integral proteins in the thylakoid membrane, with their ATPbinding domain and catalytic zinc-binding site facing the stroma (Lindahl et al, 1996;Chen et al, 2000;Sakamoto et al, 2002). FtsH6-9, 11, and 12 are also targeted to chloroplasts, whereas FtsH3, 4, and 10 are mitochondrial (Kolodziejczak et al, 2002;Sakamoto et al, 2003). DegP isomers are predicted to localize in several cell compartments.…”

Quality control of Photosystem II: The molecular basis for the action of FtsH protease and the dynamics of the thylakoid membranes

“…This bidirectionality in the processive digestion makes it possible for FtsH to promptly degrade unnecessary membrane proteins. FtsH genes exist not only in bacteria but also in photosynthetic organisms, such as cyanobacteria and higher plants [67][68][69]. An FtsH homolog in higher plants was first found by an immunological analysis with an antibody against E. coli FtsH protease, which was shown to cross-react with a protein in spinach thylakoid membranes [70].…”