The Hetero-Hexameric Nature of a Chloroplast AAA+ FtsH Protease Contributes to Its Thermodynamic Stability

Abstract

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4∶2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that ‘type B’ subunits (FtsH2 and FtsH8) were 2–3 fold more abundant than ‘type A’ (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two ‘type A’ and four ‘type B’ subunits.

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochromeb 6 fRemodeling inChlamydomonas reinhardtiiunder Stress Conditions

“…Thus, the thylakoid enzyme from C. reinhardtii shows a similar but simpler subunit composition than its counterpart from Arabidopsis, which assembles four distinct FtsH isoforms. We cannot exclude that some FtsH1 and/or FtsH2 may form homohexamer complexes, although homomeric structures are thought to be less likely to exist since they are less stable, as modeled by Moldavski et al (2012).…”

“…Transformation of these strains with an empty vector (pSL18) did not change their fluorescence kinetics, demonstrating that the FTSH1 gene alone was sufficient for complementation. Thylakoid FtsH proteases form various heterohexamers in cyanobacteria (Boehm et al, 2012) and Arabidopsis (Sakamoto et al, 2003;Yu et al, 2004;Zaltsman et al, 2005;Moldavski et al, 2012). Whereas four isoforms of thylakoid FtsH were identified in Arabidopsis (Zaltsman et al, 2005), only two thylakoid isoforms, FtsH1 and FtsH2, have been identified by proteomic studies in C. reinhardtii (Allmer et al, 2006).…”

“…Four isoforms have been identified in Arabidopsis thylakoid membranes: two of type A (FtsH1 and FtsH5) and two of type B (FtsH2 and FtsH8; Friso et al, 2004). The thylakoid enzyme is made of an active heterohexameric ring structure containing type A and B subunits whose accumulation is concerted, with a proposed stoichiometry of two type A and four type B subunits (Zaltsman et al, 2005;Moldavski et al, 2012) or three type A and three type B subunits (Yu et al, 2004;Boehm et al, 2012). Arabidopsis type A and type B subunits use different membrane insertion pathways (Rodrigues et al, 2011).…”

Subunit Organization of a Synechocystis Hetero-Oligomeric Thylakoid FtsH Complex Involved in Photosystem II Repair

“…Given the close evolutionary relationship between cyanobacteria and chloroplasts, it seems reasonable that the hetero-oligomeric complexes found in chloroplasts also contain three of each type of subunit arranged in an alternating fashion around the ring. However, recent work has concluded that chloroplast FtsH heterocomplexes might be composed of two type A and four type B subunits, but deviations from a 1:1 stoichiometry because of contamination by FtsH monomers or partially assembled FtsH complexes could not be excluded (Moldavski et al, 2012). The single-particle approach described here helps overcome this potential problem.…”

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…The stoichiometry of type A to type B subunits in an FtsH complex is estimated as 2:4 (Moldavski et al, 2012), whereas the cyanobacterial prototype comprises type A and B subunits in a 3:3 ratio with an alternating arrangement (Boehm et al, 2012). FtsH2 and FtsH5 are targeted to the thylakoid via the TAT (for twin-Arg translocation) pathway and the SEC (for secretion) pathway, respectively (Rodrigues et al, 2011).…”