Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana

Abstract

The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.

The FtsH-Inactive Protein FtsHi5 Is Required for Chloroplast Development and Protein Accumulation in Chloroplasts at Low Ambient Temperature in Arabidopsis

“…Our current results further revealed substantial changes at chloroplast proteome level in tsl2 mutant plants at 18 • C, which is associated with chloroplast development and function. Similar pale-green phenotypes with defects in chloroplast have also been found in knock-down lines of FtsHi4 (Lu et al, 2014), FtsH12 (Mielke et al, 2021) and pdNAD-MDH (Schreier et al, 2018), and in ftshi1 carrying a point mutation in FtsHi1 (Kadirjan- Kalbach et al, 2012), which is consistent with the association of FtsHi5/TSL2 with the protein complex in chloroplast development. Like all other ATP-dependent proteases, the function ATPase domain is essential for substrate recognition, unfolding and translocation to the proteolytic sites (Jessop et al, 2021).…”

To New Beginnings: Riboproteogenomics Discovery of N-Terminal Proteoforms in Arabidopsis Thaliana

“…In addition, protein synthesis from the identified dTIS was evidenced by three unique NTA peptides (18 PSMs), "TDTK DAGMDAVQR" (trypsin-digested), "TDTKDAGMDAVQRR L" (chymotrypsin-digested), and "TDTKDAGMDAVQRRLFE" (Glu-C-digested), and tryptic Nt peptide evidence previously reported in four Arabidopsis studies (Venne et al, 2015;Zhang et al, 2015Zhang et al, , 2018Mielke et al, 2021). Mutation of the dTIS corresponding to the N-terminus starting at AA position 52 and the dbTIS of IPP2 (resulting in M1 or M52>L mutation) resulted in the exclusive expression of the Nt proteoform translated from the non-mutated TIS (Figure 3A).…”

The FtsHi Enzymes of Arabidopsis thaliana: Pseudo-Proteases with an Important Function

“…Even Ycf2 was observed being part of this 2 MDa complex using transgenic lines overexpressing FTSH12 [ 58 ]. Still, the protein could not be detected in complexes isolated from wild-type and tic56-3 plastids using a combination of native gel electrophoresis and protein quantification [ 64 ] and neither after pull-down of FtsH12 using its native promoter [ 65 ]. Kikuchi and coworkers [ 57 ] determined the super-complex Ycf2-FtsH12-FtsHi1,2,4,5-pdNAD-MDH physically to interact with TIC components such as Tic214 (Ycf1), Tic100, and Tic56 as well as with the pre-protein translocation components Toc75 and Toc159 [ 59 ].…”

“…These results imply that even FtsHi4 can be substituted in the FtsH12/FtsHi complex. Gene expression of FTSHi2 and FTSHi3 was enhanced in homozygous ftshi4-2 mutants compared to WT [ 26 ], while on the protein level, the amount of FtsH12 was slightly diminished in the mutant [ 65 ].…”