Abundance of metalloprotease FtsH12 modulates chloroplast development in Arabidopsis thaliana.

Kati Mielke,Raik Wagner,Laxmi S Mishra,Pitter F Huesgen,Andreas Perrar,Fatih Demir,Christiane Funk,Peter Bozhkov

doi:10.1093/jxb/eraa550

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.

Highlights

Proteolysis is a degradative process that supplies nutrients, controls protein amounts or removes damaged or superfluous proteins, and allows post-translational protein modifications and signaling (Turk et al, 2012; Salvesen et al., 2016)
FtsH12 is expressed in non-photosynthetic plastids, located in the inner envelope with its C-terminus exposed to the stroma
Immunoblotting with a peptide-directed antibody raised against the C-terminal sequence DRVSYQPVDLRAAPLHRS of FtsH12 revealed steady abundance of this protease during leaf development, in chloroplasts, and in non-photosynthetic organs, including roots (Supplementary Fig. S3A)

Summary

Introduction

Proteolysis is a degradative process that supplies nutrients, controls protein amounts or removes damaged or superfluous proteins, and allows post-translational protein modifications and signaling (Turk et al, 2012; Salvesen et al., 2016). FtsH (filamentation temperature sensitive protein H) proteases are a family of membrane-bound metalloproteases present in eubacteria, animals, and plants These indispensable members of the M41 peptidase family (Rawlings et al., 3456 | Mielke et al.2017) consist of an AAA (ATPase associated with various cellular activities) domain and a metalloprotease domain ligating Zn2+ in the consensus sequence HEXXH (where X is any uncharged residue). Compared with non-photosynthetic organisms, plants and cyanobacteria contain more FTSH genes, with 17 members present in the model plant Arabidopsis thaliana (Wagner et al, 2012) Twelve of these enzymes contain the typical zincbinding motif required for proteolytic activity, while five carry mutations in the HEXXH motif, which render them presumably proteolytically inactive; they were termed FtsHi. The sub-cellular localization of eukaryotic FtsH proteins is restricted to organelles of endosymbiotic origin (mitochondria and chloroplasts). FtsH12 knock-out results in developmental arrest of the embryo (Meinke et al, 2008)

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