Identification, Characterization, and Molecular Cloning of a Homologue of the Bacterial FtsH Protease in Chloroplasts of Higher Plants

Abstract

In an attempt to identify and characterize chloroplast proteases, we performed an immunological analysis of chloroplasts using an antibody against Escherichia coli FtsH protease, which is an ATP-dependent metalloprotease bound to the cytoplasmic membrane. A cross-reacting protein of 78 kDa was found in the thylakoid membrane of spinach, but not in the soluble stromal fraction. Alkali and high salt washes, as well as trypsin treatment of thylakoid membranes, suggest that the chloroplastic FtsH protein is integral to the membrane, with its hydrophilic portion exposed to the stroma. The protein is not bound to any photosynthetic complex and is exclusively located in the stromally exposed regions of the thylakoid membrane. Its expression is dependent on light, as it is present in green pea seedlings, but absent from etiolated ones. An Arabidopsis cDNA was isolated, and the deduced amino acid sequence demonstrated high similarity to the E. coli FtsH protein, especially in the central region of the protein, containing the ATP-and zinc-binding sites. The product of this clone was capable of import into isolated pea chloroplasts, where it was processed to its mature form and targeted to the thylakoid membrane. The trans-bilayer orientation and lateral location of the FtsH protein in the thylakoid membrane suggest its involvement in the degradation of both soluble stromal proteins and newly inserted or turning-over thylakoid proteins.Numerous examples of protein degradation in chloroplasts have been accumulated over the years. These include degradation of unassembled proteins, apoproteins lacking their prosthetic groups or pigments, photo-or otherwise-damaged proteins, and developmentally or environmentally regulated proteins (see Ref. 1, and references therein). In particular, emphasis has been placed on the degradation of the D1 protein in connection with photoinhibitory damage of the PSII 1 reaction center (2, 3). However, despite extensive documentation of these processes, little is known about the identity of the proteases involved. Only recently, the existence of Clp protease, a homologue of a well characterized bacterial ATP-dependent serine protease (4, 5), has been demonstrated in chloroplasts of higher plants (1, 6 -8). The occurrence of Clp subunits in plants subjected to a variety of environmental conditions suggests its function in chloroplasts as a housekeeping protease.The existence of a protein related to a bacterial protease in the chloroplast, together with other prokaryotic characteristics of this organelle, led us to hypothesize that additional chloroplast proteases may resemble prokaryotic ones. This notion was recently substantiated by the finding that the chloroplast genomes of red and brown algae (9, 10) contain genes homologous to another bacterial protease, FtsH. FtsH and related proteins are found in different bacteria and yeast mitochondria; it was demonstrated to be a 74-kDa (11), ATP-dependent metalloprotease, involved in the response of Escherichia coli to heat shock (12, 13), degradation o...

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

“…Together with FtsH6, they are significantly similar to other chloroplastic and cyanobacterial FtsHs, suggesting their localization in chloroplasts. However, to date, the cellular locations of only FtsH1, VAR2, and VAR1 have been verified experimentally (Lindahl et al, 1996;Chen et al, 2000;Takechi et al, 2000;Sakamoto et al, 2002). To confirm the predicted cellular location of the other FtsH homologs, we conducted an in vivo transient expression assay using a green fluorescent protein (GFP) gene and suspension-cultured tobacco cells.…”

“…It exists in prokaryotes and organelles of higher eukaryotes and is involved in various biological functions (Langer, 2000). In higher plants, chloroplastic FtsH has been shown immunologically to reside in thylakoid membranes, and a corresponding Arabidopsis cDNA ( FtsH1 ) was isolated (Lindahl et al, 1996). Subsequently, the latter's involvement in the degradation of unassembled thylakoid proteins was suggested (Ostersetzer and Adam, 1997).…”

Purification and Characterization of Alkaline Serine Proteinase from Photosynthetic Bacterium,Rubrivivax gelatinosusKDDS1

“…16) FtsH proteinase takes part in degrading proteins whose synthesis and assembly are light-dependent. 17) Hence, the serine proteinase from Rvi. gelatinosus KDDS1 may be light-inducible or light-dependent proteinase.…”

“…Landsberg erecta) 16) and light-dependent chloroplast metalloproteinase complex associated with thylakoid membranes, FtsH, from spinash leaves. 17) SppA is a light-inducible serine-type proteinase participating in the light-dependent turnover of thylakoid membrane proteins or of peptides released from such proteins under light stress conditions. 16) FtsH proteinase takes part in degrading proteins whose synthesis and assembly are light-dependent.…”

A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress

“…Clp protease is an ATP-dependent serine protease which is a hetero-multimer complex composed of the proteolytic subunit ClpP and the regulatory ATPase ClpC, and is located in the stroma (Shanklin et al 1995;Clarke 1999), where it functions in the degradation of unassembled soluble and membrane proteins (Halperin and Adam 1996;Majeran et al 2000;Halperin et al 2001). FtsH protease is a membrane-bound ATP-dependent metalloprotease, the ATP-binding and catalytic domains of which are exposed to the stromal side of the thylakoid membrane (Lindahl et al 1996). It has been implicated in the degradation of unassembled proteins in thylakoid membranes and stroma (Ostersetzer and Adam 1997), and in the degradation of photodamaged D1 protein of photosystem II (Lindahl et al 2000).…”