Immunological Detection of Proteins Similar to Bacterial Proteases in Higher Plant Chloroplasts

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187

171

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...

Section: Discussionmentioning

confidence: 73%

mentioning

confidence: 99%

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

Lindahl

Tabak

Cseke

et al. 1996

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187

171

“…Stromal and thylakoid fractions were obtained by freezing and thawing, followed by a 1-min centrifugation at 15,000 ϫ g at 4°C (37). The resulting supernatant contained the stroma proteins, and the pellet contained the thylakoid fraction.…”

Section: Methodsmentioning

confidence: 99%

The Protein Disulfide Isomerase-like RB60 Is Partitioned between Stroma and Thylakoids in Chlamydomonas reinhardtii Chloroplasts

Trebitsh¹,

E²,

Ostersetzer³

et al. 2001

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Translation of psbA mRNA in Chlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s). RB60 is a member of a protein complex that binds with high affinity to the 5-untranslated region of psbA mRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplast psbA mRNA. Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulumlocalized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C. reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.

“…Only two of the five, SPP and TPP, were apparently involved in the routing of pPC. A thylakoid-bound homologue of the bacterial protease FtsH was recently characterized from spinach chloroplasts (38), and two stromal proteins related to other bacterial proteases were described (44). Our SPP might be involved in protein degradation, unrelated to protein sorting.…”

Section: Discussionmentioning

confidence: 99%

Purification and Properties of a Novel Chloroplast Stromal Peptidase

Koussevitzky¹,

Ne’eman²,

Sommer³

et al. 1998

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the ⌬pH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.Nuclear-encoded plastid proteins are synthesized in the cytosol. Targeting is conferred by a positively charged transit peptide rich in hydroxyamino acids (1, 2). The presequence is removed by a stromal processing peptidase (SPP) 1 upon import of the precursor (3). Some imported proteins are subsequently directed to the thylakoid membrane or lumen. Transit peptides of lumen proteins contain, in addition to the stromal-targeting domain, a thylakoid-targeting domain that is removed by TPP while crossing the thylakoid or immediately thereafter (2, 4). A single peptidase is believed to be responsible for the stromal processing of all plastid proteins (1, 2). A partially purified SPP acted on several precursors of plastid proteins (3). Two polypeptides that processed the precursors of LHCPII and other proteins were purified (5), and a gene coding for an antigenically related metalloprotease was cloned (6). The deduced protein (CPE) contained a zinc binding motif, and its relation to subunit ␤ of the mitochondrial processing peptidase was pointed out. Escherichia coli-expressed CPE was reported to process a wide range of protein precursors, including some lumen proteins (7). Two serine-protease SPPs from Chlamydomonas processed pSSU (8, 9). One catalyzed the formation of an intermediate form of SSU, and the other formed the mature protein. A consensus cleavage site, (Ile/Val)-X-(Ala/Cys)-2-Ala, was derived from comparing precursors of chloroplast proteins (10). However, this motif was absent in many cases. In general, SPP cleavage sites lack significant primary sequence similarity or a conserved secondary structure motif (1, 2, 4, 11). ...