Purification and Properties of a Novel Chloroplast Stromal Peptidase

Koussevitzky, Shai; Ne’eman, E.; Sommer, A.; Steffens, John C.; Harel, E.

doi:10.1074/jbc.273.42.27064

Cited by 41 publications

(17 citation statements)

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“…However, the Heterosigma SPP was unaffected by EDTA and was unable to cleave higher plant precursors, suggesting that it is different from the plant SPP. More than one SPP may exist in Heterosigma, as is the case for plants and Chlamydomonas (Koussevitzky et al, 1998;Ru¨fenacht and Boschetti, 2000).…”

Section: Discussionmentioning

confidence: 93%

“…It possesses a zinc-binding motif (HXXEH), a characteristic of members of the metallopeptidase family M16, critical for processing activity (Richter and Lamppa, 2003). The possibility that more than one type of plant SPP exists was introduced when another stromal processing enzyme with different properties was discovered (Koussevitzky et al, 1998), and there is evidence for four distinct SPPs in the green alga Chlamydomonas reinhardtii (Ru¨fenacht and Boschetti, 2000).…”

Section: Introductionmentioning

confidence: 97%

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Protein import pathways in ?complex? chloroplasts derived from secondary endosymbiosis involving a red algal ancestor

Chaal

Green

2005

Plant Mol Biol

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Heterokont algae such as diatoms and the raphidophyte Heterosigma akashiwo and peridinin-containing dinoflagellates such as Heterocapsa triquetra originally acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host, resulting in 'complex' chloroplasts surrounded by four and three membranes, respectively. The precursors of both heterokont and dinoflagellate chloroplast-targeted proteins are first inserted into the ER with removal of an N-terminal signal peptide, but how they traverse the remaining membranes is unclear. Using a nuclear-encoded thylakoid lumen protein, PsbO, from the heterokont alga Heterosigma akashiwo, the dinoflagellate Heterocapsa triquetra and the red alga Porphyra yezoensis we show that precursors without the ER signal peptide can be imported into pea chloroplasts. In the case of the H. triquetra and Porphyra PsbO, the precursors were processed to their predicted mature size and localized within the thylakoid lumen, using the Sec-dependent pathway. We report for the first time a stromal processing peptidase (SPP) activity from an alga of the red lineage. The enzyme processes the Heterosigma PsbO precursor at a single site and appears to have different substrate and reaction specificities from the plant SPP. In spite of the fact that we could not find convincing homologs of the plant chloroplast import machinery in heterokont (diatom) and red algal genomes, it is clear that these three very different lines of algae use similar mechanisms to import chloroplast precursors.

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Section: Discussionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 97%

Protein import pathways in ?complex? chloroplasts derived from secondary endosymbiosis involving a red algal ancestor

Chaal

Green

2005

Plant Mol Biol

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“…Sequence Analysis-Although there is probably more than one sort of SPP in plants (20), only one has been cloned (21). This SPP belongs to a family of metalloendopeptidases known as the pitrilysin family (Ref.…”

Section: Methodsmentioning

confidence: 99%

Processing of an Apicoplast Leader Sequence inPlasmodium falciparum and the Identification of a Putative Leader Cleavage Enzyme

Dooren¹,

D’Ombrain

et al. 2002

Journal of Biological Chemistry

161

167

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The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a ⌬-aminolevulinic acid dehydratase homologue via an alternative splicing event.Plasmodium spp., the causative agents of malaria, belong to a family of intracellular parasites called the Apicomplexa. Plasmodium infects approximately 300 million people annually, causing over 1 million deaths, the great majority of which are caused by one species, Plasmodium falciparum (1). P. falciparum infects both humans and mosquitoes during its life cycle, with the pathogenic part of this cycle occurring predominantly in the erythrocytes of humans. The discovery of a non-photosynthetic plastid (the apicoplast) in the Apicomplexa has opened up a new area of anti-malarial drug targets (2, 3). However, the rational development of drugs targeting the apicoplast requires knowledge of apicoplast function. Preliminary studies indicate that the apicoplast is a site of fatty acid and isoprenoid biosynthesis (4 -6), and drugs targeting these pathways have been shown to kill P. falciparum (4, 6, 7).Like plant plastids, the apicoplast contains a reduced bacterial-like genome (8), from which a small number of proteins are expressed. The great majority of apicoplast proteins, as in plant plastids, are encoded in the nucleus and must be post-translationally targeted to the plastid. In plants, nuclear-encoded plastid proteins require a cleavable, N-terminal sequence called the transit peptide, which directs these proteins across the two membranes surrounding plant plastids (for reviews, see Refs. 9 and 10). Once in the plastid stroma, this transit peptide is cleaved by a stromal-processing peptidase (SPP 1 ; Refs. 11 and 12). Apicoplasts, however, are bound by four membranes (Ref. 2, but see Ref. 13), and proteins targeted to this organelle have been shown to require a bipartite N-terminal leader sequence (5,14,15). By fusing these N-terminal leader sequences to green fluorescent reporter protein (GFP)...

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“…In vitro import assays into intact chloroplasts were performed as described previously (32,35). The import assay was conducted for 30 min at 25°C in the light in the presence of 10 mM ATP, unless otherwise indicated.…”

Section: Methodsmentioning

confidence: 99%

“…Alaska) were grown under standard conditions (32). Intact chloroplasts were isolated on Percoll gradients as described (32,33).…”

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

Journal of Biological Chemistry

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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.

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Purification and Properties of a Novel Chloroplast Stromal Peptidase

Cited by 41 publications

References 50 publications

Protein import pathways in ?complex? chloroplasts derived from secondary endosymbiosis involving a red algal ancestor

Protein import pathways in ?complex? chloroplasts derived from secondary endosymbiosis involving a red algal ancestor

Processing of an Apicoplast Leader Sequence inPlasmodium falciparum and the Identification of a Putative Leader Cleavage Enzyme

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

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