A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Vandervere, Pamela; Bennett, Thecla; Oblong, John E.; Lamppa, Gayle K.

doi:10.1073/pnas.92.16.7177

Cited by 122 publications

(67 citation statements)

References 29 publications

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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%

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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

164

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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Section: Methodsmentioning

confidence: 99%

“…This SPP belongs to a family of metalloendopeptidases known as the pitrilysin family (Ref. 21; peptidase family M16). To determine whether any of the pitrilysin proteins in P. falciparum are homologous to the plant SPPs, a phylogenetic tree of the pitrilysin family was constructed.…”

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

164

167

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“…We initially demonstrated that a soluble metallopeptidase is responsible for processing of the precursor of light-harvesting chlorophyll a/b-binding protein, and it was implicated in the maturation of the precursors of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and the acyl carrier protein as well (11). We subsequently established that a recombinant form of the enzyme expressed in Escherichia coli cleaved 10 other precursors destined for different compartments and biosynthetic pathways within the organelle, indicating that it is a general stromal processing peptidase (SPP) 1 (12,13). Moreover, we demonstrated that both native SPP and its recombinant form process the precursor of SPP in trans.…”

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confidence: 99%

Determinants for Removal and Degradation of Transit Peptides of Chloroplast Precursor Proteins

Richter

Lamppa

2002

Journal of Biological Chemistry

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The stromal processing peptidase (SPP) cleaves a large diversity of chloroplast precursor proteins, removing an N-terminal transit peptide. We predicted previously that this key step of the import pathway is mediated by features of the transit peptide that determine precursor binding and cleavage followed by transit peptide conversion to a degradable substrate. Here we performed competition experiments using synthesized oligopeptides of the transit peptide of ferredoxin precursor to investigate the mechanism of these processes. We found that binding and processing of ferredoxin precursor depend on specific interactions of SPP with the region consisting of the C-terminal 12 residues of the transit peptide. Analysis of four other precursors suggests that processing depends on the same region, although their transit peptides are highly divergent in primary sequence and length. Upon processing, SPP terminates its interaction with the transit peptide by a second cleavage, converting it to a subfragment form. From the competition experiments we deduce that SPP releases a subfragment consisting of the transit peptide without its original C terminus. Interestingly, examination of the ATP-dependent metallopeptidase activity responsible for degradation of transit peptide subfragments suggests that it may recognize other unrelated peptides and, hence, act separately from SPP as a novel stromal oligopeptidase.Chloroplasts are structurally and functionally complex organelles found in green plants and algae. They not only carry out photosynthesis but also perform other essential metabolic reactions required for plant growth and development. The vast majority of chloroplast proteins, however, is encoded by the nuclear genome. These proteins are synthesized in the cytoplasm as precursors containing an N-terminal chloroplast targeting signal, the transit peptide, that mediates post-translational import into the chloroplast. It is predicted from the genome sequence of Arabidopsis thaliana that up to 14% of nuclear genes encode precursor proteins with a transit peptide (1, 2). Thus, ϳ3500 different precursor proteins may be targeted to the chloroplast. The import pathway for these precursors depends on direct interactions between the transit peptide and import receptors of the outer envelope, translocation across the two envelope membranes, and removal of the transit peptide in the stromal compartment of the organelle (3-6).The cleavage of transit peptides during import is likely to be essential for chloroplast biogenesis and function. Given the central role of the chloroplast in plant development and the enormous amount of protein that must be imported, proteolytic processing of precursor proteins is one of the most important post-translational modifications that occur in a plant cell. Moreover, it was shown for the precursors of some highly abundant chloroplast proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, ferredoxin, and plastocyanin, that the processing step is a prerequisite to adopt their acti...

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“…The C-terminal half is responsible for outer envelope targeting and thought to function as "stop-transfer" sequence to prevent the complete translocation of the protein. After processing by the stromal processing peptidase (SPP) (27), the protein is thought to remain in the intermembrane space (i75). Here it is processed further by an IMS localized type I signal peptidase (28) to its mature form (m75) before it is integrated into the OEM (26,29).…”

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confidence: 99%

Chloroplast Omp85 proteins change orientation during evolution

Sommer

Daum

Groß

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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The majority of outer membrane proteins (OMPs) from Gramnegative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport-associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using selfassembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function.

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A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Cited by 122 publications

References 29 publications

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

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

Determinants for Removal and Degradation of Transit Peptides of Chloroplast Precursor Proteins

Chloroplast Omp85 proteins change orientation during evolution

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