Characterization and engineering of alginate lyases

Lamppa, John W.

doi:10.1349/ddlp.426

Cited by 9 publications

(16 citation statements)

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“…The data also indicate that the single terminal processing step must take place during or after integration of CFoII into the membrane, since TPP is active only on the lumenal side of stroma thylakoids (Kirwin et al, 1988). The N-terminus of the mature protein must, therefore, be translocated across the thylakoid membrane prior to cleavage, implying an integration mechanism radically different to those involved in the biogenesis of the other integral thylakoid proteins analysed to date (LHCPII and the CP24 apoprotein) which can assemble after processing to the mature size by SPP (Lamppa, 1988;Cai et al, 1993).…”

Section: Resultsmentioning

confidence: 99%

“…Comparisons with LHCPII (the only other integral thylakoid membrane protein analysed in detail) point to major differences in integration mechanism. Integration of LHCPII into the thylakoid membrane is mediated by topogenic signals in the mature LHCPII protein (Lamppa, 1988;Viitanen et al, 1988) and in vitro reconstitution assays have been used to analyse in detail the underlying mechanism. These studies have shown that integration is dependent on the presence of at least one stromal protein factor and ATP (Cline, 1986;Chitnis et al, 1987;Fulson and Cline, 1988), and is in addition stimulated by the thylakoidal delta pH (Cline et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

“…The characteristics of the transit peptide, however, differ considerably from those of other integral components of the thylakoid membrane studied to date, such as the lightharvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) and the CP24 apoprotein. These polypeptides are both synthesized with a presequence which serves to target the protein only as far as the stromal phase, after which integration into the thylakoid membrane is mediated by information present in the mature proteins (Lamppa, 1988;Viitanen et al, 1988;Cai et al, 1993). In contrast, CFoII is synthesized with a bipartite presequence which contains 'envelope transit' and 'thylakoid transfer' signals in tandem specifying, firstly, translocation of the protein across the chloroplast envelope membranes and, secondly, translocation of the N-terminus of CFoII across the thylakoid membrane .…”

Section: Introductionmentioning

confidence: 99%

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Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

Michl¹,

Robinson²,

Shackleton³

et al. 1994

The EMBO Journal

122

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The CFoII subunit of the ATP synthase is an integral component of the thylakoid membrane which is synthesized in the cytosol with a bipartite, lumen‐targeting presequence similar in structural terms to those of imported lumenal proteins such as plastocyanin. This presequence is shown to possess a terminal cleavage site for the thylakoidal processing peptidase, but no intermediate site for the stromal processing peptidase. The integration of CFoII into the thylakoid membrane of Pisum sativum has been analysed using in vitro assays for the import of proteins into intact chloroplasts or isolated thylakoids. Efficient integration into thylakoids is observed in the light and dark, and the integration process does not require the presence of either stromal extracts or nucleoside triphosphates. The uncoupler nigericin inhibits integration only very slightly, indicating that the thylakoidal delta pH does not play a significant role in the integration mechanism. In each of these respects, the requirements for CFoII integration differ notably from those determined for integration of the light‐harvesting chlorophyll‐binding protein of photosystem II. The integration mechanism also differs significantly from the two mechanisms involved in the translocation of lumenal proteins across the thylakoid membrane, since one of these processes requires the presence of stromal protein factors and ATP, and the other mechanism is dependent on the thylakoidal delta pH. This conclusion is reinforced by the finding that saturation of the translocation system for the precursor to the lumenal 23 kDa oxygen‐evolving complex protein does not affect integration of CFoII into thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

Michl¹,

Robinson²,

Shackleton³

et al. 1994

The EMBO Journal

122

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“…Composite transit peptides are restricted to hydrophilic, lumenal proteins including plastocyanin, the 16, 23 and 33 kDa polypeptides of the oxygen-evolving complex [22]. Integral proteins, such as the chlorophyll a/b and CP24 apoproteins [33,34], or the Rieske Fe/S protein of the cytochrome complex [35], generally carry stroma-targeting transit sequences and integrate via internal, uncleaved hydrophobic epitopes (e.g. [33,34]).…”

Section: The Transit Sequence Aspects Of Assemblymentioning

confidence: 99%

“…Integral proteins, such as the chlorophyll a/b and CP24 apoproteins [33,34], or the Rieske Fe/S protein of the cytochrome complex [35], generally carry stroma-targeting transit sequences and integrate via internal, uncleaved hydrophobic epitopes (e.g. [33,34]). Although CFo-II is an integral protein and does not traverse the thylakoid membrane, an intriguing feature of its transit peptide is that it resembles those of the extrinsic lumenal proteins (Fig.…”

Section: The Transit Sequence Aspects Of Assemblymentioning

confidence: 99%

The nuclear‐encoded polypeptide Cfo‐II from spinach is a real, ninth subunit of chloroplast ATP synthase

et al. 1993

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Proton-translocating F-ATP synthases from chloroplasts contain a nuclear-coded subunit, CFo-II, that lacks an equivalent in the corresponding E. coil complex. Three recombinant phages that code for the entire precursor of this subunit have been isolated from 2 gtl 1 cDNA expression libraries made from polyadenylated spinach RNA using a two-step strategy. The reading frame of 222 amino acid residues includes 147 residues for the mature protein (M r 16.5 kDa) and a transit sequence of 75 residues (M r 8.0 kDa). Secondary structure predictions indicate a bitopic protein, anchored by a single N-terminal transmembrane segment and a C-terminal hydrophilic region that probably reaches into CF~. CFo-II precursor made in vitro can be imported into isolated, intact chloroplasts and assembled into ATP synthase. This protein is a real subunit of the plastid enzyme and a distinctive characteristic of ATP synthases involved in photosynthetic processes. Unique features are (i) that the gene for CFo-II (atpG) appears to be a duplication of atpF encoding CFo-I, the homologues of the genes for subunits b" and b in photosynthetic bacteria, (ii) that it represents the first instance that one copy of the various duplicated loci found in plastid chromosomes has been phylogenetically translocated to the nucleus, and (iii) that it operates with a bipartite (import/thylakoid-targeting) transit peptide but without an intermediate cleavage site for the stroma protease, suggestive of a way of membrane integration different from that of its plastome-encoded counterpart CFo-I. With these data, the first complete sequence for a chloroplast ATP synthase of a higher plant (spinach) is available.

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Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

Cashmore

1989

The EMBO Journal

112

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Various chimeric precursors and deletions of the 33 kd oxygen‐evolving protein (OEE1) were constructed to study the mechanism by which chloroplast proteins are imported and targeted to the thylakoid lumen. The native OEE1 precursor was imported into isolated chloroplasts, processed and localized in the thylakoid lumen. Replacement of the OEE1 transit peptide with the transit peptide of the small subunit of ribulose‐1,5‐bisphosphate carboxylase, a stromal protein, resulted in redirection of mature OEE1 into the stromal compartment of the chloroplast. Utilizing chimeric transit peptides and block deletions we demonstrated that the 85 residue OEE1 transit peptide contains separate signal domains for importing and targeting the thylakoid lumen. The importing domain, which mediates translocation across the two membranes of the chloroplast envelope, is present in the N‐terminal 58 amino acids. The thylakoid lumen targeting domain, which mediates translocation across the thylakoid membrane, is located within the C‐terminal 27 residues of the OEE1 transit peptide. Chimeric precursors were constructed and used in in vitro import experiments to demonstrate that the OEE1 transit peptide is capable of importing and targeting foreign proteins to the thylakoid lumen.

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Characterization and engineering of alginate lyases

Cited by 9 publications

References 0 publications

Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

Targeting of proteins to the thylakoids by bipartite presequences: CFoII is imported by a novel, third pathway.

The nuclear‐encoded polypeptide Cfo‐II from spinach is a real, ninth subunit of chloroplast ATP synthase

Targeting of proteins to the thylakoid lumen by the bipartite transit peptide of the 33 kd oxygen-evolving protein.

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