Transport of Proteins into Cryptomonads Complex Plastids

Wastl, Jürgen; Maier, Uwe‐G.

doi:10.1074/jbc.m003125200

Cited by 59 publications

(60 citation statements)

References 27 publications

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“…Thus, irrespective of the presence or absence of an additional ER-targeting signal peptide at the N terminus, the chloroplast transit peptide of PE␣ from G. theta is capable of mediating transport of the protein into chloroplasts from higher plants. This strongly suggests that the transport information of chloroplast transit peptides was conserved during secondary endosymbiosis, demonstrated also for other chromalveolates (11,21,22). In contrast, PE␣RR, which lacks both the ER-targeting signal peptide and the chloroplast-targeting transit peptide, was apparently not imported since no processing product corresponding in size to the mature polypeptide could be detected (Fig.…”

Section: Cloning Of Nuclear-encoded Plastid Proteins Of G Theta-ourmentioning

confidence: 87%

“…5). The N-terminal ER targeting signal and the Sec61 complex catalyze guided import into the lumen between the two outermost membranes, in which the preprotein is processed leading to the exposition of the transit peptide (10,11). The next step, the passage across the second outermost membrane, is most likely dependent on the transit peptide and an ERAD-like mechanism as proposed recently (31).…”

Section: Discussionmentioning

confidence: 99%

“…All genes encode preproteins containing a bipartite topogenic signal (BTS), composed of an N-terminal signal peptide for co-translational import into the ER lumen via the Sec61 complex, followed by a transit peptide-like region mediating transport across the remaining three membranes into the plastid stroma (10,11). Additionally, more than half of the PE␣ subunits carried a twin pair of arginine residues, followed by a hydrophobic stretch, between the transit peptide and mature protein region.…”

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confidence: 99%

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Translocation of a Phycoerythrin α Subunit across Five Biological Membranes

Gould

Fan

Hempel

et al. 2007

Journal of Biological Chemistry

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Cryptophytes, unicellular algae, evolved by secondary endosymbiosis and contain plastids surrounded by four membranes. In contrast to cyanobacteria and red algae, their phycobiliproteins do not assemble into phycobilisomes and are located within the thylakoid lumen instead of the stroma. We identified two gene families encoding phycoerythrin ␣ and light-harvesting complex proteins from an expressed sequence tag library of the cryptophyte Guillardia theta. The proteins bear a bipartite topogenic signal responsible for the transport of nuclear encoded proteins via the ER into the plastid. Analysis of the phycoerythrin ␣ sequences revealed that more than half of them carry an additional, third topogenic signal comprising a twin arginine motif, which is indicative of Tat (twin arginine transport)-specific targeting signals. We performed import studies with several derivatives of one member using a diatom transformation system, as well as intact chloroplasts and thylakoid vesicles isolated from pea. We demonstrated the different targeting properties of each individual part of the tripartite leader and show that phycoerythrin ␣ is transported across the thylakoid membrane into the thylakoid lumen and protease-protected. Furthermore, we showed that thylakoid transport of phycoerythrin ␣ takes place by the Tat pathway even if the 36 amino acid long bipartite topogenic signal precedes the actual twin arginine signal. This is the first experimental evidence of a protein being targeted across five biological membranes.

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Section: Cloning Of Nuclear-encoded Plastid Proteins Of G Theta-ourmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Translocation of a Phycoerythrin α Subunit across Five Biological Membranes

Gould

Fan

Hempel

et al. 2007

Journal of Biological Chemistry

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“…Presequences of nucleus encoded plastid proteins consist of a signal peptide followed by a transit peptide-like domain (Pancic and Strotmann 1993). The functionality of both domains was proven individually in vitro in heterologous import systems (Bhaya and Grossman 1991;Chaal and Green 2005;Ishida et al 2000;Lang et al 1998;Nassoury et al 2003;Wastl and Maier 2000), and previous studies in the diatom Phaeodactylum tricornutum demonstrated the in vivo functionality of native plastid presequences:GFP fusion proteins (Apt et al 2002). Interestingly, also heterologous presequences from the diatom Odontella sinensis Kroth et al 2005) or from the dinoflagellate Symbiodinium sp.…”

Section: Introductionmentioning

confidence: 99%

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

et al. 2007

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Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function at the cleavage site of the signal peptides (ASA-FAP), which is particularly important for successful plastid targeting. Screening genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan, tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications of the ''ASAFAP''-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import, as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus of the transit peptide together with a cleavable signal peptide is crucial for plastid import.

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“…To test this, we asked if the translocators in the double membrane-bound plastids of higher plants could recognize this sequence in vivo. In vivo experiments allow a potential import into plastids to be evaluated in the context of competition with other compartments, unlike in vitro tests where only import into isolated pea chloroplasts is assessed (DeRocher et al, 2000;Wastl and Maier, 2000).…”

Section: The Leader Contains a Functional Plastid Transit Sequencementioning

confidence: 99%

Plastid ultrastructure defines the protein import pathway in dinoflagellates

Nassoury

Cappadocia

Morse

2003

Journal of Cell Science

104

143

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Eukaryotic cells contain a variety of different compartments that are distinguished by their own particular function and characteristic set of proteins. Protein targeting mechanisms to organelles have an additional layer of complexity in algae, where plastids may be surrounded by three or four membranes instead of two as in higher plants. The mechanism of protein import into dinoflagellates plastids, however, has not been previously described despite the importance of plastid targeting in a group of algae responsible for roughly half the ocean's net primary production. Here, we show how nuclear-encoded proteins enter the triple membrane-bound plastids of the dinoflagellate Gonyaulax. These proteins all contain an N-terminal leader sequence with two distinct hydrophobic regions flanking a region rich in hydroxylated amino acids (S/T). We demonstrate that plastid proteins transit through the Golgi in vivo, that the first hydrophobic region in the leader acts as a typical signal peptide in vitro, and that the S/T-rich region acts as a typical plastid transit sequence in transgenic plants. We also show that the second hydrophobic region acts as a stop transfer sequence so that plastid proteins in Golgi-derived vesicles are integral membrane proteins with a predominant cytoplasmic component. The dinoflagellate mechanism is thus different from that used by the phylogenetically related apicomplexans, and instead, is similar to that of the phylogenetically distant Euglena,whose plastids are also bound by three membranes. We conclude that the protein import mechanism is dictated by plastid ultrastructure rather than by the evolutionary history of the cell.

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Transport of Proteins into Cryptomonads Complex Plastids

Cited by 59 publications

References 27 publications

Translocation of a Phycoerythrin α Subunit across Five Biological Membranes

Translocation of a Phycoerythrin α Subunit across Five Biological Membranes

Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif

Plastid ultrastructure defines the protein import pathway in dinoflagellates

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