A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

Sulli, Chidananda; Schwartzbach, Steven D.

doi:10.1105/tpc.8.1.43

Cited by 50 publications

(40 citation statements)

References 30 publications

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“…Details of the mechanism differ, however, as in Euglena the plastid-directed leader sequences contain two hydrophobic regions separated by an S/T-rich region reminiscent of a plastid transit sequence (Henze et al, 1995;Kishore et al, 1993). In vitro studies have demonstrated that the plastid directed proteins remain inserted in the ER membranes, unlike the apicomplexans where the plastid proteins are soluble inside the ER (Kishore et al, 1993;Osafune et al, 1990;Sulli et al, 1999;Sulli and Schwartzbach, 1996). The second hydrophobic region present in the Euglena leaders is absent in apicomplexan leaders (Foth et al, 2003).…”

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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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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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“…Marker enzymes identified fractions 2-5 as containing ER membranes, fractions 6-14 as containing light and dense Golgi membranes and fractions 14-22 as containing broken and intact chloroplasts (Sulli and Schwartzbach, 1995;Sulli and Schwartzbach, 1996). For direct comparisons between gradients loaded with differing amounts of 35 S-labeled protein, pLHCPII and LHCPII amounts in each fraction were plotted as the percentage of total immunoprecipitate (pLHCPII and LHCPII) recovered from the gradient.…”

Section: Euglena Golgi To Chloroplast Transport Is Reversibly Blockedmentioning

confidence: 99%

“…Immunoelectron microscopy localized Euglena and dinoflagellate chloroplast proteins to the Golgi apparatus (Nassoury et al, 2003;Osafune et al, 1991). In vivo pulse-chase immunoprecipitation experiments have shown that Euglena chloroplast precursors are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus prior to chloroplast localization (Sulli and Schwartzbach, 1995;Sulli and Schwartzbach, 1996). In vitro studies with canine microsomes have shown that the Euglena (Sulli et al, 1999) and dinoflagellate (Nassoury et al, 2003) pre-sequence signal peptide co-translationally inserts the precursor into the ER whereas the hydrophobic domain within the transit peptide region functions as a stop-transfer sequence anchoring the protein in the membrane oriented with the N-terminus formed by signal peptidase cleavage in the lumen and the remainder of the precursor in the cytoplasm.…”

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confidence: 99%

“…35 S]O 4 and chased at 26°C by addition of a 100-fold excess of K 2 SO 4 for a final concentration of 0.1 M (Sulli and Schwartzbach, 1996). Temperature shifts were performed by diluting cultures threefold with 26°C medium or 15°C medium to continue incubation at 15°C.…”

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confidence: 99%

“…1PSSU1 was digested with AgeI-HindIII, and the 260 bp 3′ end fragment was ligated into AgeI-HindIII-digested 1NSSU (Sulli and Schwartzbach, 1996) in pBluescript II KS + creating plasmid 1PSSU encoding a oneunit pSSU including the 3′ end stop codon. Plasmid mSSU was created by digesting 1PSSU1 with AgeI-KpnI and ligating the 300 bp 3′ end fragment into AgeI-KpnI-digested SSU1 (Sulli and Schwartzbach, 1996) in pBluescript II KS + creating plasmid mSSU encoding the mature SSU including the 3′ end stop codon.…”

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Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

Sláviková

Vacula

Fang

et al. 2005

Journal of Cell Science

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Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that 35S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15°C and transport to the chloroplast resumed after transfer to 26°C. Transport of the 35S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15°C cells returned to 26°C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

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Computational Complexity

2001

Problem Solving in Automata, Languages, and Complexity

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Secondary endosymbiosis describes the origin of plastids in several major algal groups such as dinoflagellates, euglenoids, heterokonts, haptophytes, cryptomonads, chlorarachniophytes and parasites such as apicomplexa. An integral part of secondary endosymbiosis has been the transfer of genes for plastid proteins from the endosymbiont to the host nucleus. Targeting of the encoded proteins back to the plastid from their new site of synthesis in the host involves targeting across the multiple membranes surrounding these complex plastids. Although this process shows many overall similarities in the different algal groups, it is emerging that differences exist in the mechanisms adopted. ß 2001 Published by Elsevier Science B.V.

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A soluble protein is imported into Euglena chloroplasts as a membrane-bound precursor.

Cited by 50 publications

References 30 publications

Plastid ultrastructure defines the protein import pathway in dinoflagellates

Plastid ultrastructure defines the protein import pathway in dinoflagellates

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena

Computational Complexity

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