Topology of Euglena Chloroplast Protein Precursors within Endoplasmic Reticulum to Golgi to Chloroplast Transport Vesicles
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.457
ISSN1083-351X
AutoresChidananda Sulli, Zhiwei Fang, Umesh S. Muchhal, Steven D. Schwartzbach,
Tópico(s)Protist diversity and phylogeny
ResumoEuglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophylla/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46–146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglenapresequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm. Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophylla/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46–146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglenapresequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm. endoplasmic reticulum endoglycosidase Hf polymerase chain reaction light-harvesting chlorophylla/b-binding protein of photosystem II precursor to the light-harvesting chlorophylla/b-binding protein of photosystem II precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Eukaryotic cells contain a number of membrane-bound organelles. Proteins contained within the ER,1 Golgi apparatus, vacuole, plasma membrane, and secreted proteins are synthesized on membrane-bound ribosomes as preproteins containing an N–terminal extension, the signal peptide, that initiates translocation into the ER (1Corsi A.K. Schekman R. J. Biol. Chem. 1996; 271: 30299-30302Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The nascent chain is co-translationally translocated into the ER through a translocation channel that appears to be open on one side to the lipid bilayer (2Martoglio B. Hofmann M.W. Brunner J. Dobberstein B. Cell. 1995; 81: 207-214Abstract Full Text PDF PubMed Scopus (234) Google Scholar) allowing integral membrane proteins to be inserted into the membrane co-translationally by lateral migration of the signal anchor from the translocation channel into the lipid bilayer. The signal sequence is removed in the ER lumen by signal peptidase (1Corsi A.K. Schekman R. J. Biol. Chem. 1996; 271: 30299-30302Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and the proteins are transported in vesicles to their final intracellular location.(3Pryer NK. Wuestehube L.J Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (368) Google Scholar).On the other hand, the nuclear encoded proteins transported across the two envelope membranes of plant (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar), green (5Howe G. Merchant S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1862-1866Crossref PubMed Scopus (31) Google Scholar) and red algal (6Apt K.E. Hoffmann N.E. Grossman A.R. J. Biol. Chem. 1993; 268: 16208-16215Abstract Full Text PDF PubMed Google Scholar) chloroplasts, and cyanelles (7Jakowitsch J. Neumann-Spallart C. Ma Y. Steiner J. Schenk H.E.A. Bohnert H.J. Löffelhardt W. FEBS Lett. 1996; 381: 153-155Crossref PubMed Scopus (33) Google Scholar) are synthesized on free cytoplasmic ribosomes as preproteins containing an N-terminal presequence, the transit peptide, that initiates post-translational translocation across the two envelope membranes. The transit peptide interacts with receptor proteins on the outer envelope membrane, and the precursor is translocated through separate protein conducting channels spanning the outer and inner membrane at contact sites, regions where the outer and inner membrane are in close physical proximity (8Schnell D.J. Blobel G. J. Cell Biol. 1993; 120: 103-115Crossref PubMed Scopus (141) Google Scholar, 9Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (232) Google Scholar). The transit peptide is removed by a stromal peptidase (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar). Thylakoid proteins have a bipartite presequence composed of a stromal targeting domain that is removed upon chloroplast entry and a C-terminal thylakoid targeting domain (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar) that is removed during thylakoid import (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar, 5Howe G. Merchant S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1862-1866Crossref PubMed Scopus (31) Google Scholar).Euglena is a protist having complex chloroplasts with a three-membrane envelope (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar, 11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar). Euglena chloroplast precursors are synthesized on membrane-bound polysomes (12Kishore R. Schwartzbach S.D. Plant Sci. 1992; 85: 79-89Crossref Scopus (21) Google Scholar) rather than free polysomes as found for higher plant, green and red algal chloroplast precursors. In vivo pulse-chase intracellular localization studies have demonstrated that both pLHCPII (13Sulli C. Schwartzbach S.D. J. Biol. Chem. 1995; 270: 13084-13090Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and pSSU (14Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar) are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus and from the Golgi apparatus to the chloroplast. pLHCPII and pSSU are anchored in the transport vesicle with major portions if not all of the mature protein remaining in the cytoplasm (13Sulli C. Schwartzbach S.D. J. Biol. Chem. 1995; 270: 13084-13090Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar).An understanding of the novel Euglena chloroplast protein import process requires knowledge of the topology of the precursors within the membrane of the transport vesicle that fuses with the outermost of the three chloroplast envelope membranes. Vesicle membrane protein topology is determined by decoding of topogenic sequences during protein translocation into the ER (15von Heijne G. Biochim. Biophys. Acta. 1988; 947: 307-333Crossref PubMed Scopus (328) Google Scholar, 16Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Hydrophobic stop transfer membrane anchor sequences stop translocation into the ER anchoring the protein within the membrane (15von Heijne G. Biochim. Biophys. Acta. 1988; 947: 307-333Crossref PubMed Scopus (328) Google Scholar, 16Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In some cases, an uncleaved signal peptide functions as a signal anchor sequence initiating translocation and anchoring the protein in the membrane (15von Heijne G. Biochim. Biophys. Acta. 1988; 947: 307-333Crossref PubMed Scopus (328) Google Scholar,17Szczesra-Skorupa E. Browne N. Mead D. Kemper B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 738-742Crossref PubMed Scopus (103) Google Scholar). Signal peptides and topogenic sequences are conserved between prokaryotes and eukaryotes as well as between plants and animals, allowing canine microsomes to be used as an in vitro system for identifying ER targeting domains and membrane anchor sequences (15von Heijne G. Biochim. Biophys. Acta. 1988; 947: 307-333Crossref PubMed Scopus (328) Google Scholar,16Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Since N-linked glycosylation occurs within the ER lumen and alters the electrophoretic mobility of a protein, glycosylation sites inserted within a protein can be used as reporters to determine whether a domain is translocated into the ER lumen or remains on the cytoplasmic side of the membrane (16Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Previous work has shown that pSSU (14Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar) and pLHCPII (18Kishore R. Muchhal M.S. Schwartzbach S.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11845-11849Crossref PubMed Scopus (46) Google Scholar) are inserted in vitro into canine microsomes. In this study, pLHCPII deletion constructs and glycosylation reporter constructs have been used to determine the topology of pLHCPII within the ER membrane and to identify the stop transfer membrane anchor domain. The results indicate that the presequence contains an N-terminal signal peptide domain targeting the protein to the ER and a C-terminal hydrophobic stop transfer membrane anchor domain that inserts the precursor into the membrane oriented with the presequence N terminus in the lumen and the mature protein including the presequence C terminus on the cytoplasmic membrane face.DISCUSSIONEuglena is one of a group of organisms having complex chloroplasts, chloroplasts with a three- or four-membrane envelope rather than a two-membrane envelope as found for the chloroplasts of plants, red and green algae and the cyanelles of glaucocystophytes (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar,11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar, 31Gibbs S.P. Annu. N. Y. Acad. Sci. 1981; 361: 193-207Crossref PubMed Google Scholar, 32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar, 34Dodge J.D. Phycologia. 1975; 14: 253-263Crossref Google Scholar, 35Sweeney B.M. J. Phycol. 1981; 17: 95-101Crossref Scopus (14) Google Scholar, 36Gibbs S.P. Int. Rev. Cytol. 1981; 72: 49-99Crossref Scopus (115) Google Scholar). Immunoelectron microscopy (37Osafune T. Sumida S. Schiff J.A. Hase E. J. Electron Microsc. 1991; 40: 41-47Google Scholar, 38Osafune T. Sumida S. Schiff J.A. Hase E. Exp. Cell Res. 1991; 193: 320-330Crossref PubMed Scopus (34) Google Scholar) and pulse-chase intracellular localization studies (13Sulli C. Schwartzbach S.D. J. Biol. Chem. 1995; 270: 13084-13090Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar) have clearly demonstrated that protein import into Euglena chloroplasts is fundamentally different from import into chloroplasts with a double envelope membrane. Euglena precursors are co-translationally inserted into the ER, transported as integral membrane proteins from the ER to the Golgi apparatus and from the Golgi apparatus to the outermost of the three Euglena envelope membranes, and then imported through the middle and inner chloroplast envelope membranes (13Sulli C. Schwartzbach S.D. J. Biol. Chem. 1995; 270: 13084-13090Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 14Sulli C. Schwartzbach S.D. Plant Cell. 1996; 8: 43-53Crossref PubMed Scopus (49) Google Scholar, 37Osafune T. Sumida S. Schiff J.A. Hase E. J. Electron Microsc. 1991; 40: 41-47Google Scholar, 38Osafune T. Sumida S. Schiff J.A. Hase E. Exp. Cell Res. 1991; 193: 320-330Crossref PubMed Scopus (34) Google Scholar).The novel feature of Euglena chloroplast protein import, vesicular transport from the Golgi apparatus to the chloroplast envelope, appears to be an evolutionary consequence of the origin of the third envelope membrane. The ancestral Euglenoid is believed to have been a phagotrophic trypanosome-like organism that engulfed a eukaryotic algae (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar, 31Gibbs S.P. Annu. N. Y. Acad. Sci. 1981; 361: 193-207Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar, 36Gibbs S.P. Int. Rev. Cytol. 1981; 72: 49-99Crossref Scopus (115) Google Scholar). The third envelope membrane is thought to have evolved from the host's phagocytic vacuole membrane (11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar). Vacuolar protein precursors contain signal peptides and are transported from the ER to the Golgi apparatus, where they are sorted into transport vesicles for delivery to the vacuole (1Corsi A.K. Schekman R. J. Biol. Chem. 1996; 271: 30299-30302Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 3Pryer NK. Wuestehube L.J Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (368) Google Scholar). This signal peptide-dependent vacuole targeting system was probably utilized by the ancestral phagocytic trypanosome host and the endosymbiotic eukaryotic algae for vacuolar protein localization (32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar,33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar). During reduction of the endosymbiont to a chloroplast, the transfer of genes from the endosymbiont to the host nucleus required evolution of a system to transport the encoded proteins from the cytoplasm to the chloroplast. The signal peptide-dependent vacuolar targeting system coupled with the evolution of a unique Golgi sorting signal provided a specific mechanism for returning proteins to the endosymbiont evolving into a chloroplast within the phagocytic vacuole.The presequence of Euglena pLHCPII is 141 amino acids composed of an N-terminal signal peptide and a stromal targeting region containing a hydrophobic domain 57 amino acids from the predicted signal peptidase cleavage site (18Kishore R. Muchhal M.S. Schwartzbach S.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11845-11849Crossref PubMed Scopus (46) Google Scholar). In vitro studies found that Euglena pLHCPII was co-translationally inserted as an integral membrane protein into canine microsomes, and the signal peptide was cleaved (18Kishore R. Muchhal M.S. Schwartzbach S.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11845-11849Crossref PubMed Scopus (46) Google Scholar). A glycosylation site within the mature protein was not glycosylated, while a glycosylation site inserted within the presequence stromal targeting region N-terminal to the hydrophobic domain was. Upon deletion of the presequence stromal targeting region including the hydrophobic domain, the glycosylation site within the mature protein was glycosylated, indicating that this hydrophobic domain functions as a stop transfer membrane anchor sequence. Taken together, the in vivo transport studies (13Sulli C. Schwartzbach S.D. J. Biol. Chem. 1995; 270: 13084-13090Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) and these in vitro microsomal processing studies demonstrate that pLHCPII is transported from the Golgi apparatus to the chloroplast with the presequence oriented with the N terminus formed by signal peptidase cleavage in the vesicle lumen, the hydrophobic domain of the stromal targeting region anchored in the vesicle membrane, and the mature protein in the cytoplasm.The topology of a protein within an ER-derived membrane is determined by linear decoding of topogenic sequences within the protein during its translocation into the ER (15von Heijne G. Biochim. Biophys. Acta. 1988; 947: 307-333Crossref PubMed Scopus (328) Google Scholar, 16Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Inspection of the knownEuglena chloroplast protein presequences (17Szczesra-Skorupa E. Browne N. Mead D. Kemper B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 738-742Crossref PubMed Scopus (103) Google Scholar, 19Muchhal U.S. Schwartzbach S.D. Nucleic Acids Res. 1994; 22: 5737-5744Crossref PubMed Scopus (28) Google Scholar, 22Chan R.L. Keller M. Canaday J. Weil J.H. Imbault P. EMBO J. 1990; 9: 333-338Crossref PubMed Scopus (63) Google Scholar, 23Sharif A.L. Smith A.G. Abell C. Eur. J. Biochem. 1989; 184: 353-359Crossref PubMed Scopus (63) Google Scholar, 24Lin Q. Ma L. Burkhart W. Spremulli L.L. J. Biol. Chem. 1994; 269: 9436-9444Abstract Full Text PDF PubMed Google Scholar, 25Plaumann M. Pelzer-Reith B. Martin W.F. Schnarrenberger C. Curr. Genet. 1997; 31: 430-438Crossref PubMed Scopus (52) Google Scholar, 26Henze K. Badr A. Wettern M. Cerff R. Martin W. Proc. Natl. Acad. Sci. 1995; 92: 9122-9126Crossref PubMed Scopus (139) Google Scholar) indicates that as found for pLHCPII, they all contain an N-terminal signal peptide domain and a stromal targeting domain with a hydrophobic region about 60 amino acids from the predicted signal peptidase cleavage site. Since all of the presequences have the same topogenic sequences as pLHCPII, they all will be transported to the chloroplast anchored in the vesicle membrane with approximately 60 amino acids within the lumen. Upon fusion of transport vesicles with the outer chloroplast membrane, the N-terminal 60 amino acids of the stromal targeting domain will extend from the inner surface of the outer envelope membrane into the space separating the outer and intermediate chloroplast envelope membranes.The mechanism by which precursors are translocated through the intermediate and inner Euglena chloroplast envelope membranes after fusion of Golgi transport vesicles with the outermost membrane remains unclear. Sequence analysis indicates a common ancestor for the phenotypically diverse plastids of higher plants, green algae, rhodoplasts, diatoms, Euglena, and the cyanelles of glaucocystophytes (33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar, 39Martin W. Stoebe B. Goremykin V. Hausmann S. Husejaws M. Kowallik K.V. Nature. 1998; 393: 162-165Crossref PubMed Scopus (583) Google Scholar). Similar stromal targeting sequences and similar transport mechanisms are utilized for protein import into cyanelles (7Jakowitsch J. Neumann-Spallart C. Ma Y. Steiner J. Schenk H.E.A. Bohnert H.J. Löffelhardt W. FEBS Lett. 1996; 381: 153-155Crossref PubMed Scopus (33) Google Scholar) and the chloroplasts of plants (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar) green (5Howe G. Merchant S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1862-1866Crossref PubMed Scopus (31) Google Scholar) and red algae (6Apt K.E. Hoffmann N.E. Grossman A.R. J. Biol. Chem. 1993; 268: 16208-16215Abstract Full Text PDF PubMed Google Scholar), suggesting that the post-translational mechanism for protein translocation through two plastid envelope membranes evolved prior to the divergence of these different plastid types. This ancestral post-translational chloroplast protein import system was probably utilized by the photosynthetic eukaryotic algae that through endosymbiosis evolved into the Euglena chloroplast. Just as the ancestral prokaryotic preprotein translocation system was retained for thylakoid protein import during the evolution of a photosynthetic prokaryotic endosymbiont into a chloroplast (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar), the ancestral chloroplast protein import system was retained as the chloroplast envelope membranes of the photosynthetic eukaryotic endosymbiont evolved into the intermediate and inner membranes of theEuglena chloroplast (32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar). The bipartiteEuglena presequence arose by the addition of a signal peptide to the existing stromal targeting presequence (32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar), enabling the precursor to be transported first to the phagocytic vacuole-derived Euglena outer chloroplast membrane (11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar), separating the host cytoplasm from the endosymbiont, and subsequently through the intermediate and inner chloroplast membranes derived from the endosymbiont's chloroplast envelope (32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar).Proteins are imported into higher plant chloroplasts at envelope contact sites, regions of association between the outer and inner membrane translocation machinery (8Schnell D.J. Blobel G. J. Cell Biol. 1993; 120: 103-115Crossref PubMed Scopus (141) Google Scholar, 9Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (232) Google Scholar). Transmission electron microscopy has shown that the three Euglena chloroplast membranes are closely appressed in some regions reminiscent of higher plant envelope contact sites, while in other regions they are well separated (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar, 11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar). Regions of adherence between the Euglenaintermediate and inner membrane similar to the contact sites between the plant outer and inner envelope membrane have been identified in freeze etch replicas (11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar). These ultrastructural studies support the assumption that protein translocation through the Euglenachloroplast intermediate and inner membranes is mechanistically similar to post-translational protein import into the plastids of cyanelles, plants, and green and red algae.Based on the evolutionary origins of the Euglena chloroplast and available biochemical evidence, a working model has been developed for protein import into Euglena chloroplasts (Fig.4). It is proposed that the outer membrane contains a protein translocation channel that is open on one side to the lipid bilayer as found for the ER protein translocation channel (2Martoglio B. Hofmann M.W. Brunner J. Dobberstein B. Cell. 1995; 81: 207-214Abstract Full Text PDF PubMed Scopus (234) Google Scholar), while the intermediate and inner membranes contain an import apparatus functionally similar to the import apparatus of plant chloroplasts (4Cline K. Henry H. Annu. Rev. Cell Dev. Biol. 1996; 12: 1-26Crossref PubMed Scopus (187) Google Scholar, 8Schnell D.J. Blobel G. J. Cell Biol. 1993; 120: 103-115Crossref PubMed Scopus (141) Google Scholar, 9Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (232) Google Scholar). Euglena chloroplast precursors are co-translationally inserted and anchored in the ER membrane oriented with the N-terminal 60 amino acids of the stromal targeting presequence projecting into the lumen and the remainder of the precursor in the cytoplasm. This orientation is maintained within Golgi to chloroplast transport vesicle so that upon fusion of transport vesicles with the outer chloroplast membrane, the precursor will be embedded in the outer membrane with 60 amino acids of the stromal targeting presequence projecting into the intermembrane space. Interactions between the presequence and middle membrane translocation complex would alter the orientation of the presequence in the outer membrane, promoting lateral movement from the lipid bilayer into the outer membrane translocation channel by a process that is mechanistically a reversal of the lateral migration of a protein from the ER translocation channel into the lipid bilayer (2Martoglio B. Hofmann M.W. Brunner J. Dobberstein B. Cell. 1995; 81: 207-214Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Interaction between the outer and intermediate membrane translocation channels would form a contact site providing a pathway for precursor import from the cytoplasm. As the precursor moves through the intermediate membrane, the presequence would engage inner membrane import receptors forming a three-membrane contact site for translocation into the stroma. Formation of the three-membrane contact site is probably mechanistically similar to the formation of contact sites between the outer and inner envelope membranes of higher plant chloroplasts (8Schnell D.J. Blobel G. J. Cell Biol. 1993; 120: 103-115Crossref PubMed Scopus (141) Google Scholar, 9Nielsen E. Akita M. Davila-Aponte J. Keegstra K. EMBO J. 1997; 16: 935-946Crossref PubMed Scopus (232) Google Scholar). Presequence cleavage and, in the case of polyprotein precursors such as pLHCPII and pSSU, polyprotein processing by stromal proteases (21Enomoto T. Sulli C. Schwartzbach S.D. Plant Cell Physiol. 1997; 38: 743-746Crossref Scopus (16) Google Scholar) would occur during or rapidly after precursor import.Euglena is one of a number of organisms with complex plastids having three or four envelope membranes (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar, 11Lefort-Tran M. Pouphile M. Freyssinet G. Pineau B. J. Ultrastruct. Res. 1980; 73: 44-63Crossref PubMed Scopus (19) Google Scholar, 31Gibbs S.P. Annu. N. Y. Acad. Sci. 1981; 361: 193-207Crossref PubMed Google Scholar, 32Schwartzbach S.D. Osafune T. Löffelhardt W. Plant Mol. Biol. 1998; 38: 247-263Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar, 34Dodge J.D. Phycologia. 1975; 14: 253-263Crossref Google Scholar, 35Sweeney B.M. J. Phycol. 1981; 17: 95-101Crossref Scopus (14) Google Scholar, 36Gibbs S.P. Int. Rev. Cytol. 1981; 72: 49-99Crossref Scopus (115) Google Scholar). The diverse organisms with complex plastids are thought to have arisen through multiple secondary endosymbiotic associations between a heterotrophic or possibly phototrophic host and photosynthetic eukaryotes (10Gibbs S.P. Can. J. Bot. 1978; 56: 2883-2889Crossref Google Scholar, 31Gibbs S.P. Annu. N. Y. Acad. Sci. 1981; 361: 193-207Crossref PubMed Google Scholar, 33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google Scholar, 40Häuber M.M. Müller S.B. Speth V. Maier U-G. Bot. Acta. 1994; 107: 383-386Crossref Scopus (34) Google Scholar). The different mechanisms used to deliver and translocate proteins through the outermost membranes of complex plastids reflects the multiple secondary endosymbiotic events that produced the diverse organisms having complex plastids. The complex plastids of dinoflagellates are surrounded by three membranes (33Melkonian M. Verh. Dtsch. Zool. Ges. 1996; 89.2: 71-96Google
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