Topology of Euglena Chloroplast Protein Precursors within Endoplasmic Reticulum to Golgi to Chloroplast Transport Vesicles*

Chidananda Sulli, ZhiWei Fang, Umesh MuchhalDagger , and Steven D. Schwartzbach§

From the School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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 chlorophyll a/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 Euglena presequences, 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

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 (1). 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 (2) 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 (1), and the proteins are transported in vesicles to their final intracellular location.(3).

On the other hand, the nuclear encoded proteins transported across the two envelope membranes of plant (4), green (5) and red algal (6) chloroplasts, and cyanelles (7) 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 (8, 9). The transit peptide is removed by a stromal peptidase (4). 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 (4) that is removed during thylakoid import (4, 5).

Euglena is a protist having complex chloroplasts with a three-membrane envelope (10, 11). Euglena chloroplast precursors are synthesized on membrane-bound polysomes (12) 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 (13) and pSSU (14) 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 (13, 14).

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 (15, 16). Hydrophobic stop transfer membrane anchor sequences stop translocation into the ER anchoring the protein within the membrane (15, 16). In some cases, an uncleaved signal peptide functions as a signal anchor sequence initiating translocation and anchoring the protein in the membrane (15, 17). 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 (15, 16). 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 (16). Previous work has shown that pSSU (14) and pLHCPII (18) 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.

    EXPERIMENTAL PROCEDURES

Preparation of Plasmids-- A cDNA clone, 1NLHCP, encoding a 39-kDa Euglena pLHCPII composed of the 141-amino acid LHCPII presequence and a mature LHCPII was constructed in the vector pBluescript II KS+ in two steps from plasmids pNLH1 and CLH22 (18, 19). Plasmid NLH1 was cut with XbaI-BssHII, and plasmid CLH22 was cut with XhoI-BssHII to release the LHCPII-encoding fragments. The XbaI-BssHII and XhoI-BssHII fragments were ligated into XbaI-XhoI-cut pBluescript KS+, creating the plasmid 2NLHCP encoding a two-unit Euglena pLHCPII polyprotein containing the presequence and two mature LHCPIIs (N1PEP/575PEP and 811PEP) covalently joined by a decapeptide linker (19, 20). 2NLHCP was cut with SfiI, removing a 0.7-kb fragment, and religated forming 1NLHCP. The plasmid 1LHCP encoding a mature Euglena LHCPII but lacking a presequence was constructed by cutting 2LHCP (21) with SfiI to remove an LHCPII unit and then religating the cut plasmid.

Plasmid 1NLHCPglyc40, encoding a pLHCPII containing a glycosylation site at Asp40, was constructed using PCR to replace the sequence Asp40-Ile-Gln-Gln within the 1NLHCP presequence with the glycosylation sequence Asp40-Gly-Ser-Met. A PCR fragment was synthesized using Pfu polymerase (Stratagene) and 1 ng of 1NLHCP as template as described by the manufacturer using 20 pmol of the oligonucleotide CTTATGTTAACGGATCCATGGCTCCTGCAGTTA as the 5'-primer and 20 pmol of the oligonucleotide GACCATGATTACGCCAAGCG as the 3'-primer. Amplification conditions were 95 °C for 5 min, 30 cycles of 55 °C for 30 s, 75 °C for 3 min, 95 °C for 1 min, and a final cycle with the elongation time at 75 °C extended to 5 min. The PCR product was cut with HpaI-EcoRI and ligated into HpaI-EcoRI-cut 1NLHCP. The 5'-primer inserted BamHI and PstI sites into 1NLHCPglyc40, and these sites were used to construct other plasmids.

Plasmid 1NLHCPglyc40Delta 138-438, encoding a protein containing a glycosylation site at Asp40 but lacking pLHCPII amino acids 46-146, was constructed by cutting 1NLHCPglyc40 with PstI to remove the 300-base pair fragment encoding amino acids 46-146 and religating the cut plasmid. Plasmid 1NLHCPDelta 138-438 encoding a protein lacking a presequence glycosylation site and pLHCPII amino acids 46-146 was constructed by using PCR to convert Asp40 to Thr40. A PCR fragment was synthesized using Pfu polymerase (Stratagene) and 1 ng of 1NLHCPglyc40Delta 138-438 as template as described by the manufacturer using 20 pmol of the oligonucleotide CTCATAGTACTGGATCCATGGCTCCTGCAGTTA as the 5'-primer and 20 pmol of the oligonucleotide GACCATGATTACGCCAAGCG as the 3'-primer. Amplification conditions were 95 °C for 5 min, 30 cycles of 55 °C for 30 s, 75 °C for 3 min, 95 °C for 1 min, and a final cycle with the elongation time at 75 °C extended to 5 min. The 5'-primer inserts a ScaI site into the PCR fragment and converts Asp40 to Thr40. The PCR product was digested with ScaI-EcoRI and ligated into the HpaI-EcoRI-cut plasmid 1NLHCPglyc40Delta 138-438. The plasmid 1NLHCPDelta 1-138 encoding a protein lacking the pLHCPII presequence signal peptide domain (amino acids 1-42) was constructed by digesting 1NLHCPglyc40 with BamHI-EcoRI to release a fragment lacking nucleotides 1-138 encoding the first 40 amino acids of pLHCPII. The released BamHI-EcoRI fragment was ligated into BamHI-EcoRI-cut pBluescript KS+. Translation of the encoded protein starts at Met43, which was inserted by the 5'-primer used to construct plasmid 1NLHCPglyc40.

In Vitro Transcription and Microsomal Processing Reactions-- All of the cDNAs were cloned in the pBlueScript KS+ plasmid oriented for in vitro transcription from the T7 promoter. Run-off transcripts were prepared from linearized plasmids using the T7 MEGAscript (Ambion, Austin, TX) in vitro transcription kit as described by the supplier. Transcripts (0.5 µg/25-µl reaction) were translated as described previously (18) using [35S]methionine in nuclease-treated rabbit reticulocyte lysates (Promega, Madison, WI) for 60 min at 30 °C in the presence or absence of 3.6 equivalents of canine pancreatic microsomal membranes (Promega). Reactions were terminated by adding 0.1 volume of a solution containing 0.12 M methionine, 20 mM puromycin, and 3 mM cycloheximide as described (14). The amount of [35S]methionine-labeled protein synthesized was determined by precipitation with trichloroacetic acid as described previously (13).

Assay for Glycosylation by Digestion with Endo H-- Protein glycosylation was determined by a mobility shift after deglycosylation with Endo H (New England Biolabs). After terminating translation, aliquots of the translation mixture (5-10 µl) were denatured by boiling for 2 min in the presence of 1.0% SDS, 0.1% beta -mercaptoethanol and incubated with 1000-2000 units of Endo H in 50 mM sodium citrate, pH 5.5, at 37 °C for 1 h as recommended by the supplier. At the end of the incubation, the protein was precipitated with 10% trichloroacetic acid (w/v) and resuspended in SDS sample buffer (2% SDS, 60 mM Tris-HCl, pH 8.6).

Assay for Membrane Integration by Na2CO3 Extraction and Trypsin Digestion-- Na2CO3 and potassium acetate extractions were performed as described previously (14) by adjusting aliquots of in vitro translation reactions to 0.1 M Na2CO3, pH 11.5, or 0.5 M potassium acetate, incubating on ice for 30 min, and recovering the extracted membranes by centrifugation for 20 min at 150,000 × g in a Ti-70.1 rotor (Beckman Instruments). The pellet was resuspended in SDS sample buffer. Protein remaining in the supernatant was precipitated with 10% trichloroacetic acid (w/v) and resuspended in SDS sample buffer.

Protease protection assays were performed by incubating an aliquot of the in vitro translation mixture on ice for 30 min with 0.1 mg/ml trypsin (type XIII; Sigma) in the presence or absence of 0.05% Triton X-100 as described previously (14). An aliquot incubated in the absence of trypsin served as a control for endogenous protease. The reaction was terminated by the addition of phenylmethylsulfonyl fluoride for a final concentration of 10 mM. Microsomal membranes were recovered by centrifugation for 20 min at 150,000 × g in a Ti-70.1 rotor (Beckman Instruments). The pellet was resuspended in SDS sample buffer.

SDS Gel Electrophoresis-- Proteins in SDS sample buffer were loaded onto 13% SDS-polyacrylamide gels, the gels were impregnated with 1 M sodium salicylate, dried, and exposed to preflashed Kodak X-Omat AR film at -70 °C, as described previously (13).

    RESULTS

Common Features of Euglena Chloroplast Protein Presequences-- Conceptual translation of cDNAs encoding Euglena chloroplast proteins has revealed the sequence for one thylakoid protein precursor, pLHCPII (18, 19), and five stromal protein precursors, pSSU (22), the precursor to porphobilinogen deaminase (23), precursor to translational initiation factor-3 (24), precursor to fructose-1-6-bisphosphate aldolase (25), and precursor to glyceraldehyde-3-phosphate dehydrogenase (26). The presequences have highly divergent primary sequences, but they are similar in size (approximately 140 amino acids) and share a number of structural features (Fig. 1). The algorithm of von Heijne and co-workers (27) identifies the N-terminal 24-46 amino acids as a signal peptide composed of a charged N-terminal region, a central hydrophobic core of variable length and a short sequence preceding a potential signal peptidase cleavage site (Fig. 1). A second hydrophobic domain (Fig. 1) is found with a C terminus 13-31 amino acids from the presequence mature protein junction. Alignment of the presequences at the predicted signal peptidase cleavage site shows that for each presequence, the second hydrophobic domain is in the same relative position, approximately 60 amino acids, from the predicted signal peptidase cleavage site (Fig. 1). The region between the signal peptidase cleavage site and the second hydrophobic domain is enriched in hydroxylated amino acids, a feature characteristic of stromal targeting transit peptides (4). The Euglena presequences are thus bipartite, being composed of an ER targeting signal peptide domain and a stromal targeting domain containing a hydrophobic region.


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Fig. 1.   Comparison of the deduced amino acid sequence of the presequences of the known Euglena chloroplast precursors identifying common structural and targeting features. The deduced amino acid sequence for the bipartite presequences of the pLHCPII (18, 19), the pSSU (22), the precursor to porphobilinogen deaminase (pPBGD), the precursor to translational initiation factor-3 (pIF3) (24), the precursor to fructose-1-6-bisphosphate aldolase (pFba1) (25), and the precursor to glyceraldehyde-3-phosphate dehydrogenase (pGapA) (26) were aligned at their predicted (27) signal peptidase cleavage site (*). Potential hydrophobic membrane-spanning domains within the signal peptide domain and stromal targeting domain of the presequences are underlined, and hydroxylated amino acids are in boldface type. CONS is a diagrammatic representation of the consensus presequence structure with hydrophilic regions in gray, hydrophobic reigons in black, and an arrow indicating the predicted signal peptidase cleavage site.

Localization of the pLHCPII Signal Peptide to the Precursor Presequence-- Previous work has shown that canine microsomes co-translationally import and process Euglena chloroplast protein precursors (14, 18). In order to identify ER targeting and topogenic domains within the precursor and determine precursor topology within the transport vesicle membrane, import of a series of pLHCPII deletion and glycosylation reporter constructs into canine microsomes was studied. Clone 1NLHCP encodes a 39-kDa protein composed of the 141-amino acid LHCPII presequence and a mature LHCPII (Fig. 2A). LHCPII is an integral thylakoid protein containing three hydrophobic membrane-spanning domains. Based on the criteria of von Heijne (27), the first presequence hydrophobic domain is part of an N-terminal signal peptide, while the second presequence hydrophobic domain, the second hydrophobic domain of mature LHCPII, and the third hydrophobic domain of mature LHCPII are hydrophobic cores of potential internal signal peptides that can function as start and stop transfer membrane anchor sequences (15). An N-linked glycosylation site at Asp148 (Fig. 2A) is found near the presequence mature protein junction, providing a reporter for translocation of the N terminus of the mature protein across the microsomal membrane.


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Fig. 2.   Canine microsomal processing of pLHCPII presequence deletion constructs. A, schematic diagram of pLHCPII presequence deletion constructs indicating locations of potential topogenic sequences. The predicted signal peptidase cleavage site is indicated by an arrow, hydrophobic domains within the presequence and mature protein are indicated by black boxes, and the potential glycosylation site within mature LHCPII (Asp148) is indicated by a lollipop shape. B, microsomal processing and glycosylation of pLHCPII deletion constructs. mRNA transcribed from the indicated clones was translated in the absence (lane 1) or presence (lanes 2-9) of canine microsomes. After terminating translation, microsomal membranes were incubated with Endo H (EndH), washed with 0.5 M potassium acetate (Ac) or 0.1 M Na2CO3, pH 11.5 (CO3), and separated into a pellet (P) and supernatant (S) fraction or treated with trypsin (Tryp) in the presence and absence of Triton X-100 (Trit). Translation products were separated by SDS gel electrophoresis and visualized by fluorography.

Translation of 1NLHCP mRNA in the presence of canine microsomes produced a protein approximately 3.5 kDa smaller than the protein produced in the absence of microsomes, indicative of cleavage of a 35-amino acid signal peptide (Fig. 2B). Digestion of the ER-inserted 1NLHCP translation product with Endo H had no effect on the electrophoretic mobility of the synthesized protein (Fig. 2B), indicating that Asp148 was not translocated across the microsomal membrane. Na2CO3 extraction converts microsomal vesicles into open sheets releasing luminal and peripheral proteins, while integral proteins remain associated with the membranes (28). The processed 1NLHCP translation product remained associated with microsomal membranes after Na2CO3 extraction (Fig. 2B), and the translation product was digested by trypsin both in the presence and absence of detergent (Fig. 2B). These results indicate that a cleaved signal peptide initiates insertion of the 1NLHCP translation product into the membrane and that a stop transfer sequence N-terminal to Asp148 stops translocation anchoring the protein within the membrane oriented with the mature protein, including Asp148, remaining exposed on the cytoplasmic membrane face.

A chloroplast homologue of the 54-kDa subunit of signal recognition particle, cp54SRP, specifically binds the third hydrophobic domain of LHCPII during insertion into the thylakoid membrane (29). A plant LHCPII containing a bacterial signal sequence expressed in Escherichia coli is inserted into the bacterial inner membrane as an integral membrane protein anchored by at least one of the LHCPII hydrophobic domains (30). These results raise the possibility that the third transmembrane domain of mature LHCPII may be involved in insertion and orientation of Euglena pLHCPII within the ER. In order to determine whether all of the ER targeting information is contained within the pLHCPII presequence, two constructs have been prepared. Clone 1NLHCPDelta 1-138 encodes a protein whose translation starts at Met43 of pLHCPII which is 8 amino acids C-terminal to the predicted signal peptidase cleavage site (Fig. 2A). The 1NLHCPDelta 1-138-encoded protein lacks the N-terminal presequence signal peptide domain but contains the presequence stromal targeting domain (Fig. 2A). The second construct, 1LHCP, lacks the presequence encoding only the mature LHCPII (Fig. 2A).

Translation of 1NLHCPDelta 1-138 mRNA produced three peptides, and translation of 1LHCP mRNA produced two peptides (Fig. 2B). In each case, the largest has the size expected for the plasmid-encoded protein, suggesting that the additional proteins are produced through utilization of alternative initiation codons or by premature termination. The size of the 1NLHCPDelta 1-138- and 1LHCP-encoded peptides was unaltered when microsomes were present during translation (Fig. 2B) and after Endo H digestion (Fig. 2B). The 1NLHCPDelta 1-138 translation product remained associated with microsomal membranes after Na2CO3 extraction, while most of the 1LHCP translation product did not sediment with acetate- or Na2CO3-extracted membranes (Fig. 2B). The translation products from both plasmids were digested by trypsin in the presence or absence of detergent (Fig. 2B). Taken together, these results localize all of the ER targeting information to the presequence and indicate that the hydrophobic domains within LHCPII do not play a role in targeting the precursor to the ER.

The sedimentation with Na2CO3-extracted membranes, sensitivity to endogenous protease, and the absence of glycosylation at Asp148 indicate that the 1NLHCPDelta 1-138-encoded peptides are anchored in the membrane with the N terminus in the lumen and the C terminus remaining in the cytoplasm. Many integral membrane proteins contain signal anchor sequences that initiate translocation and anchor the protein within the membrane (15, 17). The algorithm of von Heijne (27) identifies the hydrophobic domain within the stromal targeting region of pLHCPII as the hydrophobic core of a signal peptide. In the 1NLHCPDelta 1-138-encoded protein, this signal peptide is near the N terminus and appears to be functioning as a signal anchor sequence initiating translocation and anchoring the proteins in the microsomal membrane. Consistent with the positive inside rule (15, 16), the less positively charged region N-terminal to the signal anchor sequence is translocated into the lumen, and the more positively charged region C-terminal to the signal anchor remains on the cytoplasmic membrane face. Signal peptides function as membrane anchor sequences when they are positioned C-terminal to another signal peptide (15), suggesting that in pLHCPII, the function of the hydrophobic domain within the stromal targeting region is to stop translocation and anchor the precursor within the microsomal membrane.

Identification of the Presequence Membrane Anchor Domain and Determination of the Topology of the Precursor within the Membrane-- In order to directly determine if the hydrophobic domain within the presequence stromal targeting region (Fig. 1) functions as a stop transfer membrane anchor sequence and to determine the orientation of pLHCPII within the membrane, glycosylation reporter and stromal targeting region deletion constructs were made, and their import into canine microsomes was studied. PCR was used to construct clone 1NLHCPglyc40 encoding a protein containing a glycosylation site at Asp40 of pLHCPII in addition to the glycosylation site at Asp148 (Fig. 3A). Translation of 1NLHCPglyc40 mRNA in the presence of microsomal membranes produced a protein whose apparent size was larger than the protein synthesized in the absence of membranes (Fig. 3B), and Endo H digestion reduced the apparent size of the protein (Fig. 3B). The deglycosylated protein was smaller than the protein synthesized in the absence of microsomal membranes, indicating that the signal peptide was cleaved. The glycosylated 1NLHCPglyc40-encoded protein pelleted with acetate- and Na2CO3-extracted membranes but was digested by trypsin (Fig. 3B). Since Asp148 is not glycosylated in the 1NLHCP-encoded protein (Fig. 2B), the 1NLHCPglyc40-encoded protein must be glycosylated at Asp40, localizing the stop transfer membrane anchor sequence to the hydrophobic domain within the presequence stromal targeting region. The deduced orientation of pLHCPII is with the presequence N terminus formed after signal peptidase cleavage in the ER lumen and the mature protein on the cytoplasmic membrane face.


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Fig. 3.   Canine microsomal processing of pLHCPII presequence deletion glycosylation reporter constructs. A, schematic diagram of pLHCPII presequence deletion glycosylation reporter constructs indicating locations of potential topogenic sequences. The predicted signal peptidase cleavage site is indicated by an arrow, hydrophobic domains within the presequence and mature protein are indicated by black boxes, and potential glycosylation sites within the presequence (Asp40) and mature LHCPII (Asp148) are indicated by a lollipop shape. B, microsomal processing and glycosylation of pLHCPII glycosylation reporter deletion constructs. mRNA transcribed from the indicated clones was translated in the absence (lane 1) or presence (lanes 2-9) of canine microsomes. After terminating translation, microsomal membranes were incubated with Endo H (EndH), washed with 0.5 M potassium acetate (Ac) or 0.1 M Na2CO3, pH 11.5 (CO3), and separated into a pellet (P) and supernatant (S) fraction or treated with trypsin (Tryp) in the presence and absence of Triton X-100 (Trit). Translation products were separated by SDS gel electrophoresis and visualized by fluorography.

To directly localize the stop transfer membrane anchor to the presequence stromal targeting region, the import into canine microsomes of two glycosylation reporters containing the presequence N-terminal signal peptide but lacking amino acids 46-146, the stromal targeting domain, was studied. Clone 1NLHCPglyc40Delta 138-438 and clone 1NLHCPDelta 138-438 encode proteins lacking most of the presequence stromal targeting domain, amino acids 46-140, and the first 6 amino acids of LHCPII (amino acids 141-146 (Fig. 3A)). Both proteins contain the glycosylation site, Asp148, found within LHCPII at the presequence mature protein junction. Clone 1NLHCPglyc40Delta 138-438 contains an additional glycosylation site at Asp40 (Fig. 3A).

Translation of 1NLHCPglyc40Delta 138-438 and 1NLHCPDelta 138-438 in the presence of microsomal membranes produced a group of proteins that have apparent molecular weights larger than the proteins produced in the absence of membranes (Fig. 3B). The apparent size increase for the 1NLHCPglyc40Delta 138-438-encoded proteins containing two potential glycosylation sites was greater than the apparent size increase for the 1NLHCPDelta 138-438-encoded proteins containing a single glycosylation site. After deglycosylation with Endo H, the 1NLHCPglyc40Delta 138-438- and 1NLHCPDelta 138-438-encoded proteins were the same size (Fig. 3B), consistent with a difference in the number of sites glycosylated. Of the two deglycosylated 1NLHCPglyc40Delta 138-438- and 1NLHCPDelta 138-438-encoded proteins, one was the same size and one was smaller than the protein synthesized in the absence of microsomal membranes, indicative of signal peptide cleavage within the microsomal lumen (Fig. 3B). Asp148 is the only glycosylation site present in 1NLHCPDelta 138-438 (Fig. 3A), and this site is present but not glycosylated in the protein encoded by 1NLHCP (Fig. 2). Glycosylation of this site only upon deletion of the stromal targeting presequence region (amino acids 46-146) identifies the hydrophobic domain within this region (amino acids 90-110) as the stop transfer sequence that prevents translocation of mature LHCPII into the ER.

Both the 1NLHCPglyc40Delta 138-438- and 1NLHCPDelta 138-438-encoded proteins remain associated with acetate- and Na2CO3-extracted membranes (Fig. 3B), indicative of integral membrane proteins. Trypsin-resistant proteins are found when digestion is performed in both the presence and absence of Triton X-100 (Fig. 3B). Protease resistance appears to be an intrinsic property of the glycosylated proteins and is not indicative of translocation of the entire protein through the membrane into the microsomal lumen.

LHCPII is an integral thylakoid membrane protein containing three membrane-spanning domains, any one of which has the potential to stop translocation anchoring the protein in the ER membrane. A higher plant LHCPII containing a bacterial signal sequence was inserted in vivo and anchored in the bacterial inner membrane by one of the hydrophobic domains of LHCPII (30). The 1NLHCPglyc40Delta 138-438- and 1NLHCPDelta 138-438-encoded proteins are functionally equivalent to the bacterial signal peptide-containing higher plant LHCPII, and it is not surprising that as found in bacteria, one of the LHCPII hydrophobic domains functions as a stop transfer membrane anchor sequence during signal peptide-dependent translocation into the ER.

    DISCUSSION

Euglena 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 (10, 11, 31-36). Immunoelectron microscopy (37, 38) and pulse-chase intracellular localization studies (13, 14) 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 (13-14, 37, 38).

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 (10, 31, 33, 36). The third envelope membrane is thought to have evolved from the host's phagocytic vacuole membrane (11, 33). 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 (1, 3). 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 (32, 33). 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 (18). 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 (18). 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 (13) 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 (15, 16). Inspection of the known Euglena chloroplast protein presequences (17, 19, 22-26) 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 (33, 39). Similar stromal targeting sequences and similar transport mechanisms are utilized for protein import into cyanelles (7) and the chloroplasts of plants (4) green (5) and red algae (6), 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 (4), 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 the Euglena chloroplast (32, 33). The bipartite Euglena presequence arose by the addition of a signal peptide to the existing stromal targeting presequence (32, 33), enabling the precursor to be transported first to the phagocytic vacuole-derived Euglena outer chloroplast membrane (11, 33), separating the host cytoplasm from the endosymbiont, and subsequently through the intermediate and inner chloroplast membranes derived from the endosymbiont's chloroplast envelope (32, 33).

Proteins are imported into higher plant chloroplasts at envelope contact sites, regions of association between the outer and inner membrane translocation machinery (8, 9). 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 (10, 11). Regions of adherence between the Euglena intermediate and inner membrane similar to the contact sites between the plant outer and inner envelope membrane have been identified in freeze etch replicas (11). These ultrastructural studies support the assumption that protein translocation through the Euglena chloroplast 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 (2), while the intermediate and inner membranes contain an import apparatus functionally similar to the import apparatus of plant chloroplasts (4, 8, 9). 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 (2). 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 (8, 9). Presequence cleavage and, in the case of polyprotein precursors such as pLHCPII and pSSU, polyprotein processing by stromal proteases (21) would occur during or rapidly after precursor import.


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Fig. 4.   Proposed model for transport of precursor proteins through the three chloroplast membranes of Euglena. Golgi transport vesicles containing chloroplast precursors oriented with approximately 60 N-terminal amino acids projecting into the lumen fuse with the outer chloroplast membrane. The presequence migrates laterally within the plane of the membrane into the outer membrane translocation channel, and the precursor N terminus engages first middle and then inner membrane receptors, forming a translocation channel spanning the three membranes at contact sites. The precursor is translocated into the chloroplast, where the presequence is removed.

Euglena is one of a number of organisms with complex plastids having three or four envelope membranes (10, 11, 31-36). 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 (10, 31, 33, 40). 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 (33-36) like the Euglena plastids, and it is likely that vesicular transport is utilized to deliver precursors to the dinoflagellate chloroplast. The dinoflagellate presequences are shorter than Euglena presequences, and they lack a membrane anchor (32, 41-43). Upon fusion of Golgi transport vesicles with the outer membrane, the precursor would be within the intermembrane space free to diffuse to the intermediate membrane engaging import receptors on the membrane surface.

Chromophyte plastids are surrounded by four membranes, and ribosomes are found on the outermost membrane (31, 36). The chloroplast protein import system of diatoms is the most extensively characterized chromophyte system. The bipartite presequence of diatom chloroplast proteins contains a functional N-terminal cleaved signal peptide that initiates co-translational precursor import into canine microsomes (44, 45). A truncated diatom precursor containing only the stroma-targeting transit peptide is post-translationally imported into pea chloroplasts (45), providing experimental evidence for the proposed common evolutionary origin of the import apparatus of the two innermost envelope membranes of complex plastids (32, 33) and the envelope import apparatus of higher plant chloroplasts, cyanelles, and red and green algae. The presequence of the ER-resident protein BiP from the diatom Phaeodactylum contains a signal peptide (46). Diatoms and other chromophytes appear to have solved the problem of traversing the additional chloroplast membranes by evolving a new signal recognition particle targeting system enabling ribosomes translating chloroplast proteins to attach directly to the outermost chloroplast envelope membrane for co-translational translocation across the outermost membranes while allowing those making proteins destined for the ER and other organelles to utilize the ancestral ER targeting system.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB-9630817.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Horticulture, Purdue University, West Lafayette, IN 47907.

§ To whom correspondence should be addressed: School of Biological Sciences, E 207 Beadle Center, University of Nebraska, Lincoln, NE 68588. Tel.: 402-472-1682; Fax: 402-472-8722; E-mail: schwartz{at}biocomp.unl.edu.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; Endo H, endoglycosidase Hf; PCR, polymerase chain reaction; LHCPII, light-harvesting chlorophyll a/b-binding protein of photosystem II; pLHCPII, precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II; pSSU, precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase..

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