From the School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588
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ABSTRACT |
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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.
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.
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 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% 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 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.
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.
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 1NLHCP
Translation of 1NLHCP
The sedimentation with Na2CO3-extracted
membranes, sensitivity to endogenous protease, and the absence of
glycosylation at Asp148 indicate that the
1NLHCP 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.
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 1NLHCPglyc40
Translation of 1NLHCPglyc40
Both the 1NLHCPglyc40
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 1NLHCPglyc40 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
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 1NLHCP
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
1NLHCPglyc40
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 1NLHCPglyc40
138-438.
The plasmid 1NLHCP
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.
-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).
70 °C, as described previously
(13).
RESULTS
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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.
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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.
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 1NLHCP
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).
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 1NLHCP
1-138- and
1LHCP-encoded peptides was unaltered when microsomes were present
during translation (Fig. 2B) and after Endo H digestion
(Fig. 2B). The 1NLHCP
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.
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 1NLHCP
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.
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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.
138-438 and clone
1NLHCP
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
1NLHCPglyc40
138-438 contains an additional glycosylation site at
Asp40 (Fig. 3A).
138-438 and 1NLHCP
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 1NLHCPglyc40
138-438-encoded proteins containing two potential glycosylation sites was greater than the apparent size
increase for the 1NLHCP
138-438-encoded proteins containing a single
glycosylation site. After deglycosylation with Endo H, the
1NLHCPglyc40
138-438- and 1NLHCP
138-438-encoded proteins were
the same size (Fig. 3B), consistent with a difference in the
number of sites glycosylated. Of the two deglycosylated
1NLHCPglyc40
138-438- and 1NLHCP
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
1NLHCP
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.
138-438- and 1NLHCP
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.
138-438- and
1NLHCP
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
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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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REFERENCES |
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