1 Martek Biosciences Corp, 6480 Dobbin Rd., Columbia, MD 21045, USA
2 Fachbereich Biologie, Universität Konstanz, Postfach M611, 78457
Konstanz, Germany
3 School of Botany, University of Melbourne, Parkville, Victoria 3052,
Australia
4 Carnegie Institution of Washington, Department of Plant Biology, 260 Panama
Street, Stanford, CA 94305, USA.
* Authors for correspondence (e-mail: kirkapt{at}martekbio.com; peter.kroth{at}uni-konstanz.de)
Accepted 23 July 2002
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Summary |
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Key words: Complex plastids, Bipartite pre-sequence, Diatom, Green fluorescent protein, Plastid import
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Introduction |
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Four membranes delineate the chromophytic plastid. The inner two appear to
be homologous to the double membrane envelope of plastids from red algae,
green algae and vascular plants. A third unique membrane completely surrounds
the plastid envelope and is thought to represent the former plasma membrane of
the endosymbiont, while the outermost membrane may have evolved from the
vacuolar/plasma membrane of the host organism. This outermost membrane has
been observed to be continuous with the endoplasmic reticulum
(Gibbs, 1981;
Ishida et al., 2000
). The
portion of this ER-like membrane directly adjacent to plastids (and which
appears to completely encase the plastid) is commonly referred to as the
chloroplast ER or CER (Bouck,
1965
).
Since many genes required for plastid function were lost from the genome of
the endosymbiont and relocated to the nuclear genome of the host organism, it
was critical to develop efficient transport of nuclear-encoded proteins across
the four membranes that delineate the plastid. Early observations suggested
that nuclear-encoded plastid polypeptides of diatoms and other chromophytic
algae required a multi-signal pre-sequence to facilitate trafficking of
polypeptides into plastids (Gibbs,
1979). Characterization of genes encoding several nuclear-encoded
plastid polypeptides from chromophytic algae
(Grossman et al., 1990
;
Pancic and Strotmann, 1993
)
predicted pre-sequences with bipartite structures. The N-terminal regions of
these pre-sequences are similar to classical signal sequences while the
C-terminal regions have characteristics of transit peptides, sequences
required for the transport of polypeptides into vascular plant plastids. In
vitro studies demonstrated that the putative ER targeting domain could
facilitate co-translational import of precursors of the fucoxanthin
chlorophyll a/c binding proteins (FCP) into isolated canine
microsomal vesicles (Bhaya and Grossman,
1991
; Lang et al.,
1998
). Furthermore, the putative transit peptide region of the
pre-sequence was sufficient to direct in vitro transport of polypeptides into
isolated pea or spinach plastids (Lang et
al., 1998
). With the development of a relatively easy means of
introducing genes into the nuclear genome of diatoms, we have started to
dissect the role of specific domains of the pre-sequence associated with
nuclear-encoded plastid polypeptides in the multi-step transport process that
delivers these proteins to their site of function within the plastid.
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Materials and Methods |
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Plasmid constructs
All GFP fusions (Fig. 1)
were inserted into the EcoRI and HindIII sites of the P.
tricornutum transformation vector pPha-T1
(Zaslavskaia et al., 2000).
This vector contains the sh ble (Zeo) gene fused with the
fcpB promoter region and the fcpA terminator region. A
multiple cloning site is flanked by the fcpA promoter region and the
fcpA terminator region. Fusion of pre-sequences to EGFP were
generated either by creating restriction sites by `full-circle'-PCR or by
fusion-PCR using fusion primers (36 bp) as described previously
(Pont-Kingdon, 1997
). For the
C-terminal addition of the 6xHis residues, individual PCR primers were
designed containing a (CAC)6TAA motif added in frame 3' of
the last four codons of the GFP fusion genes. His-tagged polypeptides were
partially purified from P. tricornutum transformants by fractionation
of cells into soluble and membrane compartments. Soluble fusion protein was
further purified by Ni-affinity chromatography (Qiagen NTA-column) and the
isolated polypeptide sequenced from its N-terminus.
|
Microparticle bombardment
Cells were bombarded using the Bio-Rad Biolistic PDS-1000/He Particle
Delivery System fitted with 1350 or 1500 psi rupture discs. The tungsten
particles M17 (1.1 µm median diameter) were coated with 0.8 µg plasmid
DNA in the presence of CaCl2 and spermidine, as described by the
manufacturer. Approximately 5x107 cells were spread in the
center one third of a plate of solid 0.5x I.O. medium 1 hour before
bombardment. The plate was positioned at the second level within the Biolistic
chamber for bombardment. Bombarded cells were illuminated for 24 hours (cells
divided once during this period) prior to suspension in 0.5 ml of sterile
0.5x I.O. medium; 100 µl of the cell suspension
(1x107 cells) were plated onto solid medium containing
100 µg/ml Zeocin. Plates were placed under constant illumination (75
µmol photon m-2 s-1) for 2-3 weeks and resistant
colonies re-streaked onto fresh solid medium containing 100 µg/ml
Zeocin.
Fluorescence microscopy
Standard microscopical analyses and confocal laser scanning microscopy were
performed using a Leica TCSNT system. Separation of GFP fluorescence and
chlorophyll autofluorescence was achieved with a 525/550 nm band pass filter
and a long pass filter of 590 nm. For 3D images, 50-90 individual scans of one
cell were saved as a raytrace script build by the program `KLM to POV', which
served to reconstruct the 3D structure by the program `POV-RAY'.
Electron microscopy
Cells of transformed Phaeodactylum strains were grown as clonal
cultures in 250 ml erlenmeyer flasks containing 100 ml of sterile K-medium
containing added silicates (Andersen et
al., 1997). The cultures were maintained at 16°C in a 16 hour
light/8 hour dark photoperiod under cool white and Grolux fluorescent lights.
50 ml aliquots of each strain were collected and centrifuged in 50 ml tubes at
1000 g for 5 minutes. The supernatant was discarded and
droplets of the remaining cell suspension were sandwiched between two freezer
hats (Type A, ProSciTech), with well depths of 100 µm, ensuring that all
air bubbles were excluded. The enclosed cell suspensions were then frozen
using the Leica EM High Pressure Freezer. The freezer hats enclosing the
tissue samples were split apart, and the hats to which the frozen cell pellets
were adhered were stored under liquid N2 in cryovials prior to
freeze-substitution. Frozen cell pellets were freeze-substituted in 0.1%
uranyl acetate in acetone at -90°C for 72 hours, and the temperature
raised to -50°C at 1°C/h. The cell pellets were scraped out of the
freezer hats and rinsed three times for 30 minutes each in 100% acetone.
Samples were then infiltrated with a graded series of HM20 low temperature
resin in acetone consisting of 10% resin (5 hours), 30% resin (overnight), 50%
resin (8 hours), 70% resin (12 hours), 90% resin (8 hours), and 100% resin (12
hours). The infiltrated samples were placed in a fresh change of 100% resin in
gelatin capsules, polymerised under UV light for an additional 48 h at
50°C, and brought slowly to room temperature. The soft sample blocks were
then hardened under UV light for a further 48 hours at room temperature.
Polymerised blocks were sectioned on a Reichert Ultracut microtome and gold
sections collected onto formvar coated 50 mesh hexagonal gold grids. Prior to
immuno-gold-labelling, the sections on grids were blocked in PBS containing
0.8% BSA and 0.01% Tween-20 for 30 minutes. Grids were then incubated,
section-side down, on 30 µl droplets of anti-GFP primary antibody, diluted
1:200 with blocking agent, for 3 hours at room temperature. The grids were
washed four times on drops of blocking agent for 10 minutes each. Rinsed grids
were then incubated on 30 µl droplets of goat anti-rabbit secondary
antibody (diluted 1:20 with blocking agent), conjugated to 15 nm gold
particles, for 12 hours at 4°C. Labelled grids were rinsed once on
droplets of blocking agent, three times on droplets of PBS and then immersed
twice for 30 seconds each time in distilled water. Negative controls were
performed by using secondary antiserum only. The grids were then air dried and
sequentially stained with uranyl acetate for 10 minutes and Triple Lead Stain
for 5 minutes (Sato, 1968
) and
viewed in a Phillips Biotwin transmission electron microscope at 100 kV.
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Results |
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The pre-sequence on the BiP:GFP chimeric protein used for these studies
contained the complete signal sequence (21 amino acids) plus the first 12
amino acids of the mature protein (Fig.
1A). The pre-sequence of the nuclear-encoded AtpC protein from
P. tricornutum is 54 amino acids
(Fig. 1B) with a putative
signal sequence from amino acids 1-16. The signal sequence domain is
characterized by an arginine at position 2 followed by a region of hydrophobic
amino acids. A transit peptide-like domain, with a high proportion of the
hydroxylated amino acids serine and threonine, extends from amino acid 17 to
54. A similar transit peptide-like sequence is present on the unprocessed AtpC
subunit of the centric diatom Odontella sinensis
(Pancic and Strotmann, 1993)
and on several other diatom plastid precursor proteins (P.G.K. and O.K.,
unpublished). All of the chimeric genes analyzed were expressed from the
fcpA promoter, which was previously shown to effectively drive
expression of GFP in P. tricornutum
(Zaslavskaia et al., 2000
;
Zaslavskaia et al., 2001
);
localization of GFP in the cell was readily visualized by fluorescence
microscopy.
ER targeting of GFP
Previous studies have demonstrated that GFP expressed in P.
tricornutum becomes localized to the cytoplasm
(Zaslavskaia et al., 2000;
Zaslavskaia et al., 2001
). As
presented in the right panel of Fig.
2A, GFP devoid of a targeting sequence (vector designation
pPTEGFP) appeared to accumulate in the cytoplasm, mostly towards the center of
the cell in the region of the nucleus. The GFP fluorescence was excluded from
plastids and vacuoles; the plastids are clearly visualized as brown-pigmented
structures in the cell (Fig.
2A, left).
|
When a DNA fragment encoding the pre-sequence of the P.
tricornutum ER-localized chaperone BiP
(Apt et al.,
1995,Apt et al.,
1995
) was fused to GFP and the chimeric gene (BiP:GFP)
expressed in P. tricornutum, GFP fluorescence was observed in a
network of membranes traversing the entire length of the cell
(Fig. 2B, right). These
membranes are thought to represent the ER. Addition of a diatom ER retention
sequence [DDEL (Apt et al.,
1995
,Apt et al.,
1995
)] to the C-terminus of the BiP:GFP fusion protein made no
observable difference in GFP localization (data not shown). Western blots did
not show detectable amounts of GFP in the culture medium of BiP:GFP-expressing
Phaeodactylum cells, indicating that also BiP:GFP without DDEL might
not be secreted out of the cell. However, an effect of the DDEL domain on ER
retardation of the BiP-GFP construct cannot be ruled out as GFP expression
varies in independent transformants, and BiP-GFP without DDEL might be
secreted/targeted to vacuoles followed by proteolytic degradation.
Confocal images of cells harboring BiP:GFP have a distinct sphere of GFP fluorescence near the middle of the cell (Fig. 3A) which, based on DAPI staining (not shown), corresponds exactly to the position of the nuclear envelope. The GFP fluorescence, in part, also delineates the position of the plastid. As shown in the 3D reconstitution of a confocal image series (Fig. 3B), a meshwork of GFP fluorescence extends throughout the cell (the position of the plastid was determined by red chlorophyll autofluorescence). There is the consistent appearance of ER membranes that associate with and form around the plastid, mostly longitudinal with respect to the plane of the cell. While results from electron microscopy suggest that diatom plastids reside within the lumen of the ER (see Introduction for references), the BiP signal sequence-targeted GFP was not observed in the compartment that isolates the plastid from the cytoplasm of the cell. These results suggest that detectable levels of BiP:GFP were not able to enter the CER segment of the intracellular membrane system.
|
To determine the exact site at which the BiP pre-sequence was cleaved, we constructed a vector encoding an N-terminal 6xHis-tagged BiP:GFP fusion. This vector was transformed into P. tricornutum cells, and GFP was observed to localize to the ER in a pattern identical to that observed for the analogous construct devoid of the 6xHis tag. The fusion protein was isolated from transformants by affinity chromatography using the Ni2+-affinity column and the N-terminus of the purified polypeptide was sequenced. The results demonstrated that the transport of GFP into the ER involved cleavage of the chimeric polypeptide after alanine at position 21 (Fig. 1A).
Plastid targeting
Expression of GFP fused to the complete pre-sequence of the
plastid-localized AtpC subunit (pATPC1GFP in
Fig. 1B) resulted in
accumulation of GFP in plastids; there is an exact congruence of GFP
fluorescence with the position of the plastid as observed by both light (the
plastid is apparent as a brown pigmented structure in
Fig. 2C) and fluorescence
microscopy (chlorophyll autofluorescence, not shown). The congruence was also
visualized in the analysis of a confocal image series in which the relative
position of GFP fluorescence was compared with that of chlorophyll
autofluorescence (data not shown).
Removal of the ER targeting domain of AtpC
In the construct AtpC8GFP, the region encoding the 14 amino acids following
the initiator methionine at the N-terminus of the AtpC1GFP fusion protein was
removed (Fig. 1B). GFP
expressed from this construct accumulated in the cytoplasm of the cell. The
pattern of accumulation was identical to that observed for GFP without a
pre-sequence (Fig. 2A); no GFP
fluorescence was apparent in either the plastid or ER. These results suggest
that plastid-targeted polypeptides must enter the ER prior to plastid
localization; the transit peptide-like domain alone is not sufficient for
routing polypeptides into plastids in vivo.
Deletions of the transit peptide
While the size of the signal sequence is very similar among a variety of
plastid pre-proteins characterized in chromophytes, the length of transit
peptide-like domains may vary considerably. As in vascular plants, it is
difficult to identify sequence motifs within transit peptides that might be
critical for import function; however, all transit peptides (in vascular
plants as well as in algae) appear to contain a high proportion of
hydroxylated amino acids (von Heijne et
al., 1989; Liaud et al.,
1997
). The AtpC1GFP chimeric gene was modified to generate
deletions in the transit peptide segment of the pre-sequence. Constructs
AtpC2GFP, AtpC3GFP and AtpC4GFP, shown in
Fig. 1B, encode a series of
AtpC:GFP deletions from the C-terminal end of the transit peptide domains. As
many as 24 amino acids of the transit peptide could be removed, with 14 amino
acids of the original transit peptide remaining, without an observable effect
on plastid localization. Hence, the majority of the transit peptide-like
domain of AtpC is not absolutely necessary for importing polypeptides into
plastids. The addition of the putative ER retention sequence, DDEL, to the
C-terminal ends of these chimeric polypeptides made no observable difference
in the level of GFP that accumulated in plastids (data not shown).
Surprisingly, the chimeric AtpC5GFP polypeptide, which contains only three
amino acids of the presumed transit peptide
(Fig. 1B), also accumulated in
the plastid, although the efficiency of transport and accumulation is not
known (the signal seems generally lower than in cells harboring the full
length AtpC:GFP construct). Interestingly, when an ER retention signal was
fused to the C-terminus of AtpC5GFP (AtpC5GFPDDEL construct), GFP fluorescence
was visualized as strands of fluorescence extending over the length of the
cells, and encircling the nucleus and plastids. This localization pattern
appeared identical to that observed for cells expressing the BiP:GFP fusion
(Fig. 2B,
Fig. 3A), suggesting that
fusion of the ER retention sequence to AtpC5GFP strongly interfered with the
transfer of the precursor polypeptide from the ER to the plastid.
Immunocytochemical localization of GFP in cells transformed with the
AtpC5GFPDDEL construct confirmed that GFP was concentrated around the nuclear
envelope (Fig. 4A) and the ER
emanating from the envelope. In a number of sections, high levels of GFP were
also visualized close to the plastid surface
(Fig. 4B); localization at the
plastid surface might reflect the position of the ER rings that were observed
to encircle plastids in cells expressing BiP:GFP.
|
To determine the position at which the AtpC precursor protein is cleaved by the signal peptidase in vivo, an AtpC4GFP construct was 6xHis tagged (fused to the C-terminus) and transformed into P. tricornutum. As in the case of the original AtpC4GFP construct, GFP expressed from the 6xHis-tag-modified construct localized to the plastid (data not shown). The tagged polypeptide was purified from transformants by Ni2+ affinity chromatography and its N-terminal sequence was found to begin with TTQ. This placed the ER-recognized cleavage site between amino acid 16 (phenylalanine) and 17 (threonine) of the AtpC pre-sequence (Fig. 1B) and shows that cleavage by the stromal peptidase does not occur, probably because the recognition site for the protease was deleted in this construct.
Interestingly, in cells harboring AtpC4GFP fused to a 6xHis tag, GFP
fluorescence accumulated within a distinct region of the plastid near its
mid-point (Fig. 5A).
Immunocyto-chemistry of the transformed cells confirmed that the
6xHis-tagged GFP was present in the plastid, and highly concentrated in
a region corresponding to the `lobe' that is positioned central to the plastid
(Fig. 5B); the lobe region is
associated with plastid division
(Borowitzka et al., 1977;
Borowitzka and Volcani, 1978
).
The presence of a 6xHis tag on plastid-targeted GFP may cause a spurious
association of the polypeptide with components of the plastid division
apparatus.
|
Deletions in the pre-sequence junction
Constructs AtpC6GFP and AtpC7GFP encode fusion proteins with deletions that
extend from the C-terminal end of the transit peptide into the C-terminal end
of the signal sequence (Fig.
1A). In both cases GFP fluorescence accumulated in structures near
the center of the cell and adjacent to the plastids
(Fig. 6A). Reconstitution of
confocal images of strains transformed with AtpC6GFP and AtpC7GFP showed that
the centrally located GFP was spatially distinct from plastids and appeared to
be within structures wrapped around the central portion of the plastid
(Fig. 6B). Analysis of GFP
localization by immunocytochemistry demonstrated that the GFP protein was
found both in mitochondria and plastids, additional analysis using the
fluorescent dye `Mito-Tracker' supports this result (data not shown). In
P. tricornutum a single, multilobed mitochondrium is typically
present in the center of the cell and directly adjacent to the plastid. The
extensive truncations of the AtpC pre-sequences associated with the AtpC6GFP
and AtpC7GFP constructs apparently resulted in a highly anomalous localization
pattern, with most GFP accumulating in mitochondria. Previous studies with
yeast have demonstrated that a variety of randomly generated pre-sequences can
promote the promiscuous entry of polypeptides into mitochondria
(Allison and Schatz, 1986;
Baker and Schatz, 1987
).
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Discussion |
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The evolution of organelles from free-living organisms following engulfment
must have involved extensive transfer of genetic material from the genome of
the endosymbiont to the nucleus of the host organism
(Martin and Herrmann, 1998).
Hence, the synthesis of many polypeptides required for diverse functions of
the symbiont moved to the cytoplasm of the host cell. These events made it
imperative that the associated organisms develop efficient pathways for
importing polypeptides into evolving plastids. Recent information has added
considerably to our understanding of the transport of polypeptides into
plastids that are delineated by a double envelope membrane (evolved via
primary endocytobiosis) (Chen and Schnell,
1999
; Heins et al.,
1998
). In vascular plants and green algae, nuclear-encoded plastid
polypeptides are translated in the cytosol of the cell and an N-terminal
transit peptide directs post-translational import of polypeptides into
plastids. In contrast, little is known about processes required for routing
polypeptides into the complex plastids that have evolved via secondary
endocytobiosis (van Dooren et al.,
2001
; Kroth,
2002
).
Complex plastids have been observed in a variety of algal lineages.
Plastids from these algae are delineated by multiple membranes (either three
or four distinct membranes, with a CER only present in chromophytes and
cryptophytes). Although strategies for routing polypeptides into complex
plastids evolved following distinct endosymbiotic events, there are functional
parallels among the different algal lineages with respect to mechanisms by
which polypeptides are routed to plastids. All known pre-sequences necessary
for the import of polypeptides into complex plastids are multipartite. For
some organisms, the secretory system is involved in the primary targeting step
(McFadden, 1999). In the
Euglenophytes, the plastids are delineated by three membranes but are
not associated with a CER (Gibbs,
1978
). Cell fractionation studies have demonstrated an association
of plastid pre-proteins with both Golgi and ER membranes, suggesting that
precursor polypeptides initially pass through the cytosolic ER with subsequent
translocation to the plastids via the Golgi system
(Sulli et al., 1999
).
Secretory-system-dependent transport has also been demonstrated for
Plasmodium (four membranes with no CER)
(Waller et al., 2000
).
Diatoms, a diverse group of organisms with four membranes that delineate
the plastid (Gibbs, 1981), are
representative of the chromophytic algae. The chromophytes include the
Bacillariophyceae (diatoms), Eustigmatophyceae, Phaeophyceae
and Chrysophyceae. Based on electron microscopy, plastids of
chromophytic algae were proposed to be completely compartmentalized within the
lumen of the ER, with those ER-like membranes immediately around the plastid
being designated CER (Bouck,
1965
; Gibbs,
1981
). Gibbs suggested that polypeptides routed into chromophytic
algal plastids must pass through the CER prior to translocation across the
plastid envelope (Gibbs,
1981
). This implied that targeting of polypeptides to plastids
would require multiple targeting signals. In accord with this hypothesis,
characterization of nuclear-encoded plastid polypeptides from various
chromophytic algae were shown to have leader sequences with two putative
targeting domains (Grossman et al.,
1990
; Apt et al.,
1994
; Apt et al.,
1995
,Apt et al.,
1995
; Kroth and Strotmann,
1999
). The first domain resembles a classic signal sequence that
appears to be required for transport into the ER
(Walter and Johnson, 1994
).
The second domain of the pre-sequence is variable in length but has
characteristics similar to those of transit peptides
(Heins et al., 1998
), which
are enriched for the hydroxylated amino acids serine and threonine.
Some experiments have been performed to help establish the functions of the
different pre-sequence domains associated with plastid-localized diatom
polypeptides. These pre-sequences could drive co-translational import of
proteins in vitro into canine microsomes
(Bhaya and Grossman, 1991;
Lang et al., 1998
), a system
extensively used to study the role of the signal sequence in transporting
polypeptides into the ER. This work demonstrated that nuclear-encoded plastid
polypeptides in the diatoms contain a functional ER targeting signal.
Furthermore, the transit peptide-like domain, found on nuclear-encoded plastid
polypeptides of diatoms, facilitated the import of polypeptides into isolated
vascular plant plastids (Lang et al.,
1998
). Together, these studies illustrate that the different
targeting signals present on nuclear-encoded plastid polypeptides of diatoms
can facilitate the transport of those polypeptides across both ER and plastid
envelope membranes. These results also demonstrate that no additional import
signals within or at the C-terminus of the mature protein are necessary for
the targeting process.
The generation of a complete in vitro system for elucidating events in the
transport and processing of plastid polypeptides in the chromophytes will be
extremely difficult as such a system would require cell disruption followed by
the isolation of, or enrichment for plastids enclosed by four intact
membranes. Wastl and Maier demonstrated that a preprotein encoded on the
genome of the periplastidic nucleomorph of Guillardia theta, and
therefore containing a transit peptide only, could be imported
post-translationally into isolated homologous complex plastids
(Wastl and Maier, 2000).
Therefore, the outer membranes must have been lost or severely disrupted
during the preparation of plastids.
To bypass the difficulties of developing an in vitro system, we have begun
to examine targeting of polypeptides to diatom plastids, as well as to other
subcellular compartments, using an in vivo system. In this in vivo system,
constructs were generated that encode fusions between the pre-sequences of
polypeptides targeted to different locations in the cell with GFP. In initial
experiments we demonstrated that the complete pre-sequence of the AtpC
precursor and of other plastid preproteins (O.K. and P.G.K., unpublished) are
sufficient to drive the import of GFP into diatom plastids in vivo. Therefore,
no other potential targeting signals within the mature region of diatom
plastid precursors are required for transport of proteins across the four
membranes that separate the stromal compartment of the plastid from the
cytoplasm. The signal sequence-like domain of the AtpC pre-sequence plus three
amino acids of the transit peptide fused to GFP directed GFP into the
plastids. Removal of the signal peptide domain from the fusion protein
resulted in cytoplasmic accumulation of GFP, while removal of most of the
transit peptide, coupled with the addition of an ER retention signal
(AtpC5GFPDDEL, Fig. 1),
resulted in accumulation of GFP in the ER. The short transit peptide present
on the AtpC5GFPDDEL construct did not prevent localization directed by the ER
retention signal. These results strongly suggest that the first step in
targeting polypeptides to plastids in chromophytic algae requires passage of
that polypeptide into the ER. The GFP that accumulated in the ER appeared as
thread-like strands present throughout the cytoplasm and extending laterally
within the cell. A sphere of fluorescence also encased the nucleus, probably
representing the nuclear envelope. Immuno-electron microscopy confirmed that
the GFP was localized to cytoplasmic ER and the nuclear envelope, which is
continuous with cytoplasmic ER. Furthermore, a distinct ring of GFP
fluorescence was shown to encircle plastids in the region of the valvar axis.
A 3D reconstruction of confocal images demonstrates that this GFP ring does
not completely encase the plastid. Based on EM studies of P.
tricornutum, other diatoms and other chromophytes, an ER-like membrane
called the CER is closely appressed to the plastid envelope membranes and
completely surrounds the organelle
(Crawford, 1973;
Borowitzka and Volcani, 1978
;
Gibbs, 1981
). The CER also
appears to be continuous with cytoplasmic ER membranes. Furthermore, the
periplastidic space between the CER and the plastid envelope membranes
contains vesicular and tubular structures that have been hypothesized to be
directly involved in transporting polypeptides from the ER to the outer
envelope membranes of the plastid (Smith-Johannson and Gibbs, 1972;
Gibbs, 1979
;
Gibbs, 1981
). We never
observed either ER- or plastid-targeted GFP in these vesicles (although it is
possible that the small size of these vesicles and relatively low GFP content
precluded their visualization). Hence the role of these vesicles in
polypeptide trafficking remains uncertain.
The transit peptide domains in chromophytic algae are variable in both size and amino acid sequence. Diatom FCP pre-sequences have transit peptides of about 15 amino acids, while those of AtpC are 37 or 40 amino acids. Interestingly, our results indicate that a significant portion of the C-terminal region of the AtpC transit peptide domain is not required for targeting GFP to plastids (Table 1); as few as three amino acids (TTQ) at the N-terminus of this domain targeted GFP into plastids. Those regions that were deleted from the transit peptide might function to improve the efficiency of the import process.
|
Localization of GFP bearing AtpC pre-sequences appears to be unaffected by the presence of an ER retention signal, except in the case of the AtpC5GFP. GFP accumulated in plastids when transformants expressing the AtpC5GFP chimeric protein were devoid of an ER retention signal. However, when the DDEL sequence was attached to the C-terminus of this fusion protein, GFP accumulated in the ER. Since the DDEL sequence had no apparent effect on fusion proteins targeted to the ER by the BiP signal sequence or to plastids by the complete AtpC pre-sequence, it is unlikely that an ER retrieval mechanism accounts for the accumulation of the AtpC5GFPDDEL polypeptide in the ER. An altered conformation of this polypeptide as a consequence of the attached DDEL sequence may interfere with plastid routing. There may be no distinction between plastid- and ER-targeted polypeptides at the first import step. The exchange of polypeptides between the cytosol and plastid compartments may involve the entire network of ER membranes rather than just the CER. Routing of plastid-localized polypeptides through the general secretory pathway offers a potentially larger surface area for the exchange of substrate between plastids and cytosol, which may be advantageous for systems in which four membrane barriers constrain polypeptide trafficking. Other signals on plastid pre-proteins may function in the subsequent targeting of polypeptides from the CER, although there is no direct evidence that links the CER to the targeting process.
The work presented in this manuscript demonstrates the power of using in
vivo analyses to dissect polypeptide trafficking in diatoms. Along with other
studies on protein targeting into complex plastids
(Sulli et al., 1999;
Waller et al., 2000
), our
results demonstrate the fundamental importance of the secretory pathway for
the establishment and maintenance of secondary endocytobiosis. We also
demonstrate that a signal sequence accounts for passage of polypeptides into
the ER lumen, while only a small portion of the transit peptide-domain appears
absolutely necessary to mediate transport across the double envelope membrane
of the plastid. Furthermore, these studies raise significant questions with
respect to the way in which polypeptides traverse the second most distal of
the four membranes that delineate plastids, the function of the carboxy
terminal region of the transit peptide and the role of the CER in polypeptide
targeting.
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Acknowledgments |
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References |
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