From the Institute of General Botany,
Friedrich-Schiller-University Jena, Am Planetarium 1, D-07743 Jena,
Germany and the ¶ Department of Genetics, The Hebrew University of
Jerusalem, Jerusalem, 91904 Israel
Received for publication, July 19, 2000, and in revised form, November 10, 2000
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ABSTRACT |
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The carotenoid biosynthetic pathway in algae and
plants takes place within plastids. In these organelles,
carotenoids occur either in a free form or bound to proteins. Under
stress, the unicellular green alga Haematococcus pluvialis
accumulates secondary carotenoids, mainly astaxanthin esters, in
cytoplasmic lipid vesicles up to 4% of its dry mass. It is
therefore one of the favored organisms for the biotechnological
production of these antioxidative compounds. We have studied the
cellular localization and regulation of the enzyme Carotenoids play major roles in oxygenic photosynthesis where they
function in light harvesting and protect the photosynthetic apparatus
from excess light by energy dissipation (1). Carotenoids that fulfill
these processes are commonly referred to as primary carotenoids,
because they are essential for the basic metabolism of the organism. In
contrast, secondary carotenoids
(SC)1 are defined
functionally as carotenoids that are not obligatory for photosynthesis
and are not localized in the thylakoid membranes of the chloroplast
(2). SC function in specific stages of development (e.g. flower, fruit), mainly for coloration or under extreme
environmental conditions. In plants, SC are often accumulated in
special structures, for instance in plastoglobuli of chromoplasts. In
some green algae, however, SC accumulate outside the plastid in
cytoplasmic lipid vesicles. One typical example is the unicellular
microalga Haematococcus pluvialis, well known for its
massive accumulation of ketocarotenoids, mainly astaxanthin and its
acylesters, in response to various stress conditions, e.g.
nutrient deprivation or high irradiation (3). Different functions of SC
in H. pluvialis such as acting as a sunshade (4), protecting
from photodynamic damage (5), or minimizing the oxidation of storage
lipids (6) have been proposed. There is growing commercial interest in
the biotechnological production of astaxanthin because of its
antioxidative properties and the increasing amounts needed as
supplement in the aquaculture of salmonoids and other seafood (7).
H. pluvialis is one of the preferred microorganisms for this
purpose because it accumulates SC at up to 4% of its dry mass (3).
The pathway of astaxanthin biosynthesis in H. pluvialis was
elucidated by inhibitor studies (8), and most of the involved genes are
cloned (6, 9, 10). In higher plants and green algae, the carotenoid
precursor, isopentenylpyrophosphate (IPP) is derived from the DOXP
pathway (synonyms are nonmevalonate or MEP pathway, Ref. 11). For SC
synthesis in H. pluvialis this was confirmed with inhibitor
studies (12). The first specific steps in carotenogenesis lead to the
formation of the tetraterpene phytoene. Following desaturation and
-carotene
oxygenase in H. pluvialis that catalyzes the introduction
of keto functions at position C-4 of the
-ionone ring of
-carotene and zeaxanthin. Using immunogold labeling of ultrathin
sections and Western blot analysis of cell fractions, we discovered
that under inductive conditions,
-carotene oxygenase was localized
both in the chloroplast and in the cytoplasmic lipid vesicles, which
are (according to their lipid composition) derived from cytoplasmic
membranes. However,
-carotene oxygenase activity was confined to the
lipid vesicle compartment. Because an early carotenogenic enzyme in the
pathway, phytoene desaturase, was found only in the chloroplast
(Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000)
Plant Physiol. 122, 1261-1268), a transport of
intermediates from the site of early biosynthetic steps in the
chloroplast to the site of oxygenation and accumulation in cytoplasmic
lipid vesicles is proposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cyclization,
-carotene is formed. The subsequent steps in the
pathway leading to astaxanthin in H. pluvialis are catalyzed
by
-carotene hydroxylase (10) and
-carotene oxygenase (CRTO,
synonym is
-carotene ketolase, BKT; for a recent review see
Cunningham and Gantt, Ref. 13 and Fig.
1).
View larger version (23K):
[in a new window]
Fig. 1.
Pathway of secondary carotenoid synthesis in
H. pluvialis. The enzymes catalyzing the late enzymatic
steps, namely -carotene oxygenase (CRTO,
-carotene
ketolase, BKT), presented in this paper, and the
-carotene
hydroxylase (CRTR-B) are indicated.
Little is known about the regulation of SC synthesis in vivo
in response to stress. The gene for CRTO, the enzyme studied in this
paper, was cloned from two different strains of H. pluvialis by Lotan and Hirschberg (14) and Kajiwara et al. (15). A
series of -carotene oxygenases (among them one from H. pluvialis), and bacterial
-carotene hydroxylases were
characterized in vitro with respect to substrate specificity
and cofactor requirements (16, 17). Moreover, conversion of
-carotene by cell extracts of H. pluvialis was reported
(18). Recently, we have studied regulation and compartmentation of
phytoene desaturase (PDS), an early enzyme of the carotenoid
biosynthetic pathway (19). The enzyme is up-regulated at the mRNA
level during SC synthesis and localized exclusively in the chloroplast.
This is consistent with the common hypothesis that in plants including
algae carotenoids are synthesized exclusively within plastids (11).
H. pluvialis is distinguished in that it accumulates large
amounts of carotenoids in lipid vesicles outside the plastid (3, 20).
This has given rise to speculation about the possible existence of a
biosynthetic pathway specific for secondary carotenogenesis that is
localized in the cytoplasm, as was supported by the existence of two
different IPP isomerases in H. pluvialis (6). However, no
extra pathway specific for SC biosynthesis in the cytosol of H. pluvialis was found at the level of PDS (19). It was therefore hypothesized that carotenoids are transported from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmic lipid vesicles). Here, we present a study of the origin of these lipid
vesicles as well as regulation and compartmentation of the SC
biosynthetic-specific ketolase CRTO in flagellates of H. pluvialis using immunolocalization and cell fractionation
techniques. Our results indicate that the last oxygenation steps in the
astaxanthin biosynthesis pathway take place outside the plastid in the
cytoplasmic lipid vesicles and is discussed relative to the role of
this sequestering structure in SC accumulation.
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MATERIALS AND METHODS |
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Cell Growth Conditions--
H. pluvialis Flotow
(No.192.80, culture collection of the University of Göttingen,
Germany; synonym: Haematococcus lacustris (Girod)
Rostafinski) was grown autotrophically in a two-step batch cultivation
system as described (21). Following precultivation for 5 days at 25 µmol of photons m2 s
1 of white
fluorescent light (Osram L36/W25, Berlin, Germany), flagellates in the
logarithmic growth phase were exposed to SC-inducing conditions
(nitrate-deprived medium and 150 µmol of photons m
2
s
1 of continuous white light) leading to accumulation of
SC in the flagellated developmental state of H. pluvialis
(21). These flagellates surrounded by a thin extracellular matrix are
more accessible to biochemical and ultrastructural analysis than the thick-walled and resistant aplanospore state.
Photon flux densities were measured using a LI-189 photometer (LI-COR, Lincoln, NE), and cell number was determined using a Cell Counter Casy 1 (Schärfe Systems, Reutlingen, Germany). At the time points specified, sample aliquots corresponding to a defined cell number were collected by centrifugation at 1,400 × g for 2 min.
Preparation of Cell Fractions--
Cell fractions were prepared
by gentle filtration rupture that produced less contamination of the
lipid vesicle fraction by light harvesting complexes (LHC) and
chlorophylls than sonication. Aliquots of cells were harvested by
centrifugation at 1,400 × g for 2 min and resuspended
in break buffer consisting of 0.1 M Tris-HCl, pH 6.8, 5 mM MgCl2, 10 mM NaCl, 10 mM KCl, 5 mM Na2EDTA, 0.3 M sorbitol, 1 mM aminobenzamidine, 1 mM aminohexanacid, and 0.1 mM
phenylmethylsulfonyl fluoride. The hyperosmotically shocked cells were
broken by passage through a 10-µm isopore polycarbonate filter
(Millipore, Eschborn, Germany). The filtrate was centrifuged at
10,000 × g for 10 min at 4 °C to yield a
chloroplast and cell debris pellet. The supernatant was transferred to
a fresh tube and centrifuged again at 10,000 × g for
10 min at 4 °C. The suspension below the lipid vesicle fraction
floating on top was transferred to a fresh tube and centrifuged at
76,000 × g for 2 h at 4 °C. The resulting
microsome pellet was separated from the supernatant fraction. All
fractions were stored at 20 °C.
Lipid Analysis--
Cell aliquots or lipid vesicle preparations
were extracted essentially as described (22). Lipids were then
separated on HPTLC plates (Merk, Darmstadt, Germany), developed for
two-thirds of the plate in chloroform/methanol/acetic acid/water,
73:25:2:4 (v/v/v/v) to separate the polar lipids and subsequently, in a second development, with hexane/diethylether/acetic acid, 85:20:1.5 (v/v/v) for the whole plate to separate the neutral lipids from the
pigments. Lipids were identified by cochromatography of standard substances and by color reaction with different spray reagents (ninhydrin for free amines of phosphatidylethanolamine (PE) and phosphatidylserine (PS); -naphthol for glyco- and
sulfolipids; molybdenium blue for phospholipids; Dragendorff's reagent
for quarternary amines, phosphatidylcholine (PC) and
diacylglyceryltrimethylhomoserine (DGTS)). Quantification of individual
lipids was performed densitometrically after visualization by Godin's
spray reagent (23) and calibration with standard substances. For
quantification of DGTS and PS, calibration data of PE and PC were used, respectively.
Treatment with Inhibitors of Carotenogenesis-- At the onset of SC-inducing conditions, diphenylamine (DPA, Sigma) was added to a final concentration of 30 µM. After 3 days, cells were washed three times, resuspended in fresh nitrate-deprived medium, and incubated for 2 more days under SC inductive conditions with 5 µM norflurazone (NF; SAN 9879; Sandoz Basel, Switzerland), 30 µM DPA or 30 µM DPA plus 5 µM NF, respectively.
Antibody Preparation-- The 17-amino acid peptide LPHCRRLSGRGLVPALA, corresponding to the C terminus (residues 304-320) in the predicted sequence of BKT (15) and residues 315-329 (with the last three amino acids missing) in the predicted sequence of CRTO (14), was chemically synthesized and purified (Alpha Diagnostics International, San Antonio, TX). The peptide was coupled to thyroglobulin by means of glutaraldehyde and used for immunization of rabbits to raise polyclonal antibodies as described (24). The raw serum was deployed without further purification.
Protein Analysis-- Cell pellets were thawed on ice, suspended in break buffer, and broken by sonication for 1 min on ice (Vibra-Cell 72405 sonication processor, Sonics & Materials, Danbury, CT; pulse mode, 0.75 s on, 1 s off, 60 watts output). Break buffer with SDS was added to yield a final concentration of 2% SDS (w/v). Solubilization, especially of hydrophobic proteins like CRTO, was carried out upon shaking at 2,000 rpm for 2 h at 20 °C. Samples were centrifuged to remove unsolubilized material, and sample loading buffer to a final concentration of 50 mM Tris-HCl, pH 6.8, 2% SDS (w/v), 10% glycerol (v/v), and 0.01% bromphenol blue was added. Cell fractions were thawed on ice and resuspended in break buffer with 2% SDS (w/v), and solubilization was performed as described for total cell extracts. Before loading cell aliquots, samples were boiled for 5 min. Proteins were separated on 12% SDS-polyacrylamide gels essentially as described (25). For Western blot analysis, the gels were electrophoretically transferred semidry onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and treated with Ponceau S for staining the protein ladder transiently. Membranes were blocked in blocking buffer containing 5% (w/v) nonfat dry milk, 1% Tween 20 (v/v), 150 mM NaCl, and 25 mM Tris-HCl, pH 7.6 at 4 °C overnight. Then the blots were challenged with anti-CRTO antibodies in blocking buffer at 1:250 dilution for 1 h at 4 °C and thereafter with secondary antibody alkaline phosphatase conjugates (Bio-Rad, Munich, Germany) used at 1:500 dilution. After the chromogenic reaction with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT), the labeling was quantified using densitometry (Scanpack 3.0, Biometra, Göttingen, Germany). Total protein content was determined by means of the detergent compatible protein assay kit (Bio-Rad, Munich, Germany).
Electron Microscopy and Immunolocalization-- For ultrastructural examination, algal cells were harvested at 550 × g for 3 min and then fixed with 0.7% glutaraldehyde, 0.8% paraformaldehyde, and 1% OsO4 simultaneously in growth medium for 25 min at 4 °C. After several washes in distilled water the specimens were dehydrated in graded ethanol series. The 70% ethanol step was performed in the presence of 3% uranylacetate for 10 min. Cells were embedded in LR Gold (London Resin, London) according to the manufacturer's instructions. Before immunogold labeling, ultrathin sections were cut as described (19) and etched to unmask antigenic determinants (26). Etching was done by floating grids section side down on 2% H2O2 for 2 min at room temperature followed by three washes on distilled water. The grids were exposed to anti-CRTO antibody at 1:100 dilution, and immunogold labeling was performed as described (19). Subsequent to poststaining with 3% aqueous uranylacetate (w/v) for 5 min and 1% aqueous lead citrate (w/v) for 20 s, immunogold-labeled sections were examined in a Zeiss EM 900 electron microscope (Carl Zeiss, Oberkochen, Germany) at 80 kV.
In Vitro Incubations--
Incubations were carried out in a
total volume of 600 µl under conditions essentially as reported (16,
17). Cell fraction aliquots of 107 cells were suspended in
break buffer (0.1 M Tris-HCl, pH 6.8, 0.3 M
sorbitol, 1 mM aminobenzamidine, 1 mM
aminohexanacid, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride) in a total volume of 300 µl. Following
the addition of 295 µl of cofactor buffer (5 mM ascorbic
acid, 1 mM dithiothreitol, 0.5 mM
FeSO4, 0.1% deoxycholate (w/v), 0.5 mM
2-oxoglutarate) and brief mixing, the reaction was initiated by
addition of 5 µl of a 1% -carotene stock solution (w/v) in
chloroform. In parallel samples, 100 µM DPA were added to
inhibit
-carotene oxygenase. Incubation was performed under
continuos stirring for 2 h in the dark at 30 °C. Reactions were
terminated by freezing the samples in liquid nitrogen.
Pigment Extraction and HPLC Analysis--
Cell pellets were
extracted quantitatively in 100% acetone at 4 °C, and the pigment
content was determined spectrophotometrically according to
Lichtenthaler (27). Fractions were freeze-dried, and carotenoids were
extracted with 200 µl of acetone (the chloroplast fraction was
extracted with 500 µl of acetone) at 4 °C. In vitro incubations were freeze-dried, and pigments were extracted with acetone, at 4 °C. Prior to HPLC analysis, samples were filtered and
20% water (v/v) was added. HPLC analysis was performed as described
(21).
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RESULTS |
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Origin of the SC-accumulating Lipid Vesicles--
Lipid profiles
of total extracts from cells drawn after 4 days of exposure to
conditions inductive for SC synthesis revealed massive accumulation of
TAG during SC synthesis (Fig. 2).
Concomitantly, the amount of most membrane lipids, especially of MGDG,
decreased whereas that of DGDG and DGTS increased slightly (Table
I). Analysis of the lipid vesicles formed
under inductive conditions revealed triglycerides as their predominant
lipid class. Membrane lipids accounted for less than 5% (w/w) of total
lipids in this fraction. No MGDG was detectable, and DGDG and DGTS made
up half of the membrane lipids in this fraction besides significant
amounts of PC and of PE.
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Coupled in Vivo Inhibitor Treatments--
Application of low
concentrations of DPA under conditions inductive for synthesis of SC
led to accumulation of -carotene instead of ketocarotenoids in
H. pluvialis (8, 21, 28). Lipid vesicles in the cytoplasm of
treated cells appeared yellow instead of red in control samples,
suggesting that
-carotene accumulated in the cytoplasm (8). To
substantiate this observation, we determined the pigment composition in
different cellular compartments. Results revealed a predominant
accumulation of
-carotene inside the lipid vesicles of DPA-treated
cells (Fig. 3). To test if this extraplastidic
-carotene can be converted to astaxanthin, DPA was
removed concomitantly with the addition of NF to inhibit carotenoid de novo synthesis at the level of phytoene desaturation.
Beside the known bleaching effect of NF leading to a reduced amount in total carotenoids, a significant decrease in the ratio of
-carotene to ketocarotenoids occurred inside the lipid vesicles (Fig. 3). The
pattern of SC in this fraction did not differ significantly from
untreated samples consisting mostly of mono- and diesters of
astaxanthin.
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Generation of Antibodies against CRTO-- Compartmentation studies and regulation analysis of the late steps in SC biosynthesis in H. pluvialis require specific antibodies against the enzymes involved. Attempts to obtain antibodies against the His-tagged C terminus of CRTO, encompassing two-thirds of the polypeptide overexpressed in Escherichia coli, were unsuccessful. Despite poor expression and isolation difficulties because of pronounced hydrophobic behavior of the protein, necessary amounts of the antigen were recovered by Ni2+-affinity chromatography and subsequent purification steps. The generated polyclonal antibodies recognized a series of proteins on Western blots and did not meet the needs for localization experiments, even after parallel immunization experiments and various purification approaches by affinity chromatography. Interestingly, we noticed an increasing oligomerization tendency of the overexpressed antigen up to the octamer, even under denaturating SDS-polyacrylamide gel electrophoresis conditions, dependent on storage time. Finally, we immunized rabbits with a 17-mer synthetic oligopeptide corresponding to the C-terminal part of the predicted structure of CRTO. Database searches revealed no counterparts of this peptide among plant amino acid sequences.
Abundance of CRTO during SC Accumulation--
The ability of the
antibodies to recognize less than 30 ng of CRTO in Western blots was
verified with the E. coli-overexpressed C-terminal part of
the enzyme (data not shown). Abundance of CRTO was examined in total
cell extracts of start samples and of samples taken 1, 2, 3, 4, and 7 days after inducing SC synthesis in the flagellates of H. pluvialis by intense illumination and nitrate deprivation (Fig.
4A). No CRTO was observed
before the second day after induction. After this time, the amount of a
34-kDa protein increased rapidly in parallel to SC accumulation (Fig.
4B). The apparent molecular mass of the recognized protein
was ~3 kDa smaller than predicted from the cDNA sequence of CRTO
(14). The preimmune serum did not detect this polypeptide (not
shown).
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Immunogold Localization of CRTO--
The antibodies against the
C-terminal 17-mer of CRTO were tested on LR Gold sections of H. pluvialis flagellates that have previously been shown to present
the best combination for structural preservation and maintenance of
antigenic structures (19). To prevent the extraction of cellular lipids
during dehydration steps and to ensure full preservation, high pressure
cryofixation in combination with cryodehydration was applied. However,
despite a number of modifications of the preparation protocol, the
structure of lipid vesicles could not be improved. Thus, an etching
technique was chosen as described (26). During the ethanolic
dehydration process before embedding, the lipid vesicles remained
intact because of lipid cross-linking by OsO4. Probing the
sections with polyclonal antibodies against different photosynthetic
proteins (19) did not reveal any signals because of masking of
antigenic determinants by the fixative. To unmask antigenic
determinants, sections were exposed to hydrogen peroxide for a defined
time span. Accessibility of antigenic determinants after etching was
confirmed with anti-LHC and anti-PDS antibodies, which detected the
corresponding polypeptides as reported previously (19). After
challenging the sections with the polyclonal antibodies raised against
the CRTO C-terminal 17-mer, two cell compartments became specifically
immunogold-labeled in the course of SC synthesis, namely the
chloroplast and, becoming dominant, the lipid vesicles (Fig.
5, Table
II). Labeling of the latter compartment
was not restricted to the periphery, but was scattered throughout the
vesicles. The only notable signal in the cytosol was obtained after 2 days of inductive conditions (15%) and was localized in close contact
to the Golgi cisternae (not shown). No specific labeling was observed
when sections were probed with preimmune serum (not shown).
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Detection of CRTO in Subcellular Fractions--
To ascertain the
results from the immunogold localization experiments, four cellular
fractions were obtained: (i) a pellet containing mainly the
chloroplast, (ii) a supernatant fraction, (iii) microsomes and
cytoplasmic membranes, and (iv) the lipid vesicles (19). The
polypeptides in each fraction were analyzed by Western blots using the
anti-CRTO antibodies. A 34-kDa polypeptide was observed in the
chloroplast membrane fraction (Fig. 6),
in the lipid vesicle fraction and, to a small extend, in the microsome fraction. Fractions shown here were derived from flagellates 7 days
after start of SC induction, thus representing the maximum of CRTO
protein in total extracts (Fig. 4).
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In Vitro Metabolism of -Carotene in Cell Fractions--
To
provide additional support for astaxanthin synthesis inside the lipid
vesicles, we investigated the ability of various subcellular fractions
to metabolize
-carotene in vitro. Cofactors and reaction
conditions were essentially as reported recently for recombinant
-carotene oxygenases from different organisms (16, 17). Cell
fractions were prepared from flagellates exposed for 3 days to SC
inductive conditions, which contained relatively low initial amount of
ketocarotenoids. We observed a conversion of
-carotene to
ketocarotenoids in the lipid vesicle fraction, but not in the
chloroplast fraction (Fig. 7). The SC
product pattern included mainly mono- and diesters of astaxanthin. The
pool sizes, i.e. the total of
-carotene and
ketocarotenoids, remained constant in all samples. Control experiments
with heat-denatured extracts (16) were not feasible because of
concomitant degradation of fraction pigments. Therefore, DPA was
applied to inhibit
-carotene oxygenase but did not completely
prevent the conversion of
-carotene in the lipid vesicle fraction.
This is consistent with results from in vivo experiments
(Fig. 3, Ref. 21).
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DISCUSSION |
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Carotenoid accumulation in plant cells requires specialized accumulation structures (29). Changes in the lipid composition during the period of induction of SC synthesis in H. pluvialis, namely prominent TAG accumulation and remarkable reduction of the chloroplast-specific lipid MGDG, reflect the microscopically visible formation of lipid vesicles (20, 30) and corresponding changes in the photosynthetic apparatus (30, 31), respectively. To elucidate whether the SC-accumulating lipid vesicles of H. pluvialis are derived from the plastid or from cytoplasmic compartments, we analyzed their lipid composition separately. As expected, TAG made up the main part of lipids in this fraction (95%). Of the membrane lipids, the plastidic MGDG was totally absent, whereas PE as a typical nonplastidial lipid was found. DGTS, the major membrane forming lipid in the SC-containing vesicles, which are surrounded by a half-membrane (20), is known to be primarily localized in nonplastidial membranes (32). The second abundant lipid DGDG was recently shown to be synthesized in the cytoplasm under nutrient starvation conditions (33). Altogether the results point to a cytoplasmic origin of the lipid vesicles presenting an oleosome-like structure (34).
Low concentrations of DPA inhibit ketocarotenoid biosynthesis by
preventing the introduction of oxygen functions and, thus, -carotene
accumulates instead of ketocarotenoids (8, 21, 28). Furthermore, Harker
& Young (8) observed in cells pretreated with DPA that in the presence
of norflurazone, a known inhibitor of phytoene desaturase, SC were
formed at the expense of
-carotene. We repeated this experiment with
our cultivation scheme where SC accumulate in the flagellated state of
H. pluvialis, thus allowing pigment analysis of the
cytoplasmic lipid vesicles after cell fractionation. Surprisingly, the
-carotene that accumulated inside these lipid vesicles was
oxygenated to ketocarotenoids. This implies that the
cytoplasmic-located lipid vesicles play a role in the synthesis of SC
in addition to their function as storage structure for these compounds.
A crucial point in understanding the regulation of secondary
carotenogenesis in H. pluvialis is the localization of the
enzymes involved. Therefore, immunolocalization using antibodies
against SC-specific enzymes was chosen to gain corresponding data. The problems that occurred during overexpression of the SC-specific CRTO
and its subsequent purification brought us in contact with its
special properties, particularly the very hydrophobic behavior of the
enzyme. Probably because of sequence similarity of CRTO to fatty acid
desaturases (13) and the relatively high antigenicity of the conserved
di-iron binding regions containing histidine residues (35), the
polyclonal antibodies generated from the overexpressed antigen showed
cross-reactivity with many other proteins. In contrast, antibodies
generated against a 17-mer synthetic oligopeptide representing the
C-terminal part of the predicted structure of CRTO, still recognizing
the CRTO polypeptide expressed in E. coli, reacted
specifically with a 34-kDa polypeptide in the protein extract of
induced H. pluvialis cells. The slight decrease in the
apparent molecular mass as compared with the size predicted (14, 15)
might indicate processing of a N-terminal transit signal peptide. From
the highly conserved structure of the -carotene oxygenases from two
different strains of H. pluvialis with respect to the
oligopeptide used for immunization and from the polyclonal nature of
the antibodies, it can be concluded that isoenzymes of CRTO should have
been recognized in our cytoimmunochemical experiments. Additionally the
similar size of CRTO observed in the chloroplast and in the lipid
vesicles did not support the existence of isoenzymes or different
-carotene oxygenases in H. pluvialis as was speculated
from the two existing sequences (6). More likely this reflects strain differences.
The observed pattern of CRTO induction in parallel to carotenoid
accumulation denotes the essential role of the enzyme in SC
biosynthesis. -carotene oxygenase mRNA levels have been shown to
exhibit similar kinetics of induction during the first 4 days of our
cultivation scheme, but thereafter they declined to 50% of the maximum
(19). This behavior was different for the earlier carotenogenic enzyme
PDS that showed a parallel change in the amounts of mRNA and of the
protein (19). Thus, beside regulation at the mRNA level,
post-translational mechanisms seem to be involved in CRTO induction.
Immunogold labeling of ultrathin sections revealed that in contrast to
PDS, which is localized in the chloroplast only, CRTO is present both
in the chloroplast and inside the SC-containing lipid vesicles.
Interestingly, the signals were not restricted to the vesicle boundary
but were distributed throughout the whole lumen of the vesicles,
consistent with the observed hydrophobic behavior of CRTO. Because this
location was confirmed by Western blot experiments in cell fractions,
we conclude that CRTO occurs in both compartments. Colocalization of
proteins and carotenoids in sequestering structures was reported for
the carotene globule protein (CGP) in Dunaliella bardawil, a
close relative of H. pluvialis (36). This protein is
restricted to the periphery of the globules and was suggested to
function in stabilizing the -carotene globule structure within the
chloroplast. A similar function beside ketolase activity is unlikely
for CRTO, because of its low abundance. The discrepancy between the
exclusive chloroplast localization of PDS that is up-regulated during
SC biosynthesis (19) and the in vivo and (exclusive)
in vitro CRTO activity in the lipid vesicles implies a
transport of carotenoid precursors, possibly of
-carotene, across
the chloroplast envelope into the cytoplasm where they are sequestered
in the lipid vesicles. However, electron microscopic investigation did
not reveal any structure for such a transport, at least not at the
level of membrane-enclosed vesicles (30).
Two enzymes are involved in the biosynthesis of astaxanthin from
-carotene,
-carotene C-4 oxygenase (ketolase) and
-ring hydroxylase. The in vivo and in vitro conversion
of
-carotene to astaxanthin in the cytoplasmic lipid vesicles also
predicts the occurrence and activity of a
-ring hydroxylase in this
compartment. This is of particular interest, because a
-carotene
hydroxylase exists in the chloroplast too, as is evident from the
formation of zeaxanthin. As the closest relatives of CRTO, fatty acid
desaturases are localized in a number of different compartments, among
them the chloroplast and microsomes (13, 37). These enzymes act on very
hydrophobic substrates. That could be the origin of the ability
of CRTO to act in the extraordinary environment of lipid vesicles. On
the other hand, this hydrophobic environment might provide an
explanation for the sustained increase of the CRTO protein despite the
decreasing mRNA level, by protecting protein from protease attacks.
A possible explanation for the mode of action of CRTO in this
compartment is that the enzyme, albeit its presence throughout the
lipid vesicle matrix, is active only at the periphery, allowing access
to cofactors needed because of the spatial closeness to ER
structures and Golgi vesicles. This hypothesis is strengthened by
electron microscopic observations on this prominent colocalization
(20).
Evolutionarily, one could imagine a desaturase engaged in chloroplast
fatty acid desaturation that was excreted into the cytoplasmic lipid
vesicles and subsequently acquired the competence to oxygenate -carotene. Although a search for transit signal peptides did not
yield a clear result, the nuclear-encoded CRTO could be transported first into the chloroplast as proposed for all plant carotenoid biosynthesis enzymes (11), including the
-carotene hydroxylase (13).
-carotene oxygenase might then be exported from the chloroplast into
the cytoplasm, possibly together with the substrate
-carotene, accumulating in the oleosome-like lipid vesicles. The tendency of the
almost complete CRTO antigen to form multimers could play an important
role, causing a changed secondary structure of the complex favorable
for transport outside the chloroplast. Additionally, the monomer might
be the active form of the enzyme that can be established only in a very
hydrophobic environment. That speculation is supported by the fact that
in vitro CRTO activity is increased by the presence of a
strong detergent, deoxycholate, in the cofactor buffer (17). Import
studies that could test our hypotheses cannot be performed with
H. pluvialis due do the reticulate structure of the
chloroplast and the difficulties associated with isolating this
compartment intact.
Further studies will focus on the carotenoid transport from the
chloroplast and the role of the lipid vesicles as storage structures in
the regulation of SC accumulation. The latter fact was brought recently
into consideration. Rabbani et al. (38) showed that in the
unicellular alga D. bardawil, the secondary -carotene
accumulation in intraplastidic lipid droplets is controlled by the
formation of this sequestering structure. Our hypothesis on the origin
of CRTO would also imply that
-carotene accumulation in D. bardawil represents the phylogenetically older type of SC accumulation in plants, conserved in chromoplasts of higher plants.
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ACKNOWLEDGEMENTS |
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We thank Prof. W. Braune who had suggested and encouraged us to work on Haematococcus. We are grateful to M. Fiedler for technical assistance, V. Mann, M.-A. Rebhahn, and M. Utting for practical support and helpful discussions. We also acknowledge M. Eckert (Jena, Dept. of Zoology) for advice regarding the antibody generation, M. Melzer (IPK Gatersleben) for helping us with cryofixation and cryodehydration, Dr. M. Ramm (Jena, Dept. of Pharmaceutical Biology) for valuable advice on thin layer chromatography and U. Johanningmeier (University of Halle) for kindly providing anti-spinach-LHC and anti-spinach-rubisco small subunit antibodies.
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FOOTNOTES |
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* Preliminary in vivo data were presented at International Congress XI on Photosynthesis in Budapest, 1998. This study was supported in part by Grant B301-69013 from the Thüringer Ministerium für Forschung, Wissenschaft und Kultur.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.
Dedicated to the occasion of the 65th birthday of Prof. Dr. Wolfram Braune.
§ Recipient of a graduate fellowship from Freistaat Thüringen and a short term fellowship from Deutscher Akademischer Austauschdienst. To whom correspondence should be addressed: Tel.: 49-3641-949225; Fax: 49-3641-949202; E-mail: kay.gruenewald@uni-jena.de.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M006400200
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ABBREVIATIONS |
---|
The abbreviations used are:
SC, secondary
carotenoids;
BKT or CRTO, -carotene oxygenase;
DGDG, digalactosyldiacylglycerol;
DGTS, diacylglyceryltrimethylhomoserine;
DOXP, 1-deoxy-D-xylulose-5-phosphate;
DPA, diphenylamine;
HPTLC, high performance thin layer chromatography;
HPLC, high
performance liquid chromatography;
IPP, isopentenylpyrophosphate;
LHC, light harvesting complex;
MEP, 2-methyl-D-erythritol-4-phosphate;
MGDG, monogalactosyldiacylglycerol;
NF, norflurazone;
PC, phosphatidylcholine;
PDS, phytoene desaturase;
PE, phosphatidylethanolamine;
PG, phosphatidylglycerol;
PS, phosphatidylserine;
TAG, triacylglycerol;
SQDG, sulfoquinovosyldiacylglycerol.
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