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201
Metabolite transporters in plastids
Ulf-Ingo Flugge
¨
Communication between plastids and the surrounding cytosol
occurs via the plastidic envelope membrane. Recent findings
show that the outer membrane is not as freely permeable
to low molecular weight solutes as previously thought, but
contains different channel-like proteins that act as selectivity
filters. The inner envelope membrane contains a variety
of metabolite transporters that mediate the exchange of
metabolites between both compartments. Two new classes
of phosphate antiporters were recently described that are
different in structure and function from the known triose
phosphate/phosphate translocator from chloroplasts. In
addition, a cDNA coding for an ATP/ADP antiporter from
plastids was isolated that shows similarities to a bacterial
adenylate translocator.
Addresses
¨ zu Koln,
¨ Lehrstuhl II, Gyrhofstr. 15,
Botanisches Institut der Universitat
¨ Germany; e-mail: uiflue@biolan.uni-koeln.de
D-50931 Koln,
Current Opinion in Plant Biology 1998, 1:201–206
http://biomednet.com/elecref/1369526600100201
Current Biology Ltd ISSN 1369-5266
Abbreviations
3-PGA 3-phosphoglycerate
AAT
ADP/ATP antiport system
DiT1
2-oxoglutarate/malate translocator
DiT2
glutamate/malate translocator
Glc6P
glucose 6-phosphate
GPT
Glc6P/phosphate translocator
PEP
phosphoenolpyruvate
PPT
PEP/phosphate translocator
TPT
triose phosphate/phosphate translocator
Introduction
Like other eukaryotic cells, plants cells contain different
organelles, amongst them plastids, a compartment that
is specific for plants. Chloroplasts, chlorophyll containing
plastids, are the site of photosynthesis during which
process atmospheric carbon dioxide is assimilated into
intermediates that are used within, as well as outside,
the chloroplasts for a variety of metabolic pathways.
Chloroplasts are also involved in nitrogen assimilation and
the synthesis of amino acids, transitory starch and a series
of secondary compounds. Plastids of non-photosynthetic
tissues have to import carbon as a source of energy and
biosynthetic pathways, for example, of fatty acids, amino
acids as well as starch. All plastids are double membranebound organelles. The inner envelope membrane is the
actual permeability barrier between the plastid and the
surrounding cytosol and the site of different metabolite
translocators that co-ordinate the metabolism in both
compartments. In this review, I describe the molecular
characterization of various transporters of plastidic mem-
branes and their role in plant metabolism, taking into
account the recent progress achieved in this field.
Export of photoassimilates from chloroplasts
The triose phosphate/phosphate translocator (TPT) mediates the export of the fixed carbon in the form of
triose phosphate or 3-phosphoglycerate (3-PGA) from the
chloroplast into the cytosol (Figure 1, no. 1). In the mature
leaves of most plants, the photosynthates exported by the
TPT are used in the formation of sucrose and amino acids
which are the main products allocated to heterotrophic
plant organs. Sucrose and amino acids are actively loaded
into the sieve element/companion cell complex by specific
H+/symporters (for reviews, see [1•,2•]).
The TPT antiport system accepts as substrates inorganic
phosphate, triose phosphates and 3-PGA which are
transported via a ping-pong type of reaction mechanism
[3,4]. Using the reconstituted system in which the concentrations of phosphate in both the internal and the external
compartments are accessible to experimental variations, it
could be demonstrated that the TPT can function not
only as an antiporter but also as a voltage-dependent ion
channel, preferentially permeable to anions [5].
The spinach TPT was the first plant membrane transport system for which the primary sequence could be
determined [6]. Meanwhile, TPT sequences from various
plants are available that all have a high similarity to
each other [7]. All TPTs are nuclear-encoded and possess
amino-terminal transit peptides that direct the adjacent
protein to the chloroplasts. In its functional state, the
TPT forms a homodimer and belongs to the group of
translocators with a 6+6 helix folding pattern, similar to
mitochondrial carrier proteins [8].
The final proof for the identity of a transporter cDNA is
the expression of the corresponding cDNA to produce the
functional protein in heterologous systems, for example
yeast cells, bacteria or oocytes. The recombinant TPT
protein, produced in yeast cells, exhibited transport
characteristics almost identical to those of the authentic
chloroplast protein [9].
The TPT is almost exclusively present in photosynthetically active tissues. To assess the role of the TPT on
photosynthetic metabolism, transgenic antisense potato
plants were created in which both the amount and the
activity of the TPT were reduced to ∼70% of the controls.
In ambient carbon dioxide and at intermediate light, there
was no significant effect on photosynthetic rates, growth
and tuber development. The starch content in the leaves
of the transformants, however, was much higher than in
wild-type plants suggesting that the daily assimilated carbon in the transformants is mainly maintained within the
Physiology and metabolism
202
Figure 1
Mal
Mal
9
Gln
MDH
OAA
OAA
Pi
Gln
GS
8
Glu
Glu
HCO –
Glu
Glu
3
PEP
Amino
acids
7
PEP
GS
GOGAT
4
Mal
Pi
Mal
6
ADP
2-OG
2-OG
10
ADP
TrioseP
TrioseP
TrioseP
Pi
OPPP
Glc6P
3
Pi
ADP-Glc
11
PEP
STARCH
AMP
2
Pi
Glc6P
5
Sucrose
1
PEP
The phosphoenolpyruvate/phosphate
translocator
e.g. Shikimic
acid pathway
Glucose
Plastid
This is in contrast to wild-type plants which generally
export the major part of the fixed carbon during the day
via the TPT. Most probably, the mobilization of the daily
accumulated starch in the antisense TPT plants proceeds
via the amylolytic starch breakdown resulting in hexoses
which are subsequently exported via a hexose translocator
(Figure 1, no.2). Interestingly, antisense TPT plants from
tobacco accumulate starch as potato plants do, but mobilize
the accumulated starch during ongoing photosynthesis.
Hexoses are exported via a glucose transporter, the activity
of which is two to three times higher in the transformants
compared to the wild-type [11]. Little is known about
this translocator that was first described twenty years ago
[12]. Caspar et al. [13] described a starch-excess mutant
from Arabidopsis, TC26, that is able to degrade starch
but obviously unable to export the products of starch
degradation from the chloroplasts via a glucose transporter
[14]. The corresponding gene has not yet been identified.
Cytosol
Current Opinion in Plant Biology
Translocators of the inner envelope membrane of plastids. 1,
Triose phosphate/phosphate translocator, TPT; this translocator
is primarily present in chloroplasts and mediates the export of
the fixed carbon in form of triose phosphates and 3-PGA. 2,
Glucose translocator; it exports the product of amylolytic starch
breakdown, glucose, from plastids. 3, PEP/phosphate translocator,
PPT; it provides plastids with PEP for the biosynthesis of PEP- and
pyruvate-derived amino acids, fatty acids, or precursors of the
shikimic acid pathway. 4, PEP/phosphate translocator present in
mesophyll chloroplasts of C4-plants. It exports PEP as substrate
for the PEP carboxylase. 5, Glucose 6-phosphate/phosphate
translocator, GPT; it imports glucose 6-phosphate into plastids
of heterotrophic tissues for the biosynthesis of starch and as
a substrate for the oxidative pentose phosphate pathway. 6,
2-Oxoglutarate/malate translocator, DiT1. 7, Glutamate/malate
translocator, DiT2. Both translocators, DiT1 and DiT2, are present
in chloroplasts and are involved in ammonia assimilation and the
refixation of photorespiratory ammonia. 8, Glutamine/glutamate
translocator; it exports glutamine from chloroplasts as a source
for assimilated nitrogen. 9, Oxaloacetate/malate translocator; this
transporter is present in chloroplasts (corresponding activities are
also found in other organelles) and mediates the indirect export of
reducing equivalents in the form of malate. 10, ADP/ATP translocator
providing plastids with ATP as driving force for biosynthetic
processes. 11, ADP-glucose/adenylate translocator; it is present
in plastids of some starch-storing tissues and provides the plastids
with ADP-glucose as substrate for starch biosynthesis. ADP-Glc,
ADP-glucose; 2-OG, 2-oxoglutarate; Glc6P, glucose 6-phosphate;
Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; GOGAT,
glutamate synthase; Mal, malate; MDH, malate dehydrogenase;
OAA, oxaloacetate; OPPP, oxidative pentose phosphate pathway; Pi,
inorganic phosphate; TrioseP, triose phosphates.
plastids and directed into the accumulation of starch. The
deficiency in the TPT activity is obviously compensated
for by mobilizing and exporting the major part of the daily
accumulated carbon during the following night period [10].
Chloroplasts and nongreen plastids from most plants
(depending on the developmental stage of the tissue)
lack the complete set of glycolytic enzymes for the
conversion of hexose phosphates and/or triose phosphate
into phosphoenolpyruvate (PEP) and, therefore, rely on
a supply of PEP from the cytosol [15,16]. In plastids,
PEP can be used for the biosynthesis of PEP- and
pyruvate-derived amino acids, fatty acids, or precursors
for the shikimate pathway. The aromatic compounds
synthesized from PEP via the shikimate pathway are not
only constituents of proteins (aromatic amino acids) but
are also utilized as precursors for the biosynthesis of a
large number of secondary metabolites (e.g. alkaloids,
flavonoids, and lignin) which are important in plant
defense mechanisms and stress responses (for review, see
[17]).
A transporter that enables the transport of PEP in plastids
has recently been identified and corresponding cDNAs
were isolated from nongreen and photosynthetic tissues
of various plants ([18••], Figure 1, no.3). All of these
clones were highly homologous to each other but the
identities with members of the TPT family were only
∼35%. Thus, these membrane transporters represent a
class of phosphate translocator that is different from the
known TPTs. Expression of functional PPT protein in
yeast cells revealed that this translocator transports inorganic phosphate preferentially — and under physiological
conditions, probably exclusively — in exchange with PEP.
Triose phosphates and 3-PGA, which are the counter
substrates for the TPT, are only poorly transported. Thus,
the translocator represents a PEP/phosphate translocator
(PPT) which is able to supply plastids with PEP even
in the presence of triose phosphates and 3-PGA. Most
probably, mesophyll chloroplasts of C4 plants possess a
PPT-like protein that mediates the export (Figure 1, no.4)
of PEP as substrate for the PEP carboxylase in the cytosol.
Metabolite transporters in plastids Flugge
¨
In chloroplasts, in which both PPT and TPT are present,
the combined action of both translocators would result in
an exchange of triose phosphates with PEP without net
phosphate transport. In heterotrophic tissues, erythrose
4-phosphate (another precursor for the shikimate pathway)
and triose phosphates can be provided by the oxidative
pentose phosphate pathway inside the plastids. Triose
phosphate as the end products of this pathway could be
exported from the plastids and converted into PEP, which
subsequently could be imported via the PPT into the
plastids.
A hexose phosphate/phosphate translocator
Nongreen plastids of heterotrophic tissues are carbohydrate importing organelles and, in the case of amyloplasts
from storage tissues, the site of starch synthesis. Due to
the absence of plastidic fructose 1,6 bisphosphatase [19],
these plastids rely on the import of hexose phosphates that
are formed from sucrose delivered from source tissues. In
nongreen tissues from most plants, glucose 6-phosphate
(Glc6P) is the preferred hexose phosphate taken up. In
amyloplasts from some tissues, however, both Glc6P and
glucose 1-phosphate can be transported and subsequently
used in biosynthetic processes [20,21].
A Glc6P/phosphate translocator (GPT) was purified from
maize endosperm and corresponding cDNA clones for
GPTs from nongreen tissues of various plants were isolated
([22••], Figure 1, no.5). Analysis of the deduced GPT
protein sequences revealed that the GPT proteins share
only ∼38% identical amino acids with members of both
the TPT and PPT families. Thus, the GPTs represent
a third group of plastidic phosphate translocators. The
expression of the corresponding cDNA in yeast cells and
the subsequent reconstitution of the produced protein in
artificial membranes revealed that inorganic phosphate,
triose phosphates and Glc6P are about equally well
accepted as counter substrates by the GPT, whereas PEP
is only poorly transported. Other hexose phosphates, such
as glucose 1-phosphate and fructose 6-phosphate, are
virtually not transported by the GPT.
Inside the plastids, Glc6P is the precursor for starch
biosynthesis during which process inorganic phosphate is
released. In addition, Glc6P can be transformed to triose
phosphates via the oxidative pentose phosphate pathway
in which redox equivalents are delivered for the reduction
of nitrite and for glutamate synthesis. Evidently, both
substrates, triose phosphates and inorganic phosphate, can
be used as counter substrates by the GPT in exchange
with Glc6P.
It is worth mentioning that, until recently, it was believed
that the transport of phosphate, triose phosphates, 3-PGA,
PEP and hexosephosphates was mediated by a single
transport system with a broad substrate specificity. Recent
findings, however, clearly show that plastids contain
instead a set of phosphate translocators with different
203
structures but overlapping specificities. Such a system
enables the efficient uptake of individual phosphorylated
substrates even in the presence of high concentrations of
other phosphorylated metabolites, that would otherwise
compete for the binding site of a single transport system.
Dicarboxylate translocators
C3-compounds exported from the chloroplasts can also
serve as a source for the formation of α-keto acids
(2-oxoglutarate). The resulting 2-oxoglutarate is imported
into the chloroplasts for the assimilation of ammonia
produced by nitrate reduction (which also requires the
uptake of nitrite) or deriving from glycine decarboxylation
during photorespiration. The stroma-located glutamine
synthetase (GS2)/glutamate synthase reaction yields glutamate that is exported into the cytosol. Two different
dicarboxylate antiport systems are involved in this process:
the 2-oxoglutarate/malate translocator (DiT1) transporting 2-oxoglutarate (but no amino acids) and a glutamate/malate translocator (DiT2, a general dicarboxylate
translocator) exporting glutamate (Figure 1, no. 6,7).
As both translocators use malate as the substrate for
counter-exchange, the resulting 2-oxoglutarate/glutamate
transport proceeds without net malate transport [23].
Glutamate as the key compound of nitrogen metabolism
in plants and other amino acids can then be further loaded
into the sieve tubes via specific amino acid transporters.
Mutants of Arabidopsis deficient in dicarboxylate transport
activities are not viable under photorespiratory conditions,
but they grow like wild-type plants in elevated carbon
dioxide, that is to say primary ammonia assimilation is
not impaired by the mutation. This phenotype is probably
caused by a defect in DiT2 [24]. The corresponding gene
has not yet been isolated.
DiT1 was identified as a 45 kDa component of the inner
envelope membrane and a corresponding cDNA clone
has been isolated [25,26]. The substrate specificities of
heterologously expressed DiT1 were almost identical to
the translocator purified from envelope membranes. DiT1
has a transmembrane topology with a 12-helix motif
resembling that of other plasma membrane transporters
from prokaryotes and eukaryotes that presumably all
function as monomers. Database searches showed that
DiT1 possesses no similarities to other proteins except for
transporters from bacteria, for example, from Helicobacter
pylori [27], that are as yet uncharacterized.
In addition to DiT1 and DiT2, chloroplasts contain
a translocator for glutamine with glutamate as the
preferential countersubstrate (Figure 1, no. 8). Glutamine
is not transported by DiT1 and is transported by DiT2
only at low rates. The glutamine/glutamate translocator
is probably not involved in the import of glutamine
produced during the refixation of photorespiratory ammonia by the cytosolic isoform of glutamine synthetase
(GS1) as previously assumed [28]. In contrast to the
204
Physiology and metabolism
chloroplast-located GS2 of mesophyll cells, expression of
GS1 is restricted to phloem companion cells that do not
contain photosynthetically active chloroplasts [29,30]. The
combined action of the glutamine/glutamate translocator
and both DiT1 and DiT2 allows for the export of
glutamine in exchange for 2-oxoglutarate with no net
glutamate transport and to supply the cell with assimilated
nitrogen.
Chloroplasts are also able to transport oxaloacetate (OAA)
even in the presence of a large excess of other dicarboxylates [31] (Figure 1, no. 9). If the import is linked to an
export of malate, this malate/OAA shuttle may play an important role in the transfer of excess reducing equivalents
from chloroplasts into the cytosol [32]. Imported OAA
can also be used for transamination to aspartate which
is, in turn, the precursor for the biosynthesis of various
amino acids [33]. In mesophyll cells of most C4-plants,
OAA, the product of the PEP carboxylase reaction, has to
be imported into the chloroplasts for reduction to malate
(Figure 1, no.9). The molecular structure of the OAA
translocators is as yet unknown.
Chloroplasts of C3-plants also contain an antiport system
for monocarboxylates, such as, glycerate and glycolate
which are both intermediates of the photorespiratory cycle
[34]. The molecular structure of this translocator also
remains to be determined.
Transport of adenylates
Chloroplasts and nongreen plastid possess an ADP/ATP
antiport system (AAT) that enables the supply of plastids
with ATP as the driving force for biosynthetic processes,
such as starch and fatty acid biosyntheses [35,36].
Kampfenkel et al. [37] isolated a partial cDNA clone from
A. thaliana that exhibited similarities to the ADP/ATP
translocase from the bacterium Rickettsia prowazekii. Isolation and characterization of the full-length clone revealed
no homology to the known mitochondrial adenylate translocators. The corresponding protein (AATP1) possesses
an amino-terminal transit peptide that was able to target
the adjacent protein to isolated chloroplasts. Interestingly,
the AATP1 protein contains 12 potential transmembrane
helices and thus belongs to the group of transporters with
a 12 helix motif as is the case for DiT1. Expression of the
AATP1 cDNA in heterologous systems and subsequent
transport measurements demonstrates the function of
AATP1 as an ADP/ATP translocator from plant plastids
[38••].
It has been claimed that ADP-glucose can be transported
via the AAT into both chloroplasts and amyloplasts
[39]. The plastidic AAT, however, is obviously not able
to transport the ADP-glucose [36,40]. Nonetheless, a
mechanism for the transport of ADP-glucose is required
in the seed endosperm of some cereals. It has been
shown recently that, in these tissues, the ADP-glucose
pyrophosphorylase is present mainly in the cytosol and
not in the plastids [41•,42]. The ADP-glucose formed in
the cytosol is presumably transported into the plastids
for starch biosynthesis via an ADP-glucose/adenylate
antiporter that is distinct from the AAT. The direct import
of ADP-glucose for starch biosynthesis circumvents the
requirement for an AAT in these tissues. It is assumed,
but not yet proven, that the Brittle 1 (Bt1) protein
(which shows a significant similarity to the mitochondrial
AAT) serves as an ADP-glucose/adenylate transporter
[43,44]. The corresponding Bt1 mutation results in an
accumulation of ADP-glucose and a reduction of starch
biosynthesis in maize, probably reflecting the lack of the
ADP-glucose/adenylate translocator.
Transport of amino acids
It is well established that chloroplasts are the major site
of synthesis of many amino acids including those of the
aspartate and the pyruvate family [33]. In addition, the
plastid-located shikimate pathway leads to the formation
of the aromatic amino acids phenylalanine, tyrosine and
tryptophan. The resulting amino acids are either used for
the plastid-located protein biosynthesis or are, in the main
part, exported into the cytosol for intercellular nitrogen
transport. Unfortunately, almost nothing is known about
plastidic translocators involved in these processes.
Facilitators of metabolite transport in outer
envelope membranes
Until recently, it had been assumed that the outer
envelope membrane of plastids is permeable to low
molecular weight solutes because of the presence of
porins. In chloroplast outer envelope membranes, a large
conductance channel (Λ6 720 pS, in 100 mM KCl) was
found [45]. The primary sequence of a porin from
nongreen plastids was recently obtained that resembles,
structurally and functionally, the known mitochondrial
porins (∼25% identical amino acids, [46,47]). More recently, the primary sequences of two outer envelope
membrane proteins were obtained (OEP16, OEP24) that
both possess channel-like activities [48••,49••]. OEP16
forms a slightly cation-selective, high conductance channel
(Λ61.2 nS, in 1 M KCl). Surprisingly, this channel is
selective for amino acids and molecules containing an
amino acid backbone, but excludes triose phosphate
and uncharged sugars. OEP 24 is a non-selective pore
((Λ62 pS, in 1 M KCl) that allows the flux of triose
phosphate, dicarboxylates or adenylates but excludes
sucrose. Neither OEP16 nor OEP24 show similarities to
bacterial or mitochondrial porins. These results indicate
the presence of selectivity filters in the outer envelope
membrane and show that the intermembrane space might
be not as freely accessible to small solutes as previously
assumed.
Conclusions
Much progress has been achieved during the past few
years on the molecular and physiological characterization
of plastidic translocators. Nonetheless, our knowledge of
Metabolite transporters in plastids Flugge
¨
the identity of the majority of these translocators is still
rudimentary. There are multiple reasons for this. Biochemical approaches in the cloning of plastidic transport
systems will become increasingly laborious as low-abundant translocators or translocators of tissues from which
plastids are difficult to obtain, are studied. In addition,
the identification of an unknown plastidic transporter by
functional complementation of mutants defective for the
transport of the corresponding metabolite does not appear
feasible. This is mainly due to mistargeting of plastidic
transporters in heterologous systems and to difficulty
in setting up appropriate screening systems. Functional
complementation of transport defective mutants, however,
has been efficiently applied for the identification of several transporters of the plasma membrane. Furthermore,
knowledge of transporter genes in other systems are
of only limited value for the identification of plastidic
transport systems by DNA-based strategies, because the
primary sequences of plastidic translocators characterized
to date have revealed only limited homologies to transporters known in other systems.
In the next few years, the various sequencing projects will
provide the sequences of complete higher-plant genomes
and will reveal numerous genes that presumably code for
plastidic envelope transporters. However, even if a gene
can be assigned as an envelope membrane transporter a
challenging task will be to validate that conclusion and
to elucidate the specific role of this particular gene in
plant metabolism. This will require the combination of
bioinformatic methods with genetical, biochemical and
physiological approaches.
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Acknowledgements
Work in the authors laboratory was funded by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Bundesministerium
fur
¨ Bildung und Forschung, and by the European Communities’ BIOTECH
Programme, as part of the Project of Technological Priority 1993-1996.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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• of special interest
•• of outstanding interest
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¨
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•
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2.
•
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glucose 6-phosphate into the plastids as a substrate for starch biosynthesis
and the oxidative pentose phosphate pathway.
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Somerville SC, Somerville CR: A mutant of Arabidopsis deficient
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Weber A, Menzlaff E, Arbinger B, Gutensohn M, Eckerskorn
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of the functional protein in yeast cells. Biochemistry 1995,
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Howitz KT, McCarty RE: Substrate specificity of the pea
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T, Steup M, Kampfenkel K:
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the plastid envelope of Arabidopsis thaliana L. Plant J 1997,
11:73-82.
This paper and [37] describe the isolation of a cDNA clone from Arabidopsis that exhibits a significant similarity to a bacterial adenylate translocator.
Heterologous expression of the functional protein revealed the identity as a
plant ADP/ATP translocator that is confined to plastidic envelopes and is
distinct from mitochondrial adenylate translocators.
39.
Pozueta-Romero J, Frehner M, Viale AM, Akazawa T: Direct
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T, Tjaden J, Henrichs G, Quick WP, Hausler
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inside and outside the amyloplasts in barley endosperm. Plant
J 1996, 10:243-250.
It has previously been thought that ADP-glucose pyrophosphorylase, as the
key enzyme of starch biosynthesis, resides exclusively in the plastids. The
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tissues and found that most of this enzyme activity in developing endosperm
of barley (and of maize [42]) is extraplastidial and is likely to be located in the
cytosol. The cytosolic localization of this enzyme in some (but not all) cereals
has a considerable impact on starch metabolism and plastid function
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Denyer K, Dunlap F, Thorbjrnsen T, Keeling P, Smith AM: The
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OE: Analysis of the maize brittle-1 alleles and a defective
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of the chloroplast envelope. FEBS Lett 1984, 169:85-89.
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Fischer K, Weber A, Brink S, Arbinger B, Schunemann
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269:25754-25760.
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Popp B, Gebauer S, Fischer K, Flugge
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UI, Benz R: Study of
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by biophysical methods. Biochemistry 1997, 36:2844-2852.
Pohlmeyer K, Soll J, Steinkamp T, Hinnah S, Wagner R: Isolation
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See annotation for [49••].
48.
••
Pohlmeyer K, Soll J, Grimm R, Hill K, Wagner R: A high
conductance solute channel in the chloroplastic outer
envelope from pea. Plant Cell 1998, in press.
These papers ([48••,49••]) describe the isolation of two outer envelope
membrane proteins (OEP16, OEP24) and the subsequent isolation of corresponding cDNAs. Evidence is presented that the heterologously expressed
proteins possess channel-like activities, and act as selectivity filters of the
outer envelope membrane. The permeability properties of the outer envelope
membrane to low molecular weight solutes thus appears to be defined by
the combined action of various channel proteins with different selectivities.
49.
••
Metabolite transporters in plastids
Ulf-Ingo Flugge
¨
Communication between plastids and the surrounding cytosol
occurs via the plastidic envelope membrane. Recent findings
show that the outer membrane is not as freely permeable
to low molecular weight solutes as previously thought, but
contains different channel-like proteins that act as selectivity
filters. The inner envelope membrane contains a variety
of metabolite transporters that mediate the exchange of
metabolites between both compartments. Two new classes
of phosphate antiporters were recently described that are
different in structure and function from the known triose
phosphate/phosphate translocator from chloroplasts. In
addition, a cDNA coding for an ATP/ADP antiporter from
plastids was isolated that shows similarities to a bacterial
adenylate translocator.
Addresses
¨ zu Koln,
¨ Lehrstuhl II, Gyrhofstr. 15,
Botanisches Institut der Universitat
¨ Germany; e-mail: uiflue@biolan.uni-koeln.de
D-50931 Koln,
Current Opinion in Plant Biology 1998, 1:201–206
http://biomednet.com/elecref/1369526600100201
Current Biology Ltd ISSN 1369-5266
Abbreviations
3-PGA 3-phosphoglycerate
AAT
ADP/ATP antiport system
DiT1
2-oxoglutarate/malate translocator
DiT2
glutamate/malate translocator
Glc6P
glucose 6-phosphate
GPT
Glc6P/phosphate translocator
PEP
phosphoenolpyruvate
PPT
PEP/phosphate translocator
TPT
triose phosphate/phosphate translocator
Introduction
Like other eukaryotic cells, plants cells contain different
organelles, amongst them plastids, a compartment that
is specific for plants. Chloroplasts, chlorophyll containing
plastids, are the site of photosynthesis during which
process atmospheric carbon dioxide is assimilated into
intermediates that are used within, as well as outside,
the chloroplasts for a variety of metabolic pathways.
Chloroplasts are also involved in nitrogen assimilation and
the synthesis of amino acids, transitory starch and a series
of secondary compounds. Plastids of non-photosynthetic
tissues have to import carbon as a source of energy and
biosynthetic pathways, for example, of fatty acids, amino
acids as well as starch. All plastids are double membranebound organelles. The inner envelope membrane is the
actual permeability barrier between the plastid and the
surrounding cytosol and the site of different metabolite
translocators that co-ordinate the metabolism in both
compartments. In this review, I describe the molecular
characterization of various transporters of plastidic mem-
branes and their role in plant metabolism, taking into
account the recent progress achieved in this field.
Export of photoassimilates from chloroplasts
The triose phosphate/phosphate translocator (TPT) mediates the export of the fixed carbon in the form of
triose phosphate or 3-phosphoglycerate (3-PGA) from the
chloroplast into the cytosol (Figure 1, no. 1). In the mature
leaves of most plants, the photosynthates exported by the
TPT are used in the formation of sucrose and amino acids
which are the main products allocated to heterotrophic
plant organs. Sucrose and amino acids are actively loaded
into the sieve element/companion cell complex by specific
H+/symporters (for reviews, see [1•,2•]).
The TPT antiport system accepts as substrates inorganic
phosphate, triose phosphates and 3-PGA which are
transported via a ping-pong type of reaction mechanism
[3,4]. Using the reconstituted system in which the concentrations of phosphate in both the internal and the external
compartments are accessible to experimental variations, it
could be demonstrated that the TPT can function not
only as an antiporter but also as a voltage-dependent ion
channel, preferentially permeable to anions [5].
The spinach TPT was the first plant membrane transport system for which the primary sequence could be
determined [6]. Meanwhile, TPT sequences from various
plants are available that all have a high similarity to
each other [7]. All TPTs are nuclear-encoded and possess
amino-terminal transit peptides that direct the adjacent
protein to the chloroplasts. In its functional state, the
TPT forms a homodimer and belongs to the group of
translocators with a 6+6 helix folding pattern, similar to
mitochondrial carrier proteins [8].
The final proof for the identity of a transporter cDNA is
the expression of the corresponding cDNA to produce the
functional protein in heterologous systems, for example
yeast cells, bacteria or oocytes. The recombinant TPT
protein, produced in yeast cells, exhibited transport
characteristics almost identical to those of the authentic
chloroplast protein [9].
The TPT is almost exclusively present in photosynthetically active tissues. To assess the role of the TPT on
photosynthetic metabolism, transgenic antisense potato
plants were created in which both the amount and the
activity of the TPT were reduced to ∼70% of the controls.
In ambient carbon dioxide and at intermediate light, there
was no significant effect on photosynthetic rates, growth
and tuber development. The starch content in the leaves
of the transformants, however, was much higher than in
wild-type plants suggesting that the daily assimilated carbon in the transformants is mainly maintained within the
Physiology and metabolism
202
Figure 1
Mal
Mal
9
Gln
MDH
OAA
OAA
Pi
Gln
GS
8
Glu
Glu
HCO –
Glu
Glu
3
PEP
Amino
acids
7
PEP
GS
GOGAT
4
Mal
Pi
Mal
6
ADP
2-OG
2-OG
10
ADP
TrioseP
TrioseP
TrioseP
Pi
OPPP
Glc6P
3
Pi
ADP-Glc
11
PEP
STARCH
AMP
2
Pi
Glc6P
5
Sucrose
1
PEP
The phosphoenolpyruvate/phosphate
translocator
e.g. Shikimic
acid pathway
Glucose
Plastid
This is in contrast to wild-type plants which generally
export the major part of the fixed carbon during the day
via the TPT. Most probably, the mobilization of the daily
accumulated starch in the antisense TPT plants proceeds
via the amylolytic starch breakdown resulting in hexoses
which are subsequently exported via a hexose translocator
(Figure 1, no.2). Interestingly, antisense TPT plants from
tobacco accumulate starch as potato plants do, but mobilize
the accumulated starch during ongoing photosynthesis.
Hexoses are exported via a glucose transporter, the activity
of which is two to three times higher in the transformants
compared to the wild-type [11]. Little is known about
this translocator that was first described twenty years ago
[12]. Caspar et al. [13] described a starch-excess mutant
from Arabidopsis, TC26, that is able to degrade starch
but obviously unable to export the products of starch
degradation from the chloroplasts via a glucose transporter
[14]. The corresponding gene has not yet been identified.
Cytosol
Current Opinion in Plant Biology
Translocators of the inner envelope membrane of plastids. 1,
Triose phosphate/phosphate translocator, TPT; this translocator
is primarily present in chloroplasts and mediates the export of
the fixed carbon in form of triose phosphates and 3-PGA. 2,
Glucose translocator; it exports the product of amylolytic starch
breakdown, glucose, from plastids. 3, PEP/phosphate translocator,
PPT; it provides plastids with PEP for the biosynthesis of PEP- and
pyruvate-derived amino acids, fatty acids, or precursors of the
shikimic acid pathway. 4, PEP/phosphate translocator present in
mesophyll chloroplasts of C4-plants. It exports PEP as substrate
for the PEP carboxylase. 5, Glucose 6-phosphate/phosphate
translocator, GPT; it imports glucose 6-phosphate into plastids
of heterotrophic tissues for the biosynthesis of starch and as
a substrate for the oxidative pentose phosphate pathway. 6,
2-Oxoglutarate/malate translocator, DiT1. 7, Glutamate/malate
translocator, DiT2. Both translocators, DiT1 and DiT2, are present
in chloroplasts and are involved in ammonia assimilation and the
refixation of photorespiratory ammonia. 8, Glutamine/glutamate
translocator; it exports glutamine from chloroplasts as a source
for assimilated nitrogen. 9, Oxaloacetate/malate translocator; this
transporter is present in chloroplasts (corresponding activities are
also found in other organelles) and mediates the indirect export of
reducing equivalents in the form of malate. 10, ADP/ATP translocator
providing plastids with ATP as driving force for biosynthetic
processes. 11, ADP-glucose/adenylate translocator; it is present
in plastids of some starch-storing tissues and provides the plastids
with ADP-glucose as substrate for starch biosynthesis. ADP-Glc,
ADP-glucose; 2-OG, 2-oxoglutarate; Glc6P, glucose 6-phosphate;
Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; GOGAT,
glutamate synthase; Mal, malate; MDH, malate dehydrogenase;
OAA, oxaloacetate; OPPP, oxidative pentose phosphate pathway; Pi,
inorganic phosphate; TrioseP, triose phosphates.
plastids and directed into the accumulation of starch. The
deficiency in the TPT activity is obviously compensated
for by mobilizing and exporting the major part of the daily
accumulated carbon during the following night period [10].
Chloroplasts and nongreen plastids from most plants
(depending on the developmental stage of the tissue)
lack the complete set of glycolytic enzymes for the
conversion of hexose phosphates and/or triose phosphate
into phosphoenolpyruvate (PEP) and, therefore, rely on
a supply of PEP from the cytosol [15,16]. In plastids,
PEP can be used for the biosynthesis of PEP- and
pyruvate-derived amino acids, fatty acids, or precursors
for the shikimate pathway. The aromatic compounds
synthesized from PEP via the shikimate pathway are not
only constituents of proteins (aromatic amino acids) but
are also utilized as precursors for the biosynthesis of a
large number of secondary metabolites (e.g. alkaloids,
flavonoids, and lignin) which are important in plant
defense mechanisms and stress responses (for review, see
[17]).
A transporter that enables the transport of PEP in plastids
has recently been identified and corresponding cDNAs
were isolated from nongreen and photosynthetic tissues
of various plants ([18••], Figure 1, no.3). All of these
clones were highly homologous to each other but the
identities with members of the TPT family were only
∼35%. Thus, these membrane transporters represent a
class of phosphate translocator that is different from the
known TPTs. Expression of functional PPT protein in
yeast cells revealed that this translocator transports inorganic phosphate preferentially — and under physiological
conditions, probably exclusively — in exchange with PEP.
Triose phosphates and 3-PGA, which are the counter
substrates for the TPT, are only poorly transported. Thus,
the translocator represents a PEP/phosphate translocator
(PPT) which is able to supply plastids with PEP even
in the presence of triose phosphates and 3-PGA. Most
probably, mesophyll chloroplasts of C4 plants possess a
PPT-like protein that mediates the export (Figure 1, no.4)
of PEP as substrate for the PEP carboxylase in the cytosol.
Metabolite transporters in plastids Flugge
¨
In chloroplasts, in which both PPT and TPT are present,
the combined action of both translocators would result in
an exchange of triose phosphates with PEP without net
phosphate transport. In heterotrophic tissues, erythrose
4-phosphate (another precursor for the shikimate pathway)
and triose phosphates can be provided by the oxidative
pentose phosphate pathway inside the plastids. Triose
phosphate as the end products of this pathway could be
exported from the plastids and converted into PEP, which
subsequently could be imported via the PPT into the
plastids.
A hexose phosphate/phosphate translocator
Nongreen plastids of heterotrophic tissues are carbohydrate importing organelles and, in the case of amyloplasts
from storage tissues, the site of starch synthesis. Due to
the absence of plastidic fructose 1,6 bisphosphatase [19],
these plastids rely on the import of hexose phosphates that
are formed from sucrose delivered from source tissues. In
nongreen tissues from most plants, glucose 6-phosphate
(Glc6P) is the preferred hexose phosphate taken up. In
amyloplasts from some tissues, however, both Glc6P and
glucose 1-phosphate can be transported and subsequently
used in biosynthetic processes [20,21].
A Glc6P/phosphate translocator (GPT) was purified from
maize endosperm and corresponding cDNA clones for
GPTs from nongreen tissues of various plants were isolated
([22••], Figure 1, no.5). Analysis of the deduced GPT
protein sequences revealed that the GPT proteins share
only ∼38% identical amino acids with members of both
the TPT and PPT families. Thus, the GPTs represent
a third group of plastidic phosphate translocators. The
expression of the corresponding cDNA in yeast cells and
the subsequent reconstitution of the produced protein in
artificial membranes revealed that inorganic phosphate,
triose phosphates and Glc6P are about equally well
accepted as counter substrates by the GPT, whereas PEP
is only poorly transported. Other hexose phosphates, such
as glucose 1-phosphate and fructose 6-phosphate, are
virtually not transported by the GPT.
Inside the plastids, Glc6P is the precursor for starch
biosynthesis during which process inorganic phosphate is
released. In addition, Glc6P can be transformed to triose
phosphates via the oxidative pentose phosphate pathway
in which redox equivalents are delivered for the reduction
of nitrite and for glutamate synthesis. Evidently, both
substrates, triose phosphates and inorganic phosphate, can
be used as counter substrates by the GPT in exchange
with Glc6P.
It is worth mentioning that, until recently, it was believed
that the transport of phosphate, triose phosphates, 3-PGA,
PEP and hexosephosphates was mediated by a single
transport system with a broad substrate specificity. Recent
findings, however, clearly show that plastids contain
instead a set of phosphate translocators with different
203
structures but overlapping specificities. Such a system
enables the efficient uptake of individual phosphorylated
substrates even in the presence of high concentrations of
other phosphorylated metabolites, that would otherwise
compete for the binding site of a single transport system.
Dicarboxylate translocators
C3-compounds exported from the chloroplasts can also
serve as a source for the formation of α-keto acids
(2-oxoglutarate). The resulting 2-oxoglutarate is imported
into the chloroplasts for the assimilation of ammonia
produced by nitrate reduction (which also requires the
uptake of nitrite) or deriving from glycine decarboxylation
during photorespiration. The stroma-located glutamine
synthetase (GS2)/glutamate synthase reaction yields glutamate that is exported into the cytosol. Two different
dicarboxylate antiport systems are involved in this process:
the 2-oxoglutarate/malate translocator (DiT1) transporting 2-oxoglutarate (but no amino acids) and a glutamate/malate translocator (DiT2, a general dicarboxylate
translocator) exporting glutamate (Figure 1, no. 6,7).
As both translocators use malate as the substrate for
counter-exchange, the resulting 2-oxoglutarate/glutamate
transport proceeds without net malate transport [23].
Glutamate as the key compound of nitrogen metabolism
in plants and other amino acids can then be further loaded
into the sieve tubes via specific amino acid transporters.
Mutants of Arabidopsis deficient in dicarboxylate transport
activities are not viable under photorespiratory conditions,
but they grow like wild-type plants in elevated carbon
dioxide, that is to say primary ammonia assimilation is
not impaired by the mutation. This phenotype is probably
caused by a defect in DiT2 [24]. The corresponding gene
has not yet been isolated.
DiT1 was identified as a 45 kDa component of the inner
envelope membrane and a corresponding cDNA clone
has been isolated [25,26]. The substrate specificities of
heterologously expressed DiT1 were almost identical to
the translocator purified from envelope membranes. DiT1
has a transmembrane topology with a 12-helix motif
resembling that of other plasma membrane transporters
from prokaryotes and eukaryotes that presumably all
function as monomers. Database searches showed that
DiT1 possesses no similarities to other proteins except for
transporters from bacteria, for example, from Helicobacter
pylori [27], that are as yet uncharacterized.
In addition to DiT1 and DiT2, chloroplasts contain
a translocator for glutamine with glutamate as the
preferential countersubstrate (Figure 1, no. 8). Glutamine
is not transported by DiT1 and is transported by DiT2
only at low rates. The glutamine/glutamate translocator
is probably not involved in the import of glutamine
produced during the refixation of photorespiratory ammonia by the cytosolic isoform of glutamine synthetase
(GS1) as previously assumed [28]. In contrast to the
204
Physiology and metabolism
chloroplast-located GS2 of mesophyll cells, expression of
GS1 is restricted to phloem companion cells that do not
contain photosynthetically active chloroplasts [29,30]. The
combined action of the glutamine/glutamate translocator
and both DiT1 and DiT2 allows for the export of
glutamine in exchange for 2-oxoglutarate with no net
glutamate transport and to supply the cell with assimilated
nitrogen.
Chloroplasts are also able to transport oxaloacetate (OAA)
even in the presence of a large excess of other dicarboxylates [31] (Figure 1, no. 9). If the import is linked to an
export of malate, this malate/OAA shuttle may play an important role in the transfer of excess reducing equivalents
from chloroplasts into the cytosol [32]. Imported OAA
can also be used for transamination to aspartate which
is, in turn, the precursor for the biosynthesis of various
amino acids [33]. In mesophyll cells of most C4-plants,
OAA, the product of the PEP carboxylase reaction, has to
be imported into the chloroplasts for reduction to malate
(Figure 1, no.9). The molecular structure of the OAA
translocators is as yet unknown.
Chloroplasts of C3-plants also contain an antiport system
for monocarboxylates, such as, glycerate and glycolate
which are both intermediates of the photorespiratory cycle
[34]. The molecular structure of this translocator also
remains to be determined.
Transport of adenylates
Chloroplasts and nongreen plastid possess an ADP/ATP
antiport system (AAT) that enables the supply of plastids
with ATP as the driving force for biosynthetic processes,
such as starch and fatty acid biosyntheses [35,36].
Kampfenkel et al. [37] isolated a partial cDNA clone from
A. thaliana that exhibited similarities to the ADP/ATP
translocase from the bacterium Rickettsia prowazekii. Isolation and characterization of the full-length clone revealed
no homology to the known mitochondrial adenylate translocators. The corresponding protein (AATP1) possesses
an amino-terminal transit peptide that was able to target
the adjacent protein to isolated chloroplasts. Interestingly,
the AATP1 protein contains 12 potential transmembrane
helices and thus belongs to the group of transporters with
a 12 helix motif as is the case for DiT1. Expression of the
AATP1 cDNA in heterologous systems and subsequent
transport measurements demonstrates the function of
AATP1 as an ADP/ATP translocator from plant plastids
[38••].
It has been claimed that ADP-glucose can be transported
via the AAT into both chloroplasts and amyloplasts
[39]. The plastidic AAT, however, is obviously not able
to transport the ADP-glucose [36,40]. Nonetheless, a
mechanism for the transport of ADP-glucose is required
in the seed endosperm of some cereals. It has been
shown recently that, in these tissues, the ADP-glucose
pyrophosphorylase is present mainly in the cytosol and
not in the plastids [41•,42]. The ADP-glucose formed in
the cytosol is presumably transported into the plastids
for starch biosynthesis via an ADP-glucose/adenylate
antiporter that is distinct from the AAT. The direct import
of ADP-glucose for starch biosynthesis circumvents the
requirement for an AAT in these tissues. It is assumed,
but not yet proven, that the Brittle 1 (Bt1) protein
(which shows a significant similarity to the mitochondrial
AAT) serves as an ADP-glucose/adenylate transporter
[43,44]. The corresponding Bt1 mutation results in an
accumulation of ADP-glucose and a reduction of starch
biosynthesis in maize, probably reflecting the lack of the
ADP-glucose/adenylate translocator.
Transport of amino acids
It is well established that chloroplasts are the major site
of synthesis of many amino acids including those of the
aspartate and the pyruvate family [33]. In addition, the
plastid-located shikimate pathway leads to the formation
of the aromatic amino acids phenylalanine, tyrosine and
tryptophan. The resulting amino acids are either used for
the plastid-located protein biosynthesis or are, in the main
part, exported into the cytosol for intercellular nitrogen
transport. Unfortunately, almost nothing is known about
plastidic translocators involved in these processes.
Facilitators of metabolite transport in outer
envelope membranes
Until recently, it had been assumed that the outer
envelope membrane of plastids is permeable to low
molecular weight solutes because of the presence of
porins. In chloroplast outer envelope membranes, a large
conductance channel (Λ6 720 pS, in 100 mM KCl) was
found [45]. The primary sequence of a porin from
nongreen plastids was recently obtained that resembles,
structurally and functionally, the known mitochondrial
porins (∼25% identical amino acids, [46,47]). More recently, the primary sequences of two outer envelope
membrane proteins were obtained (OEP16, OEP24) that
both possess channel-like activities [48••,49••]. OEP16
forms a slightly cation-selective, high conductance channel
(Λ61.2 nS, in 1 M KCl). Surprisingly, this channel is
selective for amino acids and molecules containing an
amino acid backbone, but excludes triose phosphate
and uncharged sugars. OEP 24 is a non-selective pore
((Λ62 pS, in 1 M KCl) that allows the flux of triose
phosphate, dicarboxylates or adenylates but excludes
sucrose. Neither OEP16 nor OEP24 show similarities to
bacterial or mitochondrial porins. These results indicate
the presence of selectivity filters in the outer envelope
membrane and show that the intermembrane space might
be not as freely accessible to small solutes as previously
assumed.
Conclusions
Much progress has been achieved during the past few
years on the molecular and physiological characterization
of plastidic translocators. Nonetheless, our knowledge of
Metabolite transporters in plastids Flugge
¨
the identity of the majority of these translocators is still
rudimentary. There are multiple reasons for this. Biochemical approaches in the cloning of plastidic transport
systems will become increasingly laborious as low-abundant translocators or translocators of tissues from which
plastids are difficult to obtain, are studied. In addition,
the identification of an unknown plastidic transporter by
functional complementation of mutants defective for the
transport of the corresponding metabolite does not appear
feasible. This is mainly due to mistargeting of plastidic
transporters in heterologous systems and to difficulty
in setting up appropriate screening systems. Functional
complementation of transport defective mutants, however,
has been efficiently applied for the identification of several transporters of the plasma membrane. Furthermore,
knowledge of transporter genes in other systems are
of only limited value for the identification of plastidic
transport systems by DNA-based strategies, because the
primary sequences of plastidic translocators characterized
to date have revealed only limited homologies to transporters known in other systems.
In the next few years, the various sequencing projects will
provide the sequences of complete higher-plant genomes
and will reveal numerous genes that presumably code for
plastidic envelope transporters. However, even if a gene
can be assigned as an envelope membrane transporter a
challenging task will be to validate that conclusion and
to elucidate the specific role of this particular gene in
plant metabolism. This will require the combination of
bioinformatic methods with genetical, biochemical and
physiological approaches.
5.
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¨
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¨
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7.
Fischer K, Arbinger B, Kammerer K, Busch C, Brink S, Wallmeier
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Heineke D, Kruse A, Flugge
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Acknowledgements
Work in the authors laboratory was funded by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Bundesministerium
fur
¨ Bildung und Forschung, and by the European Communities’ BIOTECH
Programme, as part of the Project of Technological Priority 1993-1996.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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•
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Fischer K, Kammerer B, Gutensohn M, Arbinger B, Weber A,
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and the oxidative pentose phosphate pathway.
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tissues and found that most of this enzyme activity in developing endosperm
of barley (and of maize [42]) is extraplastidial and is likely to be located in the
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has a considerable impact on starch metabolism and plastid function
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46.
Fischer K, Weber A, Brink S, Arbinger B, Schunemann
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D, Borchert
S, Heldt HW, Popp B, Benz R, Eckerskorn C, Flugge
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See annotation for [49••].
48.
••
Pohlmeyer K, Soll J, Grimm R, Hill K, Wagner R: A high
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These papers ([48••,49••]) describe the isolation of two outer envelope
membrane proteins (OEP16, OEP24) and the subsequent isolation of corresponding cDNAs. Evidence is presented that the heterologously expressed
proteins possess channel-like activities, and act as selectivity filters of the
outer envelope membrane. The permeability properties of the outer envelope
membrane to low molecular weight solutes thus appears to be defined by
the combined action of various channel proteins with different selectivities.
49.
••