The Plant Journal (2001) 27(4), 297±304
Association of the NADPH:protochlorophyllide
oxidoreductase (POR) with isolated etioplast inner
membranes from wheat
Sheila Engdahl1, Henrik Aronsson1, Christer Sundqvist1, Michael P. Timko2 and Clas Dahlin1,*,²
1
Department of Plant Physiology. GoÈteborg University, Box 461, SE-405 30 GoÈteborg, Sweden, and
2
Department of Biology, University of Virginia, Charlottesville, VA 22901, USA
Received 15 January 2001; revised 1 May 2001; accepted 19 May 2001.
*
For correspondence (fax +46 35 167 500; e-mail [email protected]).
²
Present address: Institute of Technology and Natural Sciences, Halmstad University, Box 823, SE-301 18 Halmstad, Sweden.
Summary
Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated
prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitroexpressed radiolabelled POR, with or without transit peptide, was used to characterize membrane
association conditions. Proper association of POR with PLBs and PTs did not require the presequence,
whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with
thermolysin, sodium hydroxide or carbonate, a ®rm attachment of the POR protein to the membrane
was found. Although the PLBs and PTs differ signi®cantly in their relative amount of POR in vivo, no
major differences in POR association capacity could be observed between the two membrane systems
when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost
abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in
the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence
of NADPH, which further demonstrates that the co-factor is essential for proper membrane association.
POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the
membranes as compared to the wild type. The results presented here introduce studies of a novel area
of protein±membrane interaction, namely the association of proteins with a paracrystalline membrane
structure, the PLB.
Keywords: etioplasts, chloroplasts, NADPH:protochlorophyllide oxidoreductase, prolamellar bodies,
prothylakoids.
Introduction
Plants grown in the absence of light lack plastids with
photosynthetically competent thylakoid membranes, and
thus also lack several of the components required for light
capture and charge separation. Plastid development in
light involves the differentiation of proplastids or etioplasts to chloroplasts. Etiolated angiosperms posses
photoreceptors and catalytic proteins that, upon illumination, rapidly signal the re-organization of the plastid from
a non-photosynthetic state to a photosynthetically active
chloroplast. The differentiation of etioplasts to chloroplasts involves the transformation of the regular prolamellar bodies (PLBs) into lamellar membrane structures,
synthesis of chlorophyll from the precursor protochlorã 2001 Blackwell Science Ltd
ophyllide (Pchlide), and a massive accumulation of
chloroplast proteins. The majority of the chloroplast
proteins are encoded by nuclear genes, synthesized in
the cytosol and imported into the plastid. The enzyme
responsible for the light-dependent step in chlorophyll
biosynthesis is the NADPH:protochlorophyllide oxidoreductase (POR; Grif®ths, 1978). This is a nuclear-encoded
protein which, together with Pchlide and NADPH, constitutes a minimal functional unit. Aggregates of such units,
connected to the formation of PLBs, can be detected
spectroscopically due to their low-temperature (77 K)
¯uorescence at 657 nm (Pchlide657). Following lightinduced transformation of Pchlide to chlorophyllide
297
298
Sheila Engdahl et al.
(Chlide), the large aggregates of the ternary Chlide±POR±
NADPH complexes disaggregate into smaller entities.
Two different POR proteins (PORA and PORB) were
demonstrated in barley by Holtorf et al. (1995) and in
Arabidopsis by Armstrong et al. (1995), and recently a
third isoform (PORC) was found in Arabidopsis (Oosawa
et al., 2000). The PORA protein is present in dark-grown
plants, but is inactivated and degraded during greening
(Reinbothe et al., 1995a). The PORB protein is also present
in dark-grown plants, but persists during greening in light.
The discovery of three PORs, one light-labile (PORA), one
light-stable (PORB) and one light-inducible (PORC), could
partly explain the paradox of varying levels of POR during
massive chlorophyll synthesis in greening etiolated plants
(Holtorf et al., 1995; Runge et al., 1996).
Nuclear-encoded proteins destined for the plastid interior, such as the inner membranes, are generally transported by a default pathway over the envelope to the
stroma and then subsequently translocated to their ®nal
suborganelle location. Import of PORA has been claimed
to require strictly plastid-localized Pchlide, and thus to
deviate from the common import mechanism (Reinbothe
et al., 1995b). Under certain conditions it has been shown
that, concomitant with decreasing amounts of Pchlide,
etioplasts rapidly lost their ability ef®ciently to import
PORA during greening. On the other hand, import of PORB
was equally ef®cient in chloroplasts and etioplasts
(Reinbothe et al., 1997). The substrate requirement of
PORA for import suggested that PORA binds Pchlide
during import, and that this complex, possibly in the
presence of NADPH, is transported to the PLB of etioplasts.
However, recent data have not supported the requirement
of Pchlide for import of pea POR (Aronsson et al., 2001) or
barley PORA (Aronsson et al., 2000; Dahlin et al., 2000).
Although our biochemical and physical knowledge of
POR has increased signi®cantly during recent years, the
exact nature of binding of the POR to the plastid inner
membranes has not been satisfactorily de®ned. It is
possible to discern two steps in the assembly pathway.
First, an attachment of POR to the membrane; and second,
a ®rmer and more protected physiological association of
POR with the membrane. After attachment POR is not
protected from thermolysin-catalysed breakdown, but
after the physiological membrane association, or binding,
thermolysin causes only limited proteolysis of POR. Given
the very strong membrane association of POR without a
typical membrane-spanning region in the amino acid
sequence, the ®nal membrane-association reactions of
POR with the etioplast inner membranes merits further
investigation. We present here the ®rst detailed analysis of
the membrane-targeting mechanism of POR, leading to a
physiological membrane association with isolated etioplast inner membranes of wheat.
Results
Membrane association of radiolabelled POR with or
without the transit peptide in plastid lysates
Puri®ed etioplasts and chloroplasts from wheat were lysed
in 10 HK buffer (see Experimental procedures). The lysates
were used directly for membrane-association assays, or
centrifuged further to obtain stroma-free inner membranes
(EPIMs). Initially, the assays were performed in the presence of 10 mM MgATP and 1 mM NADPH with radiolabelled POR, either with (pPOR) or without (D-TP) the transit
peptide. Thermolysin post-treatment of EPIMs, with or
without stroma, resulted in a protease degradation product (POR-DP) of approximately 4 kDa smaller than the
mature size of »36 kDa (Figure 1). Similar treatment of
chloroplast inner membranes resulted in a POR-DP of
approximately 34 kDa. This is the same approximate size
of POR-DP as observed in green pea thylakoids under
physiological conditions (Dahlin et al., 1995). We therefore
conclude that the POR-DP also represents correctly assembled POR in EPIMs, but in this case the paracrystalline
structure of the PLBs has a slight effect on the accessibility
of the protease. The appearance of the 32±34 kDa protease
product is not dependent on, nor inhibited by, the transit
peptide. The absence of stroma caused no change on the
membrane targeting of POR (Figure 1).
POR associates with both PLBs and PTs
Prolamellar body and prothylakoid (PT) fractions were
isolated and characterized using SDS±PAGE and ¯uorescence spectroscopy. The protein composition of the
membrane fractions revealed a qualitatively similar pattern, but with higher content of POR in PLBs compared to
PTs (Figure 2, inset). Low-temperature ¯uorescence spectra of the isolated PLBs and PTs revealed major peaks at
657 and 633 nm, respectively (Figure 2). The strong
Figure 1. Membrane association of radiolabelled POR with inner
membranes of etioplasts (E) and chloroplasts (C).
The assays were performed in the presence of 1 mM NADPH with (+S) or
without (±S) stroma. All samples included 10 mM MgATP and 20 mM
MgCl2, and were treated with thermolysin after the assays, giving a PORDP of approximately 32±34 kDa. pPOR, precursor POR protein containing
the transit peptide; D-TP, mature POR protein without the transit peptide;
molecular weight in kDa of POR with (44) or without (36) the transit
peptide; TM, translation mixture. Left, pPOR; right, D-TP.
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
Association of POR with PLB and PT membranes
Figure 2. Characterization of puri®ed PLB and PT preparations from
etiolated wheat.
Isolated membrane fractions were used for SDS±PAGE analysis (inset) or
low-temperature (77 K) ¯uorescence emission spectra. Excitation
wavelength 440 nm. Spectra were normalized by multiplying the PT
spectrum 7.8 times to give the same maximum peak height. Arrow
indicates position of POR.
dominance of ¯uorescence at 657 nm of the PLB fraction
indicates highly puri®ed PLBs with an intact regular
structure. Together, these methods con®rmed a proper
separation of PLBs and PTs into highly puri®ed lamellar PT
and paracrystalline PLB membrane fractions (Lindsten
et al., 1988). Fluorescence measurements were chosen for
routine evaluation of the purity of PLB- and PT-enriched
fractions before each experiment.
When inner membrane fractions were used in the
association assays together with radiolabelled pPOR or
D-TP, we found that both membrane types were capable of
binding (Figure 3). After thermolysin treatment, the puri®ed PLB and PT membranes retained a protease-resistant
form (POR-DP), similar to inner membranes in lysates.
PLB, which in vivo contain most POR (Ryberg and Dehesh,
1986), also had a great capacity to bind POR in vitro in our
experiments. Surprisingly, we also found an almost similar
amount of thermolysin-resistant POR in PTs after the
assay, even if the PTs in vivo contain only very low
amounts of POR.
The nature of the membrane association of POR was
assayed with NaOH and Na2CO3. Following binding reacã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
299
Figure 3. Association of radiolabelled POR with or without the transit
peptide with PLBs and PTs.
(a) Protein association assays with PLBs and PTs were conducted with
the mature POR (D) lacking the transit peptide, or with the precursor POR
(p) containing the transit peptide, in the presence of 1 mM NADPH,
10 mM MgATP, and 20 mM MgCl2. After assays, samples were treated
with (+th) or without (±th) thermolysin.
(b) Fluorograms from membrane-association assays of the precursor
pPOR protein containing the transit peptide. Assays were performed in
the presence of 1 mM NADPH, 10 mM MgATP and 20 mM MgCl2. After
assays, samples were treated with 50 mM NaOH (OH), 50 mM Na2CO3
(CO3), thermolysin (+th) or import buffer only (±th). Samples treated with
thermolysin (+th) show a POR-DP with a molecular weight approximately
2±4 kDa lower than the mature POR of 36 kDa. Molecular weight in kDa
of POR with (44) or without (36) the transit peptide. TM, translation
mixture.
tions of pPOR to isolated PLBs and PTs, the samples were
post-treated with sodium hydroxide (50 mM) or carbonate
(50 mM). After this treatment of the samples we found
pPOR still attached to the membrane, indicating that pPOR
is tightly associated with the membrane (Figure 3b). As can
be seen in Figure 3(b), a substantial amount of the pPOR
used in the assay is unable to attain the mature size in the
absence of the stroma processing peptidase.
Exogenous NADPH and ATP stimulate in vitro
association with isolated PLBs and PTs
Analyses were performed to elucidate if NADPH is
required for a physiological membrane±POR association,
and to ®nd out if there is a difference between PLBs and
PTs in this respect. During the puri®cation procedure of
PLB and PT membranes, NADPH is present to stabilize the
membranes and avoid a decay of the spectral properties
(Lindsten et al., 1988; Ryberg and Sundqvist, 1988). In a
concentration series of 0.25±3 mM NADPH, we found that
the association of pPOR with PLBs and PTs occurred even
at the lowest concentration of 0.25 mM NADPH added
(data not shown). In Figure 4(a) the membrane association
of POR is compared with and without the addition of 1 mM
exogenous NADPH. POR attached both to PLBs and PTs
when thermolysin was not present. However, after ther-
300
Sheila Engdahl et al.
Figure 4. Membrane association of radiolabelled pPOR with PLBs and
PTs requires NADPH and ATP.
(a) Assays were performed in the presence (+N) or absence (±N) of 1 mM
NADPH.
(b) PLBs were prewashed repeatedly in NADPH-free buffer prior to the
association assay. Numbers indicate sequential washing steps. Assays in
(a) and (b) contained 10 mM MgATP and 20 mM MgCl2.
(c) Association assays were performed in the absence of ATP. Translation
mixtures were ®ltered through Sephadex G-25 to remove excess
nucleoside triphosphates. Import buffer containing 10 mM MgATP and
20 mM MgCl2 (+A) or 20 mM MgCl2 only (±A) was added prior to
assembly reaction. Membranes were post-treated with (+th) or without
(±th) thermolysin. Samples treated with thermolysin (+th) show a POR-DP
with a molecular weight approximately 2±4 kDa lower than the mature
POR of 36 kDa. pPOR, radiolabelled precursor POR containing the transit
peptide. Molecular weight in kDa of POR with (44) or without (36) the
transit peptide. TM, translation mixture.
molysin post-treatment the POR-DP was found only in
PLBs and not in PTs when no NADPH was added. Due to
the structural differences between the two types of membrane, it was suspected that the difference could be due to
a washing effect: NADPH could prevail longer in the threedimensional network of PLBs than in the plain PTs.
Repeated washing of PLBs was performed to verify if the
binding was an intrinsic property of the membrane.
Washing of PLBs with NADPH-free buffer abolished the
thermolysin-resistant association in the PLBs after a single
washing step, but had no effect on POR attachment even
after repeated washing steps (Figure 4b).
To investigate the possible ATP requirement for the
membrane-association reaction of POR, the assays were
performed using samples with or without ATP. As seen in
Figure 4(c), the addition of MgATP to ATP-depleted
Figure 5. Association of mutated radiolabelled POR protein with isolated
PLBs and PTs.
POR protein carrying mutation in (a) the NADPH binding site (06) or (b)
the predicted active site (24) compared to wild-type mature POR (D)
lacking the transit peptide. Assays were performed in the presence of
1 mM NADPH, 10 mM MgATP and 20 mM MgCl2. Samples were posttreated with (+th) or without (±th) thermolysin. Samples treated with
thermolysin (+th) show a POR-DP with a molecular weight approximately
2±4 kDa lower than the mature POR of 36 kDa. Molecular weight in kDa
of mature POR (36) is given. TM, translation mixtures.
assembly mixtures clearly stimulated the proper association of POR with both PLBs and PTs.
Mutations in the substrate-binding and co-factor binding
sites reduce membrane association
POR protein, altered in the amino acid sequence either in
the predicted nucleotide-binding or active site (ALA06 and
ALA24, respectively; cf. Dahlin et al., 1999), were used for
membrane association studies with isolated PLBs and PTs.
As seen in Figure 5(a), almost no labelling was found on
the ¯uorograms following thermolysin post-treatment
when a protein mutated in the NADPH-binding site was
used. That is, replacement of charged amino acids to
alanine in the NADPH-binding site abolished assembly.
Charges in these regions of POR are of crucial importance
for the association with the membrane. When the assay
was performed with POR mutated in the active site, only
small amounts of the POR-DP could be found (Figure 5b).
Control experiments were performed with ALA06, ALA24
D-TP and pPOR proteins treated with thermolysin in the
absence of membranes. The results showed that the
mutated POR proteins under these conditions had the
same protease resistance as the wild-type POR (data not
shown). That is, those mutations per se in POR did not
affect the protease susceptibility.
Discussion
During recent years, large efforts have been made to
elucidate the assembly mechanism of lumenal and integral
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
Association of POR with PLB and PT membranes
thylakoid proteins into functional structures in chloroplasts. Current data suggest the existence of several
speci®c pathways for the insertion or trans-thylakoid
transfer of thylakoid proteins. Some of these can be traced
back to mechanisms utilized in the export of bacterial
proteins or co-translational transport of ER proteins. Our
data on the assembly of pea POR to PLB- and PT-enriched
fractions from wheat imply that POR does not utilize any of
the pathways outlined for thylakoid proteins (cf. Keegstra
and Cline, 1999; Robinson et al., 1998). No distinction
between the assembly conditions of POR into thylakoids or
inner etioplast membranes from wheat has been found.
For instance, proper binding to both membrane systems
includes an absolute requirement for NADPH, stimulation
by ATP, but no requirement for stroma. Similar observations have been made during the association of pea pPOR
with thylakoid membranes from light-grown pea (Dahlin
et al., 1995).
In the present paper, we have used a heterologous
system with POR having its origin in pea and the puri®ed
PLBs and PTs originating from wheat. The pea POR has
characteristics of both PORA and PORB, and is therefore
suitable for an exploratory study on etioplast inner membrane binding. However, PLBs and PTs are dif®cult to
purify from pea, as the production of etiolated leaf material
is low and methods for puri®cation have not been
developed. Furthermore, isolated pea PLBs have not
been characterized, although it can be supposed that the
major features are similar to those of wheat.
To our knowledge, this is the ®rst time the association
reaction of a protein has been reconstructed in vitro with
an isolated and puri®ed paracrystalline membrane structure such as the PLB. These results thus have a general
importance, in addition to outlining the assembly of POR
to etiolated inner membranes. It may be anticipated that
the tubular structure of the PLB may pose a problem for
detecting proper membrane association. However, the
32 kDa protease-degradation product of radiolabelled POR
following thermolysin post-treatment (Figure 1) could also
be observed when isolated etioplast inner membranes
were treated with thermolysin (LuÈtz and ToÈnissen, 1984). In
the same work, it was also shown that thermolysin has
only a limited effect on the proper PLB structure.
POR has been shown to associate speci®cally with the
plastid inner membranes and, for example, the envelope
membranes of pea chloroplasts do not bind POR (Dahlin
et al., 1995). This strongly indicates that the binding of POR
to PLBs and PTs is a speci®c process. The membraneassociation assays of POR with isolated PLBs and PTs led
to several interesting observations. The POR expressed
from cDNA clones from etiolated plants associated with
inner membranes of chloroplasts (Figure 1; Dahlin et al.,
1995) and etioplasts (Figure 1). This suggests that identi®cation mechanisms (receptors) and/or other components
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
301
of the assembly apparatus for POR may be functionally
similar between etioplast inner membranes and thylakoid
membranes of chloroplasts. Logically, this re¯ects the
in vivo situation, which requires that POR is present in
inner membranes of both etioplasts and chloroplasts
(Sundqvist and Dahlin, 1997).
The basis for the formation of PLBs in etioplasts is not
well understood. PLBs contain POR as the dominating
protein, but we do not know if the association of POR
occurs directly with growing PLBs or takes place primarily
in PTs, which may then transform into PLBs. A comparison
can be made between the attachment of POR to EPIMs,
and the integration or thylakoid transfer of several other
chloroplast proteins. Insertion of LHCP (Yalovsky et al.,
1990) and transfer across the thylakoid membranes of
OE33 (Hashimoto et al., 1997) and newly synthesized D1
protein (Mattoo and Edelman, 1987) occur primarily in the
single stroma lamellae. The proteins thereafter translocate
to the granal region. Alternatively, an enrichment, for
example LHCP molecules, may induce the formation of
stacked regions. A similar mechanism may operate during
the development of proplastids to etioplasts in dark-grown
seedlings. Proplastids in dark- or light-grown plants contain invaginations from the inner envelope and a few
lamellar membranes, but generally lack PLBs (Whatley,
1977). Following transport of POR into the proplastids, the
protein associates with the inner lamellar membranes. A
massive accumulation of POR, together with a relatively
high content of MGDG (Leese and Leech, 1976; Ryberg
et al., 1983), stimulates the formation of PLBs (Murakami
and Ikeuchi, 1982). The inner membranes then ef®ciently
incorporate POR and the small PLBs grow into a large PLB
structure.
The physical nature of the interaction of POR with inner
membranes is confusing. Sequencing data (Schulz et al.,
1989; Spano et al., 1992) and in vitro assembly studies
(Dahlin et al., 1995; Teakle and Grif®ths, 1993) in chloroplasts have suggested that POR is a peripheral protein
located on the stromal side of the thylakoid membrane.
Despite this, it is also a protein tightly bound to the
membrane, as previously shown by salt and detergent
washes of immobilized PLBs (Grevby et al., 1989). This is
further con®rmed in Figure 3(b), where post-treatment with
NaOH and Na2CO3 following association, in the presence of
NADPH, had a very limited effect on the removal of
assembled pPOR. The location of the protein to the stromal
side of the inner membranes is clearly seen. POR must be
accessible to thermolysin, as the action of this protease
results in a 2 or 4 kDa smaller POR-DP product.
The assembly of pPOR into PTs appears to be strictly
dependent on the presence of exogenous NADPH (Figure
4a). The assembly of pPOR into PLBs could occur even
when the samples were deprived of exogenous NADPH.
However, as the preservation of the regular ultrastructure
302
Sheila Engdahl et al.
of isolated PLBs has been shown to depend on the
presence of NADPH (Ryberg et al., 1988), the PLBs (and
PTs) were isolated in the presence of NADPH. During the
study of NADPH dependence, NADPH was excluded from
the last centrifugation step. The loss of the assembly after
an extra washing step indicates that the retained binding
was not an intrinsic property of the PLB membrane, but
more probably an effect of retained NADPH in the tubular
PLBs from the isolation procedure. This retention did not
occur with PTs. However, repeated washing did not
eliminate the capacity for POR to associate with PLB
membranes, but only abolished the proper binding as
revealed by the thermolysin post-treatment (Figure 4b).
Attachment, but no binding, occurred in either PLBs or PTs
of the POR protein ALA06 mutated in the NADPH-binding
site, not even in the presence of 1 mM NADPH (Figure 5a).
This strengthens the interpretation that the PLB can retain
a substantial amount of NADPH, which suf®ces for binding
of POR, even in the absence of exogenous NADPH. This
suggests a strict NADPH requirement for proper association of POR with both PLBs and PTs. A requirement of
NADPH for correct folding of POR is possible. POR
mutated in the active site (ALA24) had a severely hampered capacity to bind to both PLBs and PTs, but small
amounts of the protein still managed to assemble in the
membranes (Figure 5b). This suggests that membrane
association of POR can take place, albeit at a signi®cantly
lower ef®ciency, in the absence of bound pigment. Similar
observations have been made in chloroplasts where
membrane-bound pigments stimulated the integration of
LHCP into the thylakoids (Dahlin and Timko, 1994).
In conclusion, both pPOR and POR (as assayed with the
D-TP translation product) appear properly to associate with
the two types of inner membranes ± the PTs and PLBs ±
present in etioplasts. The binding process is dependent on
the presence of NADPH and ATP, but does not require that
the ternary complex of Pchlide, POR and NADPH has
formed before membrane association. In addition, the
existence of PLB as a cubic-phase membrane system does
not present any hindrance for physiological membrane
association of POR.
Experimental procedures
Plant material
Wheat grains (Triticum aestivum L. cv. Kosack, from Weibull,
Landskrona, Sweden) were imbibed in tap water overnight and
sown in commercially available soil (Yrkesplantjord, HammenhoÈg,
Sweden). The seedlings were grown in darkness or in a greenhouse at 24°C for 6 days.
Isolation of plastids and etioplast inner membranes
Etioplasts and chloroplasts were isolated by a combination of
differential centrifugation and Percoll gradient centrifugation
(Cline, 1986). Puri®ed plastids were resuspended in a small
volume of import buffer (0.33 M sorbitol, 50 mM Hepes±KOH
pH 8.0) and counted in a haemocytometer. Lysates were prepared
by resuspending plastids in a suitable volume of a buffer of
10 mM Hepes±KOH and 10 mM MgCl2 pH 8.0 (10 HK buffer) for
5 min. Stroma-free EPIMs and thylakoids were obtained by a
4200 g centrifugation of the lysates for 8 min. The membranes
were resuspended in import buffer, centrifuged as above, and
again resuspended in a small volume of import buffer.
Isolation and puri®cation of PLBs and PTs were performed in
the presence of 0.3 mM NADPH according to Lindsten et al. (1988).
To preserve the PLB structure and the Pchlide657 form after
isolation, PLBs and PTs were resuspended in import buffer
containing 0.3 mM NADPH. The purity of fractions was assayed
with Coomassie Blue-stained SDS±PAGE gradient gels (10±15%)
and low-temperature ¯uorescence measurements using an SLM
Aminco 8100C spectro¯uorometer (SLM Instruments, Urbana, IL,
USA).
Preparation of radiolabelled precursor protein
Radiolabelled proteins were produced in vitro from the Bluescript
vector containing the coding sequence for the pPOR (Spano et al.,
1992) and different pAlter vectors, containing the coding
sequences for the wild-type mature POR (D-TP), and the mutant
proteins ALA06 and ALA24 (carrying, respectively, the charge-toalanine mutations RD120,121A and DK270,273A; Dahlin et al.,
1999; Wilks and Timko, 1995) from pea. The linearized plasmids
were used in a coupled transcription/translation wheatgermextract system (TNT) from Promega (Madison, WI, USA), using
[3H]leucine (5.8 PBq mol±1) and T7 RNA polymerase. Before use
the translation mixture was diluted threefold with import buffer
and adjusted to 30 mM unlabelled leucine.
Protein analysis
The total amount of protein was measured (Bradford, 1976) prior
to association and SDS±PAGE. Bovine serum albumin (Sigma, St
Louis, MO, USA) was used as standard protein. Samples were
solubilized in CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, 2.5% w/v, ®nal concentration) before
addition of the colour reagent (B. Wiktorsson, Department of Plant
Physiology, GoÈteborg University, Sweden, personal communication).
Membrane association assays
Isolated PLB and PT membranes or EPIMs and thylakoids were
assayed for proper membrane association during incubation with
the radiolabelled POR constructs. Exogenous MgATP (10 mM),
containing 20 mM MgCl2, and NADPH (1 mM) were present during
the incubation. In samples with no MgATP added, the translation
mixture containing radiolabelled protein was ®ltered through a
Sephadex G-25 column in order to remove excess nucleoside
triphosphates. All handling and membrane association assays
were performed in weak green light or in darkness. If not stated
otherwise, samples were post-treated with or without thermolysin
(100 mg ml±1) while kept on ice for 30 min. The post-treatment
was inhibited by EDTA (®nal concentration 25 mM), the samples
were centrifuged, and the pellets (consisting of membrane-bound
radiolabelled POR) were dissolved in a small volume of import
buffer containing EDTA (®nal concentration 25 mM). The nature of
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
Association of POR with PLB and PT membranes
the membrane association of POR was assayed with posttreatment with NaOH (®nal concentration 50 mM) and Na2CO3
(®nal concentration 50 mM). Proteins were separated by 12.5%
SDS±PAGE using the buffer system of Laemmli (1970) and
prepared for ¯uorography and exposed to X-ray ®lm at ±80°C.
Acknowledgements
The authors thank Ann Marie MaÊnqvist and Nancy Ilenius for
skilful technical assistance, and Margareta Ryberg and Agneta
Lindsten for valuable discussions. Financial support from the
Adlerbertska foundation to H.A., the Hvitfeltds stipend foundation
and Helge Axelssons foundation to S.E., and grants from the
Swedish Natural Science Research Council to C.S., The Carl
Trygger Foundation for Scienti®c Research to C.D., and the US
DoE to M.P.T. are gratefully acknowledged.
References
Armstrong, G.A., Runge, S., Frick, G., Sperling, U. and Apel, K.
(1995) Identi®cation of NADPH: protochlorophyllide oxidoreductases A and B: a branched pathway for light-dependent
chlorophyll biosynthesis in Arabidopsis thaliana. Plant Physiol.
108, 1505±1517.
Aronsson, H., Sohrt, K. and Soll, J. (2000) NADPH: protochlorophyllide oxidoreductase uses the general import route into
chloroplasts. Biol. Chem. 381, 1263±1267.
Aronsson, H., Sundqvist, C., Timko, M.P. and Dahlin, C. (2001)
Characterisation of the assembly pathway of the pea NADPH:
protochlorophyllide (Pchlide) oxidoreductase (POR) and with
emphasis on the role of its substrate Pchlide. Physiol. Plant.
111, 239±245.
Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248±254.
Cline, K. (1986) Import of proteins into chloroplasts: membrane
integration of a thylakoid precursor protein reconstituted in
chloroplast lysates. J. Biol. Chem. 261, 14804±14810.
Dahlin, C. and Timko, M.P. (1994) Integration of nuclear-encoded
proteins into pea thylakoids with different pigment contents.
Physiol. Plant. 91, 212±218.
Dahlin, C., Sundqvist, C. and Timko, M.P. (1995) The in vitro
assembly of the NADPH-protochlorophyllide oxidoreductase in
pea chloroplasts. Plant Mol. Biol. 29, 317±330.
Dahlin, C., Aronsson, H., Wilks, H.M., Lebedev, N., Sundqvist, C.
and Timko, M.P. (1999) The role of protein surface charge in
catalytic activity and chloroplast membrane association of the
pea NADPH:protochlorophyllide oxidoreductase (POR) as
revealed by alanine scanning mutagenesis. Plant Mol. Biol.
39, 309±323.
Dahlin, C., Aronsson, H., Almkvist, J. and Sundqvist, C. (2000)
Protochlorophyllide-independent import of two NADPH:
Pchlide oxidoreductase proteins (PORA and PORB) from
barley into isolated plastids. Physiol. Plant. 109, 298±303.
Grevby, C., Engdahl, S., Ryberg, M. and Sundqvist, C. (1989)
Binding properties of NADPH-protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and
immobilized prolamellar bodies. Physiol. Plant. 77, 493±503.
Grif®ths, W.T. (1978) Reconstitution of chlorophyllide formation
by isolated etioplast membranes. Biochem. J. 174, 681±692.
Hashimoto, A., Ettinger, W.F., Yamamoto, Y. and Theg, S.M.
(1997) Assembly of newly imported oxygen-evolving complex
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304
303
subunits in isolated chloroplasts: sites of assembly and
mechanism of binding. Plant Cell, 9, 441±452.
Holtorf, H., Reinbothe, S., Reinbothe, C., Bereza, B. and Apel, K.
(1995) Two routes of chlorophyllide synthesis that are
differentially regulated by light in barley (Hordeum vulgare
L.). Proc. Natl Acad. Sci. USA, 92, 3254±3258.
Keegstra, K. and Cline, K. (1999) Protein import and routing
systems of chloroplasts. Plant Cell, 11, 557±570.
Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature, 227,
680±685.
Leese, B.M. and Leech, R.M. (1976) Sequential changes in the
lipids of developing proplastids isolated from green maize
leaves. Plant Physiol. 57, 789±794.
Lindsten, A., Ryberg, M. and Sundqvist, C. (1988) The polypeptide
composition of highly puri®ed prolamellar bodies and
prothylakoids from wheat (Triticum aestivum) as revealed by
silver staining. Physiol. Plant. 72, 167±176.
LuÈtz, C. and ToÈnissen, H. (1984) Effects of enzymatic cleavage on
prolamellar bodies and prothylakoids prepared from oat
(Avena sativa) etioplasts. Israel J. Bot. 33, 195±209.
Mattoo, A.K. and Edelman, M. (1987) Intramembrane
translocation and posttranslational palmitoylation of the
chloroplast 32 kDa herbicide-binding protein. Proc. Natl Acad.
Sci. USA, 84, 1497±1501.
Murakami, S. and Ikeuchi, M. (1982) Biochemical characterization
and localization of the 36 000 dalton NADPH:protochlorophyllide oxidoreductase in squash etioplasts. Prog. Clin. Biol.
Res. 102, 13±23.
Oosawa, N., Masuda, T., Awai, K., Fusada, N., Shimada, H., Ohta,
H. and Takamiya, K. (2000) Identi®cation and light-induced
expression of a novel gene of NADPH-protochlorophyllide
oxidoreductase isoform in Arabidopsis thaliana. FEBS Lett.
474, 133±136.
Reinbothe, C., Apel, K. and Reinbothe, S. (1995a) A light-induced
protease from barley plastids degrades NADPH:protochlorophyllide oxidoreductase complexed with chlorophyllide. Mol.
Cell. Biol. 15, 6206±6212.
Reinbothe, S., Runge, S., Reinbothe, C., van Cleve, B. and Apel, K.
(1995b) Substrate-dependent transport of the NADPH:
protochlorophyllide oxidoreductase into isolated plastids.
Plant Cell, 7, 161±172.
Reinbothe, C., Lebedev, N., Apel, K. and Reinbothe, S. (1997)
Regulation of chloroplast protein import through a
protochlorophyllide-responsive transit peptide. Proc. Natl
Acad. Sci. USA, 94, 8890±8894.
Robinson, C., Hynds, P.J., Robinson, D. and Mant, A. (1998)
Multiple pathways for the targeting of tylakoid proteins in
chloroplasts. Plant Mol. Biol. 38, 209±221.
Runge, S., Sperling, U., Frick, G., Apel, K. and Armstrong, G.A.
(1996) Distinct roles for light-dependent NADPH:protochlorophyllide oxidoreductases (POR) A and B during greening in
higher plants. Plant J. 9, 513±523.
Ryberg, M. and Dehesh, K. (1986) Localization of NADPHprotochlorophyllide oxidoreductase in dark-grown wheat
(Triticum aestivum) by immune-electron microscopy before
and after transformation of the prolamellar bodies. Physiol.
Plant. 66, 616±624.
Ryberg, M. and Sundqvist, C. (1988) The regular ultrastructure of
isolated prolamellar bodies depends on the presence of
membrane-bound NADPH-protochlorophyllide oxidoreductase. Physiol. Plant. 73, 218±226.
Ryberg, M., Sandelius, A.S. and Selstam, E. (1983) Lipid
304
Sheila Engdahl et al.
composition of prolamellar bodies and prothylakoids of wheat
etioplasts. Physiol. Plant. 57, 555±560.
Schulz, R., SteinmuÈller, K., Klaas, M., Forreiter, C., Rasmussen, S.,
Hiller, C. and Apel, K. (1989) Nucleotide sequence of a cDNA
coding for the NADPH-protochlorophyllide oxidoreductase
(PCR) of barley (Hordeum vulgare L.) and its expression in
Escherichia coli. Mol. Gen. Genet. 217, 355±361.
Spano, A.J., He, Z., Michel, H., Hunt, D.F. and Timko, M.P. (1992)
Molecular cloning, nuclear gene structure, and developmental
expression of NADPH: protochlorophyllide oxidoreductase in
pea (Pisum sativum L.). Plant Mol. Biol. 18, 967±972.
Sundqvist, C. and Dahlin, C. (1997) With chlorophyll pigments
from prolamellar bodies to light-harvesting complexes. Physiol.
Plant. 100, 748±759.
Teakle, G.R. and Grif®ths, W.T. (1993) Cloning, characterization
and import studies on protochlorophyllide reductase from
wheat (Triticum aestivum). Biochem. J. 296, 225±230.
Whatley, J.M. (1977) Variations in the basic pathway of
chloroplast development. New Phytol. 78, 407±420.
Wilks, H.M. and Timko, M.P. (1995) A light-dependent
complementation system for analysis of NADPH: protochlorophyllide oxidoreductase. Identi®cation and mutagenesis of two
conserved residues that are essential for enzyme activity. Proc.
Natl Acad. Sci. USA, 92, 724±728.
Yalovsky, S., Schuster, G. and Nechushtai, R. (1990) The
apoprotein precursor of the major light-harvesting complex of
photosystem II (LHCIIb) is inserted primarily into stromal
lamellae and subsequently migrates to the grana. Plant Mol.
Biol. 14, 753±764.
ã Blackwell Science Ltd, The Plant Journal, (2001), 27, 297±304