From the
An intronless nuclear gene, psbT, isolated from cotton,
encodes a putative 11-kDa protein (PSII-T) highly homologous in its C
terminus to the N terminus of the partially sequenced PSII-T protein
from spinach photosystem II. Analysis of the deduced amino acid
sequence of cotton PSII-T revealed the presence of potential
chloroplast stroma and thylakoid targeting domains and a putative
mature PSII protein of 3.0 kDa, composed of only 28 amino acid
residues. The cotton PSII-T 11-kDa precursor was synthesized in
vitro in a wheat germ extract translation system, and the
translation product was used in assays for protein imports into
isolated pea chloroplasts. It was shown that the cotton PSII-T
precursor was imported into the chloroplasts, processed to a mature
form of approximately 3.0 kDa, agreeing with the predicted size from
amino acid sequence analysis, and localized to the lumenal side of the
thylakoid membrane, thus defining a new nuclear encoded lumenal protein
and the smallest polypeptide of PSII reported to date. Processing of
the PSII-T precursor occurred in two steps and involved the formation
of a stromal intermediate of approximately 7.5 kDa, as predicted from
primary structure analysis.
In oxygen-evolving photosynthetic organisms, photosystem II
(PSII)
Many of these low
mass PSII polypeptides are encoded in the nuclear genome in plants and
algae, as judged by their absence in the four sequenced chloroplast
genomes
(1) . Nuclear encoded chloroplastic proteins are
synthesized as higher molecular weight precursors with N-terminal
extensions (chloroplast transit peptides), which direct their import
across the chloroplast envelope into the stroma and are cleaved by a
stromal processing protease
(6) . Thylakoid membrane proteins
like the light-harvesting chlorophyll a/b binding
proteins are processed by the stromal protease to their mature form and
are directed to the thylakoid membrane by targeting signals contained
in the mature protein. Alternatively, thylakoid lumenal proteins such
as the oxygen-evolving complex polypeptides, OE33, OE23, and OE17,
contain bipartite presequences composed of the stroma targeting domains
and thylakoid targeting domains, which direct them across the thylakoid
membrane into the thylakoid lumen
(7) . The stroma targeting
domain is cleaved by the stromal protease, and the resulting
intermediate is translocated into the thylakoid lumen where a thylakoid
processing protease cleaves the thylakoid transfer domain resulting in
the mature protein
(7, 8, 9, 10) .
Stroma targeting peptides vary in size from
One of the nuclear encoded low molecular weight PSII proteins, the
``5-kDa polypeptide,'' was first isolated from spinach
thylakoids and purified to homogeneity by Ljungberg et al.(2) and found to be an extrinsic PSII membrane protein. An
analog from wheat has also been isolated; the partial N-terminal amino
acid sequence for both polypeptides has been determined for the first
29 and 10 amino acids, respectively
(4) ; and the protein species
has been named PSII-T and their genes psbT(1) .
We
report here the isolation and characterization of a nuclear gene from
cotton encoding a putative 11-kDa protein, highly homologous in its
C-terminal region to the 5-kDa PSII-T proteins from spinach and wheat
and containing potential stroma and thylakoid targeting domains. It is
further demonstrated that the PSII-T precursor is imported into
isolated pea chloroplasts, where it is localized to the lumenal side of
the thylakoid membrane with a final size of approximately 3 kDa.
Maturation of the precursor polypeptide to its final form occurs in two
steps and involves the formation of a stromal intermediate.
Restriction endonucleases, the Klenow fragment of polymerase
I, and T4 DNA ligase were purchased from Boehringer Mannheim, and T3
RNA polymerase was purchased from Stratagene. Wheat germ extract was
obtained from Promega Corp. Radiolabeled
[
From the
remaining 450 µl of the import reaction mixture, chloroplasts were
pelleted at 1000
Import assays in the dark were conducted in a 300-µl
total volume at 25 °C in tubes protected from light by aluminum
foil. Chloroplasts were separated into two aliquots and incubated with
(D+) and without (D-) thermolysin as described above.
Following protease inactivation with EDTA, unbroken chloroplasts were
reisolated as before and washed with 5 mM EDTA in IB, pelleted
at 1000
A stromal cleavage
recognition site resembling the loose consensus
VXA
In order
to determine whether the PSII-T precursor is imported into the
chloroplast and localized to the thylakoid lumen, as suggested by
targeting elements in the putative transit peptide, in vitro protein import of PSII-T into intact chloroplasts was investigated
using the in vitro translation product synthesized from RNA,
produced in turn from the T3 polymerase transcription of the
recombinant plasmid carrying the 613-nucleotide insert.
The results from the import assays are
shown in Fig. 3A. In the presence of light and ATP,
import is evident, as judged by the disappearance of precursor and the
appearance of a processed final product in both the nonprotease- and
protease-treated light import assays (Fig. 3A, lanes4 and 5). The molecular weight of the processed
product was estimated to be approximately 3 kDa, which agrees with the
size of the mature product predicted previously from primary structure
analysis of the PSII-T precursor. The absence of PSII-T precursor in
the nonprotease-treated light import lane indicates that import
occurred efficiently.
To determine the localization of
the 3-kDa product, chloroplast fractionation experiments were
performed. Import assays were carried out in the light as before, and
after lysis, the chloroplasts were fractionated and the soluble
fraction (S) was separated from the thylakoids by differential
centrifugation. The thylakoid membrane fractions underwent three
different treatments after imports and subfractionations: 1) not
treated with protease (M-), 2) treated with protease (M+),
3) treated with protease after thylakoid membranes were disrupted with
Triton X-100 (M + TX).
Radiolabeled protein is not detected in
the soluble fraction (Fig. 3A, lane6). In the membrane fraction that was not treated with
protease, the 3-kDa product is clearly present (lane7) and is also present in the protease-treated membrane
fraction (lane8), indicating that the 3-kDa product
is localized in the inner side of the thylakoid membrane and is
therefore protected from protease action. Protease treatment after the
membranes had been disrupted with Triton X-100, resulted in the
disappearance of the 3-kDa band (lane9),
demonstrating that the 3-kDa polypeptide is now sensitive to protease
digestion and confirming that it is localized to the thylakoid lumen.
It is concluded from the results obtained from the chloroplast
protein import experiments that the PSII-T precursor can be imported
into pea chloroplasts in vitro and processed to a final
product of 3 kDa, as the amino acid sequence analysis predicts.
Furthermore, chloroplast fractionation experiments demonstrate that the
mature PSII-T product is localized on the lumenal side of the thylakoid
membrane.
The larger
intermediate species observed on the gels (i1), has a
molecular mass of 7.5 kDa, similar to the molecular mass of the
predicted intermediate containing the mature protein and the thylakoid
targeting domain. The smaller intermediate (i2) was estimated
to be approximately 3.8 kDa, close to the size estimated for both the
predicted thylakoid targeting domain (4.3 kDa) and stroma targeting
domain (3.57 kDa).
In vitro protein import
experiments with radiolabeled PSII-T precursor showed that the cotton
PSII-T precursor is imported into isolated pea chloroplasts and that it
is processed to the predicted 3.0-kDa mature form. Although the spinach
PSII-T was isolated from PSII preparations of thylakoid
membranes
(2) , it was not determined whether the protein was
facing the stromal or the lumenal side of the membrane. Chloroplast
fractionation experiments presented here demonstrate that PSII-T is
localized on the lumenal side of the thylakoid membrane, thus defining
a new nuclear encoded lumenal protein. It may be a component of the
oxygen-evolving complex that is known to reside in the thylakoid
lumen
(4) .
Time course import assays showed that maturation
of the PSII-T precursor to its final form occurs through a two step
process. First, a stromal intermediate is formed, which, upon
translocation into the thylakoid membrane, is processed to the mature
protein, consistent with the processing of other lumenal proteins
(OE33, OE23, and OE17). The sizes of the stromal intermediate
(Fig. 4, i1) and mature forms are those predicted from
primary structure analysis of the PSII-T precursor. The second
intermediate of approximately 3.8 kDa is transiently present during the
time course importation (Fig. 4, i2). Since the exact stroma
processing site is not known, it is difficult to assign this species to
either the predicted stroma targeting or thylakoid targeting domain of
3.57 and 4.43 kDa, respectively. The fact that this species appears on
the time course gels after the stromal intermediate suggests that it
represents the thylakoid targeting domain. However, the identity of
this species has not been rigorously established.
We are very appreciative of the advice and assistance
of the laboratory of Dr. K. Cline, University of Florida, and in
particular Dr. R. Henry in carrying out the protein import experiments.
(
)
is composed of three major complexes:
the light-harvesting antennae, the reaction center core, and the
oxygen-evolving complex
(1) . A number of intermediate molecular
weight polypeptides (>10 kDa) composing these complexes have been
extensively studied and include the light-harvesting chlorophyll
a/b binding proteins, the D1 and D2 proteins of the
reaction core, and the oxygen-evolving (OE) complex polypeptides OE33,
OE23, and OE17. Improvements in the resolution of polypeptides by
SDS-PAGE techniques in the past few years has enabled the isolation of
a number of low molecular weight proteins from PSII, mostly of unknown
function and ranging in size from 3.6-10
kDa
(2, 3, 4, 5) .
30 to
100
amino acids, and comparison of a large number of chloroplast precursors
has defined the sequence VXAA as a loose consensus motif for
stromal processing, with cleavage occurring between the two alanine
residues
(12) . The thylakoid transfer domain, on the other hand,
contains a region very similar to the signal peptide of secreted
proteins in prokaryotes and eukaryotes and contains a strongly
conserved motif AXA preceding the processing site
(11) .
S]methionine (>1000 Ci/mmol) and
C-radiolabeled low molecular weight protein standards were
from Amersham Corp., and thermolysin was from Sigma.
Gene Isolation, Sequencing, and Amino Acid Sequence
Analysis
Construction of cDNA and genomic libraries from cotton
(Gossypium hirsutum cv. Coker 201) have been described
previously
(13) . psbT cDNA was obtained from a
light-induced cDNA library and used to screen a cotton phage 2001
genomic library, from which a genomic clone was isolated. The genomic
clone was mapped, sequenced by dideoxy DNA sequencing, and analyzed for
open reading frames. The deduced amino acid sequences were examined for
sequence homologies with known proteins in the PIR and SWISS-PROT data
banks using the Intelligenetics Suite software system.
Construction of in Vitro Transcription Plasmid
The
1933 nucleotide psbT genomic clone, which was found not to
contain introns, was digested with AvaII and NdeI,
and the resulting 613-nucleotide fragment (delineated with arrows in Fig. 1) was blunt ended with the Klenow fragment of
polymerase I. The blunt ended fragment was then ligated to plasmid
pBluescript II KS+ (Stratagene) at the EcoRV site.
Figure 1:
Nucleotide sequence of the cotton
psbT gene and deduced amino acid sequence of the encoded
protein. The partial cDNA sequence is underlined, and
differences from the gene sequence are shown in lowercaseletters. Putative CAAT box, TATA box, and polyadenylation
signal AATAAT are shaded. GATA motifs, found in other plant
light-regulated genes, are also shaded. Two arrows,
in the 5`-untranslated region and 3`-untranslated region, indicate the
AvaII and NdeII sites, respectively, which were used
for the subcloning of the in vitro transcription template (see
``Materials and Methods''). The nucleotide and amino acid
sequence have been submitted to GenBank and PIR,
respectively, under the accession number
X54092.
In Vitro Transcriptions and
Translations
Recombinant plasmid carrying the 613-nucleotide
fragment was tested for insert orientation by restriction analysis. The
plasmid was linearized downstream of the inserted fragment with
NotI, and the linearized template was transcribed in vitro using T3 RNA polymerase. The psbT RNA template was
isolated and translated in vitro in the wheat germ system in
the presence of [S]methionine, using
Promega's technical manual for in vitro translation.
Translations were analyzed by Tricine-SDS-PAGE
(14) and
fluorography.
Preparation of Chloroplasts and Thylakoids
Intact
chloroplasts were isolated from 9-10-day-old pea seedlings
(Pisum sativum L. cv. Laxton's Progress 9) as described
by Cline
(15) . Chloroplasts were resuspended in import buffer
(IB) (50 mM Hepes/KOH, pH 8.0; 0.33 M sorbitol) and
kept on ice until used. Chloroplast lysates were prepared by osmotic
lysis at 0 °C in 10 mM Hepes/KOH, pH 8.0, containing 10
mM MgCl. Thylakoids were separated from the
soluble chloroplast fraction by centrifuging lysates at 3200
g for 8 min at 4 °C and were washed twice with import
buffer containing 10 mM MgCl
.
Chloroplast Protein Import Assays and Chloroplast
Fractionation
Import of radiolabeled PSII-T precursor into
isolated chloroplasts was conducted in an illuminated water bath at 25
°C, for 15 min
(15) . Import reactions in the light were
conducted in a total volume of 600 µl. Reactions were initiated by
adding 100 µl of radiolabeled precursor, diluted 4 with 30
mM nonradioactive methionine, to 400 µl of chloroplasts,
corresponding to 200 µg of chlorophyll, and 100 µl of 60
mM Mg-ATP, pH 8.0. Import assays were stopped by transfer of
the tubes to ice. Intact chloroplasts were reisolated from 150 µl
of the import reaction mixture by centrifugation on 35% Percoll at 4
°C, 3200
g for 8 min. The chloroplasts were washed
with 1
IB, pelleted again at 1000
g for 5 min,
resuspended in 50 µl of 20 mM EDTA and 50 µl 2
SDS-PAGE sample buffer, and frozen at -20 °C.
g, resuspended in 0.5 ml of IB, and
treated with 25 µl of 2 mg/ml thermolysin for 40 min on ice. The
protease was inactivated by adding 250 µl of 50 mM EDTA in
IB. Intact chloroplasts were reisolated by centrifugation on 35%
Percoll containing 5 mM EDTA, washed with 5 mM EDTA
in IB, and pelleted as above. Chloroplasts were lysed in 75 µl of
10 mM Hepes/KOH, pH 8.0, 10 mM MgCl
,
followed by addition of an equal volume of 2
import buffer.
One-fourth of the lysate was frozen and represents protease-protected
proteins imported into the chloroplasts (L+). The rest was
fractionated into a soluble fraction (S) and membranes by
centrifugation at 3200
g for 8 min. The membrane
fraction was resuspended in 750 µl of IB, divided into three
aliquots, and pelleted by centrifugation. One aliquot was resuspended
in 37 µl of 20 mM EDTA and frozen (M-). The other
two aliquots were treated with 30 µl of thermolysin in IB (0.33
mg/ml final concentration) in the presence (M + TX) and absence
(M+) of Triton X-100 (1% final concentration). Thermolysin was
inactivated with 7 µl of 250 mM EDTA in import buffer (50
mM final concentration). Membranes were solubilized by adding
an equal volume of 2
SDS-PAGE sample buffer
(14) and
were immediately boiled to prevent degradation by residual protease
activity.
g for 10 min, and resuspended in 50 µl of
20 mM EDTA and 50 µl of 2
SDS sample buffer.
Protease Sensitivity of PSII-T Precursor
20 µl
of radiolabeled precursor diluted 5 with 30 mM
methionine was treated with 3 µl of 2 mg/ml thermolysin for 40 min
at 4 °C, and the protease was inactivated with 3 µl of 250
mM EDTA in IB. An equal volume of 2
SDS sample buffer
was added, and the tube was placed in boiling water for 5 min. To
insure that protease sensitivity did not result from SDS denaturation
prior to inactivation of protease by SDS/heating, the reaction was
repeated with thermolysin and EDTA added together just prior to
heating.
Time Course Import Assays
For time course import
assays, two reactions were set up of total volume 4.8 ml, each
containing 3.2 ml of chloroplasts (0.5 mg/ml), 800 µl of 60
mM ATP, and 800 µl of radiolabeled PSII-T precursor
diluted 4 with nonradioactive 30 mM methionine.
Chloroplasts were prewarmed for 5 min at 25 °C in an illuminated
water bath prior to the addition of precursor. The reactions were
terminated at 0, 0.5, 1.0, 2.0, 3.0, 5.0, and 10.0 min by transferring
600-µl aliquots to tubes containing 10 µl of 0.2 M
HgCl
on ice for 5 min. The two sets were treated with and
without protease, as described above, and the final pellets were
resuspended in 45 and 60 µl of 20 mM EDTA, respectively.
Analysis of Samples
All samples were visualized by
Tricine-SDS-PAGE
(14) and fluorography. Aliquots of each assay
were precipitated with cold (-20 °C) acetone (95% final
concentration), and centrifuged at 10,000 g for 10
min. The pellets were dried and solubilized in SDS-sample buffer, and
aliquots were subjected to electrophoresis and fluorography as
described in the figure legends. For time course assays, chlorophyll
concentration was determined following acetone
precipitation
(16) , and equal amounts of protein were
electrophoresed for each time point by adjusting the volume of sample
buffer used to resuspend each pellet. Precipitation with acetone and
use of the Tricine-SDS-PAGE system was necessary to resolve mature
PSII-T, due to its small size and its co-migration with chlorophyll-SDS
micelles in conventional SDS-PAGE.
RESULTS
Isolation and Characterization of the Cotton psbTGene
The nucleotide sequence of the 1933-nucleotide psbT genomic clone and the derived amino acid sequence are shown in
Fig. 1
. The gene includes a putative coding region of 315
nucleotides containing no introns that would translate to a protein of
105 amino acids with molecular mass of 11 kDa. The cDNA sequence is
underlined, and only three differences with the genomic
sequence were found, all in the 3`-untranslated region. Potential
regulatory elements in the 5` upstream and 3` downstream untranslated
regions are indicated in Fig. 1. The most likely functional CAAT
and TATA sequences are at positions -213 and -114, relative
to the putative translation initiation site, respectively. Between
these boxes, there are two elements, AAGATAATA (position -177)
and CTGATAAGA (position -127) that strongly resemble a conserved
GATA motif, AATGATAAGG, that has been found in this region in many
light-regulated genes
(17) . An AT-rich box found in several
other plant genes
(17) is also present. A putative AATAAT
polyadenylation signal is indicated.
Analysis of Protein Sequence
Homology between the Cotton Putative PSII-T Protein and
the Spinach PSII 5-kDa Protein
The deduced amino acid sequence
of psbT was examined for statistically significant homologies
with other proteins, and homology was found between the C terminus of
cotton PSII-T and the partially sequenced (29-amino acid) N terminus of
the spinach 5-kDa protein from PSII
(4) as shown in
Fig. 2
. Between the region spanning the last 28 C-terminal amino
acids of cotton PSII-T and the N terminus of the spinach sequence there
is at least 70% identity and two conservative replacements. It is
likely that the two unidentified residues (X) in the spinach
sequence are cysteine residues, as in the cotton deduced sequence, that
are not preserved during protein sequencing. The 5-kDa value of the
spinach protein may be an overestimation due to anomalous behavior of
small proteins on SDS-PAGE, caused in part by the co-migration of
lipids in thylakoid membrane protein extracts
(5) .
Figure 2:
Amino
acid sequence comparison between the putative PSII-T cotton protein and
the PSII 5-kDa protein from spinach. The twotoplines represent the cotton PSII-T precursor, and the
bottomline represents the N-terminal sequence of the
mature spinach protein. X, undetermined residue in that
sequence; ?, the possible unsequenced C-terminal region of the
protein. The region of homology is presented in boldfacetype; residue identity and conservative replacements are
shown by straight and dottedlines,
respectively. The putative stroma and thylakoidal processing sites of
PSII-T precursor are marked by arrows1 and
2, respectively. The corresponding putative recognition sites
VVANAA and ATA are shown with underlines. The potential
thylakoid transfer domain is indicated by a singleunderline.
Targeting Domains
Examination of the 75-amino acid
presequence segment revealed a potential stroma targeting domain and a
thylakoid targeting domain (Fig. 2). The first 24-amino acid
region of PSII-T contains features of stroma targeting domains
(11) in that it has a high content of serine and threonine
residues (12.5% Ser, 21.8% Thr), contains no acidic amino acid, and is
composed of an uncharged N-terminal segment followed by a region
containing four arginine residues (Fig. 2).
A
(12) , could be presumed to be the
VVANA
A sequence with hypothetical cleavage site between the two
Ala residues (Fig. 2, double underline). This would
result in a putative stroma targeting domain of 32 amino acids with a
molecular mass of 3.57 kDa and a putative PSII-T intermediate form of
73 amino acids with a molecular mass of 7.5 kDa. Within this putative
intermediate resides a sequence strongly resembling the thylakoid
transfer domains of chloroplast lumenal proteins (Fig. 2,
singleunderline). It contains a short N-terminal
segment of two arginines and a glutamate, followed by a 10-amino-acid
hydrophobic segment, and a C terminus of 9 residues with the putative
thylakoid processing site ATA
(11) . Cleavage occurring
after the ATA would yield a putative thylakoid targeting domain of 45
amino acids with molecular weight of 4.43 kDa and a putative mature
protein of 28 amino acids with molecular weight of 3.0 kDa.
In Vitro Import of Radiolabeled PSII-T Precursor into
Isolated Pea Chloroplasts
Import assays were conducted in the
light and in the presence of exogenously supplied ATP. Chloroplasts
were then treated with thermolysin, to remove precursors bound to the
envelope. Control assays were conducted in the dark without exogenously
supplied ATP to verify ATP-dependent import, since ATP is required for
translocation across the chloroplast envelope into the stroma
(18) and in some cases for transport of lumenal proteins across
the thylakoid membrane
(19) . Following import termination,
chloroplasts were divided into two fractions, and one fraction was
treated with thermolysin.
Figure 3:
A, invitro protein
import of PSII-T precursor into isolated pea chloroplasts. Lane1, initial translation product (TP); lane2, dark control minus thermolysin (D-);
lane3, dark control plus thermolysin
(D+); lane4, import in the light minus
thermolysin (L-); lane5, import in
the light plus thermolysin (L+); lane6, chloroplast soluble fraction (S); lane7, thylakoid membrane fraction minus thermolysin
(M-); lane8, thylakoid membrane
fraction plus thermolysin (M+); lane9,
thylakoid membrane fraction plus thermolysin in Triton X-100 (M + TX). On the leftside:
p, precursor; m, mature protein. 30 µl of
D-, D+, L- and 15 µl of the S, L+, M-,
M+, M + TX final pellet suspensions were precipitated with
cold (-20 °C) acetone (95% final concentration) and
centrifuged at 10,000 g for 10 min, and the pellets
were dried and resuspended in 30 µl and 10 µl of SDS sample
buffer, respectively. 10-µl aliquots were loaded in each lane. For
D-, D+, L-, and S, lanes contain 10% of the
chloroplasts or subfractions recovered from each assay. For L+,
M-, M+, and M + TX, lanes contain 20% of the
chloroplasts or subfractions recovered from each assay. B,
treatment of PSII-T precursor with thermolysin in the absence of
chloroplasts. Lane1, translation product; lane2, translation product plus thermolysin; lane3, translation product plus EDTA-treated thermolysin and
SDS sample buffer.
Under dark conditions, import of substantial
labeled PSII-T precursor is not observed, as judged by the presence of
full-length PSII-T precursor in the nonprotease-treated import assays
(Fig. 3A, lane2). The faint lower
band suggests minimal import due to preexisting ATP in the reaction
assay (ATP carried over from translation reactions, which was 50
µM or less, or endogenous chloroplast ATP). Upon treatment
with protease, the precursor and several lower molecular weight bands
are observed (Fig. 3A, lane3). The
presence of protease-protected full-length precursor suggests that a
portion of precursor molecules has completely entered the chloroplast
without entering the stroma sufficiently for processing to occur. The
presence of smaller size bands suggests that another portion of
precursor molecules has been imported only partially and to various
extents, the part extending outside the chloroplast envelope being
degraded by the protease, the remaining part inside the chloroplast
being protected. Protease-protected precursor is not a result of
insensitivity to thermolysin since PSII-T treated with the protease was
completely degraded, as shown in Fig. 3B, lane2. To exclude the possibility that residual thermolysin
was still active after EDTA treatment and repurification of
chloroplasts, the effectiveness of EDTA inactivation is shown in
Fig. 3B, lane3. As can be seen, the
PSII-T precursor treated with thermolysin and SDS sample buffer is not
degraded after exposure to EDTA.
Time Course Import Assays
It has been shown that the thylakoid lumen proteins OE33,
OE23, and OE17 are targeted to the thylakoid lumen by a two step
process, which includes the formation of a stromal intermediate after
cleavage of the stroma targeting domain by the stromal
protease
(8, 10) . To examine whether the processing of
PSII-T resembles that of the other lumenal proteins, time course import
assays with PSII-T precursor were performed. Import reactions were
conducted as before and were stopped at various time points soon after
initiation of the import process by the addition of HgCl,
which completely halts protein import into chloroplasts and all
intrachloroplast activities
(20) . Two sets of time course import
assays were conducted, and one set was treated with protease after
treatment with HgCl
. Chloroplasts were reisolated, washed,
and analyzed as described under ``Materials and Methods.''
The results of the nonprotease-treated time courses are shown in
Fig. 4
(uppergel). As the PSII-T precursor
disappears with time, two intermediate species appear, accumulate, and
disappear, followed by the appearance and accumulation of the mature
form. Time course import assays treated with protease (Fig. 4,
bottomgel) showed a similar band pattern, confirming
that these species are inside the chloroplast.
Figure 4:
In vitro time course of protein
importation of PSII-T. Import reactions were conducted in the presence
of light and 10 mM ATP as described under Materials and
Methods`` and terminated at the designated time points with
HgCl. Uppergel, minus protease treatment
of chloroplasts; bottomgel, plus protease treatment
of chloroplasts. Final pellet suspensions from each time point were
acetone-precipitated as described under ''Materials and
Methods,`` and the equivalent of 5 µg of chlorophyll was
loaded in each lane. On the left side, p, i1,
i2, and m refer to precursor, intermediate 1,
intermediate 2, and mature protein,
respectively.
Under the conditions
used, the import assay is partially a pulse-chase experiment.
Continuous binding of new precursor molecules is not observed during
the entire 10-min period, and intermediate molecular weight species
disappear with time. However, the mature radioactive protein
accumulates during the time course of the reaction, as shown by its
higher than expected level of radioactivity. (There are 6 Met residues
in the precursor and only 1 in the mature protein, yet equal amounts of
chloroplast protein were loaded in the gel lanes.)
DISCUSSION
The results presented in this paper demonstrate that the
nuclear cotton gene, psbT, encodes an 11-kDa protein that is
the precursor of the PSII-T polypeptide, one of the low molecular
weight proteins from photosystem II. PSII-T was first isolated from
spinach PSII preparations as a 5-kDa polypeptide, and the N-terminal
sequence was determined for 29 amino acids. A very high degree of
homology exists between the spinach sequence and the C-terminal cotton
PSII-T sequence. In cotton, PSII-T mature protein has a molecular
weight of 3.0 kDa, as predicted by amino acid sequence analysis, making
it the smallest polypeptide from PSII reported so far. It is possible
that the spinach analogue is smaller than determined and its assigned
size of 5 kDa is an overestimation, due to technical limitations of the
SDS-PAGE of very small proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.