Plant Cell, Vol. 13, 193-205, January 2001, Copyright © 2001, American Society of Plant Physiologists
The Gene for the Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase (Rubisco) Small Subunit Relocated to the Plastid Genome of Tobacco Directs the
Synthesis of Small Subunits That Assemble into Rubisco
Spencer M. Whitneya and
T. John Andrewsa
a Molecular Plant Physiology, Research School of Biological Sciences, Australian
National University, P.O. Box 475, Canberra 2601, Australia
Correspondence to:
T. John Andrews, john.andrews{at}anu.edu.au (E-mail), 61-2-6125-5075 (fax)
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ABSTRACT |
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To assess the extent to which a
nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed
preendosymbiotic location, we relocated the RbcS gene for the small subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid
RbcS transgenes, both with and without the transit presequence, were equipped with 3'
hepta-histidine–encoding sequences and psbA promoter and terminator elements. Both
transgenes were transcribed abundantly, and their products were translated into small subunit
polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either
the transit presequence was not translated or the transit peptide was cleaved completely. After
assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine
sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole
Rubisco hexadecamer by Ni2+ chelation. Small subunits produced by the plastid transgenes
were not abundant, never exceeding 
1% of the total small subunits, and they differed from
cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of
transplastomic small subunits might be caused by inefficient translation or
assembly.
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INTRODUCTION |
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The process of symbiogenesis that has given
rise to organelles has involved the progressive migration of most of the genes of the original
prokaryotic endosymbiont's chromosome to the nucleus of the host (Gray 1992 
;
Whatley 1993 ). This has occurred to differing extents in different organelles and in
different hosts. The RbcS gene for the small subunit of the chloroplast photosynthetic enzyme
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a much studied example of a gene that has
so migrated. In green algae and higher plants, RbcS has been transferred from the ancestral
plastid's genome to become a nuclear multigene family (Rodermel 1999 ). On the other
hand, in nongreen eukaryotic algae, rbcS has remained in its presumed ancestral location as
the second gene in the plastid rbcLS operon (except in the case of some dinoflagellates that
apparently lack RbcS altogether) (Delwiche and Palmer 1996 ).
A transferred
gene for a plastid protein, or a subunit thereof, must become equipped with regulatory and
transcriptional elements suitable to its new nuclear location and, in addition, requires a sequence
that encodes a transit peptide to target the polypeptide to the plastid. Furthermore, the precursor
polypeptide must be able to be synthesized on cytoplasmic ribosomes, folded with the aid of
cytoplasmic chaperones, transported through the envelope membrane system of the plastid, relieved of
its transit peptide, refolded with the aid of plastid chaperones, and finally assembled in the plastid
into the appropriate protein complex, which also may contain other nucleus- and/or plastid-encoded
subunits. These several requirements, which have been studied in varying degrees of detail for the
RbcS genes of higher plants and green algae, must coexist with the requirements imposed by
the function of the subunit in its ultimate complex (Rodermel 1999 ; Roy and Andrews 2000 ).
Genetic transformation of the genomes of organelles provides a means of
artificially reversing the endosymbiotic migration of genes for organelle proteins to the nucleus.
This enables analysis of the extent to which nuclear genes and their products, now acclimated to the
eukaryotic expression system and the cytoplasmic folding and transport machineries, have retained the
ability to be expressed in their presumed original prokaryotic compartment. As an example, we describe
here the relocation of the RbcS gene from the nucleus to the plastid genome of tobacco. Two
forms of the RbcS gene, one with and one without the presequence that encodes the transit
peptide, were equipped with plastid regulatory elements and inserted in the tobacco plastome. Both
functioned in the plastid environment, and their products were assembled correctly into Rubisco.
However, the amounts of small subunits synthesized in the chloroplast were small compared with the
products of the nuclear RbcS gene family. This indicates that acclimation to a nuclear
location has compromised, in ways not understood, the ability of this gene to be expressed abundantly
in the chloroplast.
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RESULTS |
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Relocation of RbcS to the Plastid
Genome
A tobacco cDNA encoding RbcS, both with and without its transit
peptide–encoding sequence and with a hepta-His–encoding sequence inserted immediately
5' to the terminator codon, was equipped with promoter and terminator sequences derived from the
plastid psbA gene. The construct lacking the transit sequence also had an Ala codon inserted
between codons 1 and 2 of the mature peptide. These cassettes were inserted into a vector derived from
pRV112a (Zoubenko et al. 1994 ) between flanking regions that direct insertion of the
cassette into a specific site in the inverted repeat regions of the tobacco plastid genome (Fig 1). These plasmids (ptpSSuH with the transit sequence and pSSuH without)
were introduced into tobacco chloroplasts biolistically (see Methods).
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Figure 1.
Plasmids Used for
Insertion of RbcS Genes into the Inverted Repeat Region of the Tobacco Plastid
Genome.
(A) Sequences of the SSu cassettes of ptpSSuH (upper nucleotide sequence), which
directs the synthesis of the SSu precursor, and pSSuH (lower sequence), which directs the synthesis of
the mature SSu with an alanyl insertion at residue 2 (in parentheses). The translated amino acid
sequence is shown above the upper nucleotide sequence. The region corresponding to the mature SSu is
shown in boldface. Deletions are shown as dashes; dots represent identity. The nucleotide
sequence includes the psbA promoter and 5' UTR (nucleotides 17 to 243), the
psbA terminator (nucleotides 822 to 1043), and rbcS (with and without the transit
peptide–encoding region) with a C-terminal hepta-His–encoding sequence (nucleotides 244 to
807).
(B) Plasmids pSSuH and ptpSSuH. To generate these plasmids, we substituted the
SacI-HindIII (sites underlined in [A]) fragments of the cassettes for the SacI-HindIII fragment
of pRV112a (Zoubenko et al. 1994 ) by using the multistep procedure described in Methods.
Long dashed lines indicate the homologous flanking regions that direct introduction of the
RbcS and aadA genes into the inverted repeats. These flanking regions constitute
nucleotides 101,145 (141,481) to 102,309 (140,317) and 102,316 (140,310) to 104,085 (138,541) of
inverted repeat A (inverted repeat B in parentheses) of the tobacco plastid genome (GenBank accession
number
Z00044). The tgf, RbcS, and PpsbA probes used in DNA gel
blot and RNA gel blot analyses and the annealing sites of the primers (TRNV, AADAr, SSuHis3, and
PRV5P) used to amplify and sequence the inserted genes are indicated. H, HindIII; H7,
hepta-His–encoding sequence; N, NcoI; p, rrn promoter and 5' UTR;
P, psbA promoter and 5' UTR; S, SalI; Sc, SacI; t, rps16
terminator; T, psbA terminator; tp, RbcS transit
presequence.
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Ten leaves were bombarded
with each plasmid, and five and 12 spectinomycin-resistant plantlets were obtained for pSSuH and
ptpSSuH, respectively. After tissue from all plantlets was subjected to another round of regeneration
on spectinomycin-containing medium, polymerase chain reaction (PCR) analysis (see Methods) was used to
identify two SSuH and four tpSSuH plantlets as genuine transformants. Both SSuH transformants (SSuH2
and SSuH5) and two of the tpSSuH transformants (tpSSuH4 and tpSSuH7) were cloned and regenerated twice
more on spectinomycin-containing medium before growing to maturity in soil.
Because DNA gel
blots of total leaf DNA from the T0 plants digested with HindIII showed no trace of the
7.7-kb fragment observed with the wild type (Fig 2A), we concluded that
the transformants were homoplasmic. For DNA digested with HindIII (Fig 2A) and other restriction enzymes (data not shown), the sizes of the fragments
detected by the tgf probe (which anneals to a sequence adjacent to the point of insertion of the
transgenes [ Fig 1B]) corresponded to those expected for correct
insertion of the transgenes into the target site in the inverted repeat regions. Correct insertion
also was confirmed by PCR (Fig 2B; see
Methods).
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Figure 2.
Analysis of DNA from Homoplasmic Transformants and a Nontransformed
Control.
The mature T0 transformants, tpSSuH4 (lane 1), tpSSuH7 (lane 2), SSuH2 (lane
3), and SSuH5 (lane 4), and a nontransformed control (lane 5) were analyzed.
(A) DNA gel
blot of HindIII-digested leaf DNA probed with the 32P-labeled 787-bp tgf fragment (see
Methods). The sizes of the bands detected were assigned by comparison with an unlabeled DNA ladder.
The point of insertion of the transgenes occurs within a 7.7-kb HindIII fragment of the tobacco
plastome. These restriction sites lie outside of the flanking regions incorporated into the
transforming plasmids shown in Fig 1B. Insertion of either transgene
introduces a new HindIII site that reduces the size of the fragment detected by the tgf probe (Fig 1B) to 3.8 kb.
(B) PCR amplification of the 1613-bp (with
transit presequence) and 1446-bp (without presequence) DNA fragments by using the primers AADAr and
PRV5P (see Fig 1B). A 1-kb DNA ladder (Promega) is shown in lane
6.
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Both Plastid RbcS Transgenes Were Strongly
Transcribed
Because both transgenes contained the 5' untranslated region (UTR) derived
from the plastid psbA gene, the PpsbA probe (Fig 1B) hybridized to the mRNA produced by the endogenous psbA gene and to that produced by the
transgenes (Fig 3A). This provided a means of comparing the amount of
message produced by the transgenes with the abundant psbA message. In young leaves from the
transformants tpSSuH4 and tpSSuH7, the message produced by the RbcS transgene with the
transit sequence was approximately two-thirds as abundant as that of the psbA mRNA. In
similar leaves of the transformants SSuH2 and SSuH5, in which the transit sequence was missing, the
transgene produced approximately one-third as much message as the psbA gene in both cases.
The greater abundance of the message with the transit sequence was reproduced in RNA gel blots probed
with the RbcS probe (Fig 3B). This probe also recognizes the
message produced by the nuclear RbcS gene family, which acts as the internal control in this
case. For both tpSSuH transformants, the chloroplast RbcS message exceeded the nuclear
RbcS message by at least fivefold (Fig 3B). In contrast, the
SSuH2 and SSuH5 transformants had approximately one-half as much chloroplast message as nuclear
message (Fig 3B).
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Figure 3.
Detection
of mRNA Produced by the Transgenes.
(A) RNA (10 µg) from nontransformed tobacco (lane
1) and the T1 transformants tpSSuH4 (lane 2), tpSSuH7 (lane 3), SSuH2 (lane 4), and SSuH5
(lane 5) was hybridized with a 239-bp SacI-NcoI fragment corresponding to the psbA promoter
and 5' UTR (PpsbA [ Fig 1B]).
(B) Duplicate
blot hybridized with a 377-bp NcoI-SalI fragment corresponding to the coding sequence of the mature
small subunit (RbcS [ Fig 1B]).
The positions of the mRNAs
for psbA, nucleus-encoded RbcS (RbcSn), and plastid-encoded His-tagged
RbcS with (tpRbcSH) and without (RbcSH) the transit presequence are
indicated.
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The Plastid RbcS Transgenes
Directed the Synthesis of Small Subunits, and These Were Assembled into Hexadecameric
Rubisco
Fusion of hepta-His to the C terminus of the tobacco small subunit increases its
molecular mass from 14.6 to 15.6 kD. The molecular mass of the His-tagged precursor of the small
subunit is 21.3 kD. Overexpression of these constructs in Escherichia produced His-tagged polypeptides
of these expected sizes in the soluble fractions of the cell extracts (Fig 4A). Although it has been reported that tobacco small subunits and precursors
expressed in Escherichia were located predominantly in inclusion bodies (Klein and Salvucci 1992 ), we observed that substantial amounts of the His-tagged versions of these polypeptides could
be isolated from the soluble fraction by Ni2+-nitrilotriacetic acid (Ni-NTA) agarose
chromatography (Fig 4A).
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Figure 4.
Expression of Plastid-Encoded RbcS Transgenes and Assembly of the
Products into the Rubisco Holoenzyme.
(A) Coomassie blue–stained SDS gel of protein
extracted from young leaves of nontransformed tobacco (NT), the T1 transformants tpSSuH4
and SSuH2, and Escherichia (E. coli) BL21 (DE3) expressing the plasmids pETSSuH and
pETtpSSuH. The insoluble protein (P) was pelleted, and His-tagged protein complexes (E) were isolated
from the supernatant fraction as described in Methods. For the leaf extracts, each lane was loaded
with protein (pelleted or His tagged) that had been derived from 0.5 cm2 of leaf. For the
Escherichia extracts, the His-tagged polypeptides in the soluble fractions constituted 5.4%
(pETtpSSuH) and 2% (pETSSuH) of the total soluble protein as determined by the dye binding method
(Pierce Coomassie Plus).
(B) Immunoblot of an analogous gel, loaded with leaf samples
only, probed with PentaHis antibody that recognized the His tag.
(C) Immunoblot probed
with tobacco Rubisco antibody.
The positions corresponding to the 14.6-kD mature small subunit
(SSu), the 15.6-kD hepta-His–tagged mature small subunit (SSuH), and the 21.3-kD
hepta-His–tagged small subunit precursor with the N-terminal transit peptide (tpSSuH) are
indicated. M, molecular mass marker lane showing soybean trypsin inhibitor (20.1 kD) and
 -lactalbumin (14.4 kD).
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His-tagged small subunits produced by the
tobacco plastid transformants were soluble and assembled into Rubisco hexadecamers (Fig 4). No small subunits were detected in the particulate fraction by either the
PentaHis antibody (Fig 4B) or the anti-Rubisco antibody (Fig 4C). (The traces of immunoreactive material detected in the pellet lanes of all
three samples by the anti-Rubisco antibody [ Fig 4C] did not correspond
to His-tagged or untagged small subunits or precursors and are likely to result from trace
contamination of the tobacco Rubisco preparation used to raise the antibody.) His-tagged Rubisco was
affinity purified and concentrated from the soluble fractions of the leaf extracts by using Ni-NTA
agarose. SDS-PAGE of these preparations revealed three bands with molecular masses of 53, 15.6, and
14.6 kD, corresponding to the Rubisco large subunit, His-tagged small subunit, and untagged small
subunit, respectively (Fig 4A, Fig 4B, and
Fig 4C; large subunit region of the gels not shown). All three
bands were recognized by the anti-Rubisco antibody. Because no trace of a 21.3-kD band was detected by
Coomassie blue or either antibody in the sample derived from the tpSSuH transformant, we conclude that
either the transit peptide was not incorporated into the initial translation product or it had been
cleaved completely and apparently normally. For both types of transformants, the intensities of the
15.6- and 14.6-kD bands when stained with Coomassie blue appeared to be approximately in the ratio
1:7, implying that only a single small subunit in the hexadecamer bore a His tag. Immunoblotting with
the anti-Rubisco antibody (Fig 4C) followed by densitometry also showed
an approximate 1:7 ratio between His-tagged and untagged small subunits. Sedimentation of the affinity
purified preparations through a sucrose gradient, as described by Whitney et al. 1999 ,
confirmed that all of the His-tagged small subunits were assembled in hexadecameric Rubisco complexes.
A single, rapidly sedimenting, UV light–absorbing peak corresponding to Rubisco hexadecamers was
observed. SDS-PAGE followed by immunoblotting with the PentaHis antibody showed no traces of
unassembled His-tagged small subunits in fractions higher in the gradient than this peak, even after
30-fold concentration by absorption onto Ni-NTA agarose (data not
shown).
Plastid-Synthesized Small Subunits Were Not Abundant but Accumulated as the
Leaves Aged
In both types of transformants, the numbers of His-tagged small subunits present
were too low for each Rubisco hexadecamer to contain one His-tagged small subunit. Therefore, most
hexadecamers were not bound by the affinity resin. The proportion of His-tagged small subunits to
total small subunits was measured by purifying the total Rubisco content of leaves of different ages
by conventional procedures (Whitney et al. 1999 ) and measuring its content of His tags by
immunoblotting with the PentaHis antibody. (The antibody to tobacco Rubisco was not well suited to
this purpose because the two small subunit bands it recognized were too closely spaced for accurate
densitometry when the band corresponding to the untagged small subunit was in vast excess.)
Calibration was achieved by separating a range of concentrations of an affinity-purified Rubisco
standard on the same gel and assuming that one-eighth of the small subunits in the standard were His
tagged (Fig 5). In younger leaves, Rubisco in the tpSSuH transformant
had a much higher proportion of its small subunits His tagged than did Rubisco in the SSuH
transformant. However, His-tagged small subunits increased in abundance more rapidly in the latter
transformant than in the former as the leaves aged. Eventually, in older leaves, both transformants
had similar contents of His-tagged small subunits. Even so, only 1.2% of the small subunits were
the products of the transgenes (Fig 5C).
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Figure 5.
Extent of Assembly of Plastid-Encoded Small Subunits into Hexadecameric
Rubisco.
(A) Coomassie blue–stained SDS gel loaded with equal amounts (11.5 pmol)
of total Rubisco (His tagged plus untagged) purified from leaves of the tobacco transformants SSuH2
and tpSSuH4 by a combination of polyethylene glycol precipitation and Suc gradient centrifugation, as
described previously (Whitney et al. 1999 ). Separate purifications were performed for
leaves at different positions on the plants (counting downward from the apical meristem). At the time
of sampling, the SSuH2 plants had 18 leaves and the tpSSuH4 plants had 16 leaves. Total Rubisco
content of these preparations was measured by binding of
14C-2'-carboxyarabinitol-1,5-bisphosphate (Butz and Sharkey 1989 ;
Ruuska et al. 1998 ).
(B) Immunoblot of an analogous gel probed with the
PentaHis antibody. The content of His-tagged small subunits was measured by densitometry of the
immunoblots by comparison with a series of standards containing 0.25 to 1.2 pmol of His-tagged small
subunits separated on the same gel. These standards were derived by separating His-tagged Rubisco
holoenzyme from the purified Rubisco preparation from the tpSSuH transformant by using Ni-NTA agarose,
as described in Methods. The Rubisco content of this standard preparation was measured by binding of
14C-2'-carboxyarabinitol-1,5-bisphosphate. Because each hexadecamer in this
preparation must have at least one His-tagged small subunit and the preparation was derived from a
total Rubisco preparation with, on average, much less than one His-tagged small subunit per
hexadecamer, we assumed that His-tagged hexadecamers contained only one His-tagged small subunit.
Densitometry of immunoblots (anti-Rubisco antibody) of SDS-PAGE gels run with the standard preparation
confirmed the assumed 1:7 stoichiometry between His-tagged and untagged small subunits (see Fig 4C). Densitometry of these standards after immunoblotting with the
PentaHis antibody produced a linear calibration (R2 = 0.99) passing through
the origin.
(C) The content of His-tagged small subunits expressed as a fraction of the
total small subunits calculated from the total Rubisco content.
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Turnover of
Plastid-Synthesized Small Subunits after Assembly into Rubisco
Pulse–chase labeling with
35S was used to investigate how quickly newly synthesized small subunits were turned over.
Autoradiography of SDS-PAGE gels loaded with total protein (insoluble and soluble) from leaf extracts
of a tpSSuH4 plant showed an intensely labeled band near 30 kD whose label decreased rapidly during
the chase period. Although we have no other evidence regarding the identity of this protein, its size
and rapid turnover are consistent with it being the D1 protein of photosystem II, and we have labeled
it as such (Fig 6). Other prominently labeled bands correspond to the
large and untagged small subunits of Rubisco, and the label in these remained constant during the
chase period, consistent with little or no turnover. In the total protein gel, no discrete band of
label corresponded to the His-tagged small subunit. However, isolation and concentration of His-tagged
Rubisco from the soluble fraction of the labeled extracts by using Ni-NTA agarose, followed by
SDS-PAGE and autoradiography, allowed ready identification of the His-tagged small subunit band,
together with more intense bands for the large and untagged small subunits (Fig 6A). Loss of label from all subunits derived from these His-tagged hexadecamers
was noticeably faster than from total Rubisco but much slower than from the D1 protein. Within this
fraction, the His-tagged and untagged small subunits turned over at the same rate and more rapidly
than the large subunits (Fig 6B).
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Figure 6.
Turnover of Rubisco Subunits and
the D1 Subunit of Photosystem II Measured by 35S Pulse–Chase
Labeling.
(A) Autoradiographs of SDS gels loaded with total leaf protein and isolated
His-tagged Rubisco (see Methods), equivalent to 1.8 and 14.5 cm2 of leaf, respectively.
Bands corresponding to the D1 protein and the Rubisco large (LSu), small (SSu), and His-tagged small
(SSuH) subunits are indicated.
(B) Densitometric measurement of 35S labeling
in the various subunits during the chase period. LSu (closed circles), SSu (closed squares), and D1
(closed triangles) band densities were measured in the total protein lanes. Because the SSuH bands in
these lanes were too faint for accurate measurement, they were measured in the His-tagged Rubisco
lanes, in which a lower background allowed a 10-fold greater amplification of the intensities of the
labeled bands. The LSu (open circles) and SSu (open squares) bands also were measured in these
lanes.
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The N Termini of
Small Subunits Synthesized in the Plastid Were Blocked
The SDS-PAGE bands corresponding to
His-tagged and untagged small subunits in affinity-purified Rubisco preparations from the
transformants were subjected to N-terminal Edman microsequencing (see Methods). The untagged small
subunit bands from both tpSSuH4 and SSuH2 plants yielded the sequence X-Q-V-W-P-P-I/Y predicted by the
nucleotide sequence of the tobacco RbcS gene family (Muller et al. 1983 ). The
residue released in the first cycle, X, was not the predicted Met, however, but gave rise to a peak
that chromatographed between the phenylthiohydantoin derivatives of Phe and Glu. This is consistent
with it being N-methyl-Met, as has been reported for the small subunits of several higher
plants (Grimm et al. 1997 ), including tobacco (Ying et al. 1999 ). A smaller
additional peak that was released during the first cycle and eluted after the Glu derivative may be
the sulfoxide form of N-methyl-Met.
The His-tagged small subunit bands from the same
gels were subjected to similar N-terminal sequencing. No sequence was returned for either
transformant, except for a trace level of the same sequence returned for the untagged small subunit
that may be attributed to cross-contamination arising from the adjacent untagged band on the gel. The
result was the same when the samples were treated with formic acid to cleave a formyl group, if
present, from an N-terminal Met residue (see Methods). Presumably, the N termini of the products of
both plastid transgenes were blocked with a group(s) other than formyl.
Attempts to obtain
information about the nature of the N-terminal blockage of the tpSSuH small subunits by using
electrospray ionization mass spectrometry (ESI-MS; see Methods) were unsuccessful. Wild-type
small subunits yielded two prominent molecular ions with similar abundances corresponding to subunit
masses of 14,530 and 14,573 D. The latter corresponds to an N-terminally methylated polypeptide with
the mature sequence shown in Fig 1A (lacking the hepta-His peptide and
the inserted Ala-2). The former presumably corresponds to an unresolved mixture of small subunits with
closely similar masses encoded by other members of the RbcS gene family (Muller et al. 1983 ). The small subunit fraction from tpSSuH plants yielded spectra identical to the wild type.
Larger ions consistent with the presence of the His tag could not be detected, presumably because of
their low abundance. Prior concentration of the His-tagged Rubisco from the transformants by Ni-NTA
agarose chromatography before isolation of the small subunits resulted in unreliable mass spectra,
despite extensive dialysis. Poor ESI-MS results with Ni-NTA– purified proteins have been
reported previously (Neylon et al. 2000 ).
Plastid-Synthesized Small
Subunits Lacking the C-Terminal Hepta-His Sequence Also Were Not Abundant
Preliminary
transformations with the pC3 plasmid (which encodes a mature small subunit with an Ala insertion at
residue 2 but without a His tag [see Methods]) yielded homoplasmic transformants that resembled the
SSuH transformants in having approximately one-third as much transgene message as psbA
message (data not shown). Sequencing of the N terminus of the small subunit band from SDS-PAGE gels of
Rubisco isolated from these transformants yielded only the wild-type sequence with no trace of Ala in
the second position. However, blockage of the N terminus of the plastid-synthesized small subunit (see
above) would prevent detection of the transgene's product in this way. ESI-MS of the small
subunit fraction also detected only the same prominent pair of molecular ions as observed with the
wild-type small subunits (see above) and no larger or smaller species (<2%) consistent with the
Ala-2 insertion or the removal of residues from the N terminus. We conclude that, even in the absence
of the His-tag, plastid-synthesized small subunits constitute <2% of the total small
subunits.
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DISCUSSION |
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Relocation of Genes for Rubisco Subunits
between Nuclear and Plastid Genomes
The experiments we report here complement those in which
the rbcL gene was deleted from the tobacco plastome, equipped with a transit presequence, and
inserted into the nuclear genome (Kanevski and Maliga 1994 ). Relocation of the
RbcS gene from the nucleus to the plastid is somewhat more complicated because the
RbcS gene family cannot be deleted from the nucleus. However, use of a C-terminal hepta-His
sequence allowed us to distinguish between the products of the plastid RbcS transgenes and
the nuclear RbcS genes. The results of the two types of relocation experiments were similar.
The transferred gene functioned in its new location, and its products were assembled correctly into
Rubisco in the chloroplast. This finding suggests that both of the Rubisco subunits are physically
capable of being synthesized in both cytoplasmic and plastidic compartments. However, in both cases,
the products of the transgenes were not very abundant.
The two relocation experiments differ in
an important detail: synthesis of the large subunit (nearly 80% of the Rubisco protein by weight)
makes much larger demands on the capacity for protein synthesis, folding, transport, and assembly than
does synthesis of the small subunit. Because Rubisco is by far the most abundant leaf protein,
relocation of the synthesis of its large subunit to the cytoplasm places an extraordinary demand on
the protein-synthesizing and protein-folding machineries of this compartment and on the mechanisms by
which the precursor protein is transported into the chloroplast. These processes are not naturally
adapted to a flux of this magnitude; therefore, it is not surprising that the nuclear
rbcL transgene directed the synthesis of only 3 to 10% of the normal Rubisco complement of
the wild-type chloroplast (Kanevski and Maliga 1994 ). Relocation of RbcS to the
plastid genome is not likely to involve capacity problems of this kind. No transport of the product is
required, and the protein synthesis capacity needed to produce small subunits in amounts
stoichiometric with large subunits would be little more than 20% of that required for the large
subunits themselves. Therefore, the reasons for the scarcity of the products of the plastid
RbcS transgenes in assembled Rubisco (never more than 1.3% of the total small subunit
content; Fig 5) must be sought
elsewhere.
Transcript Abundance
It seems unlikely that the scarcity of the
mRNA transcripts of the plastid RbcS transgenes can be the cause of the low expression level
of the plastid-synthesized small subunits. The abundance of the transcript with the transit
presequence rivaled that of the psbA message (one of the most abundant mRNAs in the
chloroplast [ Gruissem et al. 1988 ]) and was manyfold greater than that of the nuclear
RbcS transcript (Fig 3). In young leaves, the chloroplast
RbcS message lacking the transit presequence was two- to 10-fold less abundant than its
homolog with the presequence (Fig 3). This could be caused by either
slower transcription of the shorter transgene or greater instability of its transcript. Nevertheless,
this transcript was still quite abundant. Therefore, the limitation(s) that impaired accumulation of
the plastid-encoded small subunits is not likely to be related directly to the transcription rate of
the transgenes or the stability of their mRNA products. Translational or post-translational
limitations seem to be indicated.
Translational Efficiency
Inefficient
translation of the transcripts of the plastid transgenes would be a simple explanation for the
scarcity of plastid-encoded small subunits. A restriction at the level of translation (indicated by
dashed arrow 1 in Fig 7) could be caused by an inappropriate structure
of the mRNAs or unfavorable codon use and would accord with suggestions that translational control is
a dominant means of regulating the expression of plastid genes (Staub and Maliga 1994 ; Mayfield et al. 1995 ; Rodermel 1999 ).
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Figure 7.
Scheme
of the Synthesis and Assembly of Rubisco in Tobacco Transformants with a Plastid Transgene Expressing
the Small Subunit Precursor (pre-SSu).
The dashed arrows indicate steps that may be rate
limiting for the production of chloroplast-synthesized small subunits. Arrow 1 indicates the rates of
engagement of the RbcS mRNA with the chloroplast ribosomes and its subsequent translation.
The arrows labeled 2 indicate possible access routes of the newly synthesized small subunits to the
Rubisco assembly pathway. See text for details. Chaperone complexes are indicated by the paired
bracket-shaped symbols. BSD2, product of the BUNDLE SHEATH DEFECTIVE 2 gene (Brutnell et al. 1999 ); Cpn60 and Cpn21, chloroplast chaperonins 60 and 21; DnaJ, Hsp70, GrpE, and ClpC,
chaperone proteins thought to be involved in import and assembly of Rubisco (Brutnell et al. 1999 ; Keegstra and Cline 1999 ); LSu and SSu, large and small subunits of
Rubisco; N-methylase, enzyme responsible for methylating the -amino group at
the N terminus of the mature small subunit after cleavage of the transit peptide (Grimm et al. 1997 ; Ying et al. 1999 ); SPP, stromal processing peptidase; TIC and
TOC, translocons of the inner and outer chloroplast membranes (Keegstra and Cline 1999 ); tp, transit peptide.
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We chose to use
promoter and 5' and 3' UTR elements derived from the plastid psbA gene for our
constructs because of previous success in obtaining abundant expression in chloroplasts of a foreign
uidA gene flanked by these elements (Staub and Maliga 1993 , Staub and Maliga 1994 ). However, the factors that control the translation rate of plastid genes are not fully
understood. There is evidence that the 5' UTR, in particular, has sequences that bind protein
factors that regulate the initiation of translation (Mayfield et al. 1995 ; Somanchi and Mayfield 1999 ). It is possible that mismatch between the 5' UTR and the
protein-coding sequence might lead to an mRNA structure that occludes such a binding site.
Additionally, an inappropriate secondary structure of the fused mRNA species might directly impair
engagement with the ribosome independent of the involvement of trans-acting factors.
Similarities between the translational efficiencies in Escherichia and Chlamydomonas chloroplasts of
transgenes fused to randomly mutated 5' UTR elements can be interpreted in this way because
these two organisms are not likely to have identical trans-acting factors (Fargo et al. 1999 ). Therefore, the efficiency of translational initiation might vary unpredictably according
to the foreign sequence fused to the psbA 5' UTR. We will test these possibilities in
future studies using 5' UTR elements derived from other plastid genes.
In young expanding
leaves, the plastid RbcS transgene with the transit presequence directed the synthesis of
five times as much small subunit as did the transgene without the presequence, but the difference
diminished as the leaves aged (Fig 5). If both mRNA species were
translated with similar inefficiency, this difference might be explained by the greater abundance of
the mRNA with the transit presequence in the young leaves (Fig 3).
The rate of translation of the plastid RbcS genes also might
be impaired by nonoptimal codon use. However, the codon bias of RbcS appears to differ from
the consensus codon use of plastid genes in tobacco (Nakamura et al. 1999 ) and
dicotyledons generally (Ausubel 1988 ) no more significantly than does the codon use of
uidA, a gene that is expressed abundantly in tobacco chloroplasts (Staub and Maliga 1993 , Staub and Maliga 1994 ). Moreover, Morton and Levin 1997 suggested that
the sensitivity of protein synthesis in plastids of higher plants to codon bias has diminished during
evolution, perhaps as a result of the increasing copy number of the plastid genome and, consequently,
increasing levels of mRNAs. These issues could be addressed experimentally by resynthesis of the
RbcS genes to match the codon bias of abundantly expressed plastid genes such as
rbcL and psbA.
Processing of the Small Subunit Precursor
No
trace of uncleaved small subunit precursor could be detected in tobacco transformants with the
transgene incorporating the transit presequence (Fig 4). There could be
two explanations for this.
First, it is possible that the presequence might not have
been translated, initiation occurring exclusively at the ATG codon corresponding to the Met residue at
the N terminus of the mature polypeptide. (There is a GGA sequence 13 to 15 bp upstream that might
have served as a ribosome binding site [ Fig 1A].) Although our present
evidence does not exclude this possibility, it seems unlikely. In Escherichia transformed with a
construct that had the same transit presequence and, therefore, the same possibility for translation
of the message to commence at the first Met residue of the mature subunit, there was no sign of such a
product, initiation at the ATG codon at the 5' end of the transit presequence being preferred
(Fig 4). Because chloroplasts and Escherichia have similar translational
machineries, translation might be expected to initiate similarly. Our use of promoter and 5' UTR
sequences identical to those of the psbA gene strengthens the expectation that translation
would initiate at the same ATG codon as it does in psbA, that is, at 5' end of the
transit presequence.
Second, if the transit presequence was translated as expected,
cleavage of the transit peptide from the small subunit precursor so produced by the stromal processing
peptidase must have been complete (Fig 7). This would not be surprising
because the translation product would be identical (except for the C-terminal hepta-His tag) to the
cytoplasmically synthesized small subunit precursor imported into the chloroplast. However, this
scenario does not readily explain why the N terminus of the mature small subunit produced by the
nuclear RbcS is methylated whereas its plastid-encoded analog is blocked in a manner not
determined (see Results). Either translational initiation is different with the plastid RbcS
as discussed above or the plastid-synthesized precursor is denied access to the methylase responsible
for modification of the N terminus of the mature small subunit exposed after cleavage of the precursor
(Ying et al. 1999 ). Perhaps the methylase requires a particular context for efficient
recognition of the cleaved precursor. This might be provided by docking of the methylase with the
translocons of the inner chloroplast membranes complex or its associated chaperones (Fig 7).
The N termini of the mature small subunits produced by both transgenes
were blocked to Edman sequencing (see Results). If translation of the message produced by the transit
sequence–containing gene initiated at the Met residue corresponding to the mature N terminus (as
discussed above), then a similar modification mechanism might have applied to the products of both
transgenes. If the N-terminal Met or formyl-Met residue was removed post-translationally, as is
sometimes the case with chloroplast gene products (Michel et al. 1988 ; Houtz et al. 1989 ), the Ala or Gln residue thus exposed at the N terminus might have conflicted
with the specificity of the N-methylase, perhaps triggering another type of N-terminal
modification mechanism. For example, acetylation of the N terminus is observed with the Rubisco large
subunit (Houtz et al. 1989 ), the D1 and D2 proteins of photosystem II, and the chlorophyll
a binding protein, Cpa-2 (Michel et al. 1988 ; Sharma et al. 1997 ). Clearly, the mechanisms by which the N termini of plastid-synthesized proteins are modified,
and the generality of such modifications, need further investigation.
Assembly of
Small Subunits into Rubisco
Rubisco molecules with at least one (and probably only one)
plastid-synthesized small subunit (i.e., L8S7SH) were somewhat less
stable than L8S8 molecules. The former lost 35S label slowly during
the chase period whereas the latter retained it completely (Fig 6). Perhaps the presence of even one His tag is sufficient to destabilize the whole hexadecamer slightly.
However, the turnover rate of L8S7SH was slow compared with that of
the D1 protein. Although enhanced turnover might contribute to the scarcity of plastid-synthesized
small subunits, it cannot by itself completely explain it.
It is possible, however, that the
plastid-synthesized small subunits or their precursors might be subject to rapid turnover before
assembly into Rubisco. Although unassembled His-tagged small subunits would be isolated by the
Ni2+ chelation procedure, such rapid degradation might keep their steady state pool sizes
so small that they would escape detection in pulse-labeling experiments (Fig 6). Rapid degradation of unassembled small subunits or precursors is consistent
with their absence from the particulate fraction (Fig 4). Such
degradation might be specific to small subunits synthesized in the chloroplast if these subunits were
distinguishable in some way from the cytoplasmically derived precursors and subunits.
It is
conceivable that the C-terminal hepta-His sequence might slow translation or impair the folding of the
nascent small subunits or precursors sufficiently to bring them to the attention of degradative
protease(s) thought to be responsible for the disposal of unassembled small subunits that accumulate
in excess of those required to assemble with large subunits (Rodermel 1999 ). However, even
in the C3 transformants in which the RbcS transgene lacked the hepta-His–encoding
sequence, the plastid-synthesized small subunits still constituted <2% of the total small subunits
(see Results). This finding argues against the hepta-His sequence being the sole reason for the low
levels of plastid-synthesized small subunits.
Plastid-synthesized small subunits or precursors
might be distinguishable from their cytoplasmic analogs if the two types of subunits were kept
separate during the assembly of Rubisco. It is possible that, in the normal assembly pathway,
unassembled small subunit precursors and small subunits are always sequestered by chaperones unless
the small subunit supply exceeds the supply of large subunits (in which case the unsequestered excess
would be rapidly degraded). Ivey et al. 2000 identified domains in the transit peptide of
the small subunit with affinity for the Hsp70 chaperone of chloroplasts, consistent with suggestions
that this chaperone may function as an ATP-dependent translocase that draws the precursor across the
envelope membranes. Once inside the chloroplast, the precursor might be cleaved of its transit
peptide, methylated at its newly exposed N terminus, and handed on to other chaperones in the Rubisco
assembly sequence without ever existing in significant amounts in an unassociated state in the stroma
(Fig 7). Plastid-synthesized small subunit precursors or small subunits
might have difficulty joining this "inside track" to assembly, particularly if they had
partially misfolded after leaving the plastid ribosomes (dashed arrows labeled 2 in Fig 7). Therefore, they would mostly fall victim to the degradative protease(s). If
this is the case, increasing the rate of translational production of small subunits in the chloroplast
may not greatly promote the assembly of these subunits into Rubisco.
Sequestration of
unassembled small subunits in chaperone complexes also might provide an explanation for the lack of
N-terminal methylation of the plastid-synthesized small subunits. Only unassembled small subunits are
capable of acting as the substrate for the methylase (Grimm et al. 1997 ; Ying et al. 1999 ). If, as suggested above, the most effective substrate for methylation is the
small subunit/chaperone/TIC complex (Fig 7), unsequestered small
subunits might be methylated inefficiently, leaving them vulnerable to a different kind of N-terminal
processing that leads to blockage of Edman sequencing.
Conclusions
Rubisco
small subunits can be synthesized within the chloroplast as well as in the cytoplasm.
Chloroplast-synthesized small subunits assemble successfully into L8S8 Rubisco
complexes. However, the N termini of the plastid-encoded small subunits are modified incorrectly, and
the combined synthesis/assembly process is inefficient. Slow translation of the abundant chloroplast
RbcS message could be one reason for the inefficiency. Another might be the limited access of
chloroplast-synthesized small subunits or precursors to a preferred assembly pathway enjoyed by their
cytoplasmically derived analogs. Such a pathway might connect import of the precursor, cleavage,
post-translational modification, and assembly via a sequence of chaperone complexes without the
release of significant amounts of the precursors or small subunits at any stage (Fig 7).
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METHODS |
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Construction of Transformation
Plasmids
A tobacco (Nicotiana tabacum) RbcS gene was amplified from pTSSU2
(Hudson et al. 1992 ) by using the primers SSu5C (5'-
GGGTCTAGAGTCGACGAGGTTAACGCCATGGCACAGGTGTGG-CCACC-3', which introduces an NcoI site
[underlined] and has an Ala codon [italic] inserted between codons 1 and 2 of the mature small
subunit) and SSu3B (5'-GGGTCTAGAGTCGACTTAGTAGCCTTCTGGCTT-3', which introduces
XbaI [underlined] and SalI [italic] restriction sites). The uidA gene in pJS25 (Staub and Maliga 1993 ) was replaced with the 383-bp NcoI-XbaI RbcS fragment of the
amplified RbcS gene to produce pJSTSSu. An 846-bp SacI-DraI cassette from pJSTSSu, comprising
the psbA promoter–RbcS-psbA terminator sequence, was ligated into
SacI/SmaI-digested pBluescript II KS+ (Stratagene) to give pBlueSSu. The SacI-HindIII cassette from
pBlueSSu was inserted into the multiple cloning site of pRV112a (Zoubenko et al. 1994 ) to
give the preliminary plastid-transforming plasmid pC3. This plasmid was used for preliminary
transformations only. For most of the present work, variant RbcS genes with 3'
hepta-His–encoding sequences were substituted into it.
To construct the hepta-His variants
of RbcS, RNA was purified from leaves of tobacco cultivar Petit Havana (N,N) by
using the SV Total RNA isolation system (Promega, Madison, WI). Using avian myeloblastosis virus
reverse transcriptase (Promega), we reverse transcribed 0.4 µg of RNA with primer TPRIME
(5'-GGGAGCTCGCTA-GCGGATCCTTTTTTTTTTTTTTTTT-3'). A full-length RbcS gene
(including the transit peptide–coding sequence) was amplified using primers SSuTP5
(5'-TAACCATGGCTTCCTCAGTTCT-3' [NcoI site underlined]) and SSuHis3
(5'-TAGTCGACTTAgtggtggtggtggtggtggtgA-TATCCTTCTGGCTTGTAGGCAATA-3',
introducing SalI [underlined] and EcoRV [italic] sites and with a hepta-His–encoding sequence
[lowercase] immediately 3' to the complement of the RbcS stop codon [boldface]) and
cloned into pGEM-T Easy (Promega) to give ptpSSuHT. An RbcS gene encoding only the mature
small subunit with a C-terminal hepta-His extension and an additional Ala at codon 2 was amplified
from ptpSSuHT by using primers SSu5C and SsuHis3 (Fig 1). The amplified
product and ptpSSuHT were digested with NcoI and SalI, and the corresponding 398- and 569-bp fragments
were ligated into NcoI-SalI–digested pC3 to give the transforming plasmids pSSuH and ptpSSuH,
respectively (Fig 1). The plasmids were sequenced by BigDye terminator
cycle sequencing (Applied Biosystems, Foster City, CA) using the primers PRV5P
(5'-CTTCGGAATCGGTAGTCA-3') and AADAr (5'-GAATGTCATTGC-GCTGCCATTCTCCA-3';
Fig 1B). The sequence obtained from ptpSSuH (Fig 1A) is identical (except for the hepta-His–encoding sequence) to that
inferred from a previously reported RbcS genomic sequence from tobacco (Mazur and Chui 1985 ). The sequence from pSSuH had the expected deletion of the transit sequence and insertion
of an Ala codon at position 2 and, in addition, one polymerase chain reaction (PCR)–induced
silent substitution within the coding sequence (Fig 1A). The amino acid
sequence of the small subunit encoded by pC3 is identical to that shown for pSSuH except for the
omission of the C-terminal hepta-His peptide and a substitution of Val for Ile at residue 8 (not
shown).
Analogous plasmids for transforming Escherichia coli BL21 (DE3), pETtpSSuH and
pETSSuH, were constructed by inserting the NcoI-SalI fragments from pSSuH and ptpSSuH, respectively,
into pET28a(+) (Novagen, Madison, WI).
Plastid Transformation and Plant
Growth
Tobacco (cultivar Petit Havana [N,N]) leaves were transformed using the
biolistic method (Svab and Maliga 1993 ). Tissue from spectinomycin-resistant plantlets was
put through two additional rounds of regeneration on regeneration medium (Carrer et al. 1993 ) containing 500 µg/mL spectinomycin before transformed plantlets were identified by PCR
analysis of leaflet DNA isolated according to Saghai-Maroof et al. 1984 using two
different pairs of primers: SSuHis3 with TRNV (5'-CAGGCTCGAACTGATGACTTCCACC-3') and AADr
with PRV5P (Fig 1B). Two independently transformed lines of pSSuH (SSuH2
and SSuH5) and ptpSSuH (tpSSuH4 and tpSSuH7) transformants were maintained through two additional
rounds of regeneration. Homoplasmicity then was confirmed by DNA gel blot analysis (Sambrook et al. 1989 ) of HindIII-digested leaf DNA probed with a 32P-labeled (Amersham Megaprime
labeling kit) 787-bp HindIII-BamHI fragment of pRV112a derived from the flanking region adjacent to
the insertion site (tgf; Fig 1B). Correct insertion into the
plastid genome was confirmed by PCR using the primers AADAr (Fig 1B) and
PRV5' (5'-CCGAAGAGTAACTAGGACCAAT-3'). The latter primer anneals to a sequence in the
tobacco plastome just beyond the right flanking region (Fig 1B) of the
transforming plasmids (nucleotides 101,000 to 101,021 of inverted repeat A and 141,605 to 141,626 of
inverted repeat B in the update of the sequence of Shinozaki et al. 1986 provided in
GenBank accession number
Z00044). Another transformed line (C3) was obtained using
the pC3 plasmid, and its homoplasmicity was confirmed after several rounds of regeneration by the same
procedures.
Regenerated transformed plantlets (and nontransformed controls) were transferred to
5-liter pots of soil and grown to maturity in an air-conditioned glasshouse. Pots were watered daily,
and a complete nutrient solution was applied three times per week.
RNA Gel
Blotting
Total RNA was extracted from young expanding leaves with the TriPure isolation reagent
(Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions.
RNA pellets were dissolved in water and immediately electrophoresed, blotted, and cross-linked to
Hybond N+ membranes (Amersham) as described by Kucharski and Maleska 1998 . Duplicate blots
were hybridized with two 32P-labeled DNA fragments derived from pBlueSSu (PpsbA
and RbcS; Fig 1B). Membranes were exposed to a storage
phosphor screen, and computer-generated images were analyzed using ImageQuant software (Molecular
Dynamics [Sunnyvale, CA] PhosphorImager 400S).
Extraction of Rubisco from Tobacco
Leaves
Tissue from young mature leaves (5 cm2) was frozen in liquid N2
and then ground in ice-cold glass homogenizers (Wheaton, Millville, NJ) in 1 mL of extraction buffer
(0.1 M Tris-HCl, pH 8.0, 0.3 M NaCl, 20 mM imidazole, 5 mM MgCl2, and 1 mM
phenylmethylsulfonyl fluoride). The extracts were centrifuged at 13,000g for 2 min at
4°C, and the pellet fraction was washed with three 1-mL volumes of extraction buffer before
resuspension in SDS-containing sample buffer (NuPAGE; Novex, San Diego, CA) to a final volume of
0.2 mL and applied to NuPAGE electrophoresis gels (see below). His-tagged ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco) complexes were isolated from the supernatant fraction as described
below.
Extraction of Rubisco Small Subunits and Precursors from Escherichia
Cells
Escherichia BL21 (DE3) cells transformed with plasmids pETtpSSuH and pETSSuH were grown
at 28°C on a rotary shaker (150 rpm) in 0.3 liters of Luria-Bertani medium containing 30 µg/mL
kanamycin. When cell absorbance (A600) reached 0.6,
isopropyl-ß-D-thiogalactopyranoside was added to 0.5 mM. After 150 min, the cells
(A600 1.3 to 1.4) were harvested by centrifugation at 3300g for 10
min at 4°C and resuspended in 10 mL of ice-cold extraction buffer. The cells were lysed by two
passes through a prechilled French pressure cell at 140 MPa. The extract was centrifuged at
33,000g for 10 min at 4°C. His-tagged small subunits or precursors were isolated from the
supernatant fraction as described below.
Affinity Purification of His-Tagged Rubisco
Hexadecamers or Small Subunits
Total Rubisco (untagged plus His tagged, purified from leaves
according to Whitney et al. 1999 ) or the soluble protein fraction from extracts of leaves
or Escherichia cells was mixed with Ni2+-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen,
Valencia, CA) by rotation at 4°C for 2 hr. The resin was collected into a Wizard minicolumn
(Promega) and washed with 40 to 100 bed volumes of extraction buffer. Bound protein was collected by
successive applications of 0.5 bed volumes of elution buffer (0.1 M Tris-HCl, pH 8.0, 0.3
M NaCl, and 200 mM imidazole). These washes were collected by centrifugation for
10 sec at 13,000g.
Electrophoresis and Immunoblotting
Procedures
Proteins were separated by SDS-PAGE using NuPAGE 4 to 12% Bis-Tris gels (Novex)
buffered with Mes according to the supplier's instructions. Protein bands were visualized by
staining with Coomassie Brilliant Blue G 250 by using the Gelcode Blue reagent (Pierce Chemical Co.).
Separated proteins were transferred to nitrocellulose (Hybond C; Amersham) by using an Xcell
transfer cell (Novex). After blocking for 45 min with 3% (w/v) BSA (Sigma) in 10 mM Tris-HCl, pH 7.5,
150 mM NaCl (TBS), membranes were probed for 1 hr with antiserum raised in rabbits against Rubisco
purified from tobacco according to Servaites 1985 (diluted 1:20,000 in TBS) or the
PentaHis antibody preparation (Qiagen; diluted 1:1000 in TBS containing 3% [w/v] BSA). Membranes
were then probed with appropriate secondary antibodies conjugated to alkaline phosphatase
(Bio-Rad; diluted 1:2000 with TBS). Immunoreactive polypeptides were visualized by fluorescence
with AttoPhos (JBL Scientific, San Luis Obispo, CA) by using a FluorImager (Molecular Dynamics). Band
densities were measured with ImageQuant software.
35S Pulse–Chase
Labeling
Ten leaf discs (0.97 cm2) from young fully expanded leaves of transformant
tpSSuH4 growing in natural illumination (maximum irradiance = 1300 µmol
m-2 sec-1) were sampled, layered immediately on 5
mL of Murashige and Skoog 1962 nutrient solution, pH 7.2, containing 10 mM
NaHCO3, 0.25% (v/v) Silwet-L77 (Lehle Seeds, Round Rock, TX), and 400 µCi of
Trans35S-label (ICN, Costa Mesa, CA), and vacuum infiltrated for 90 sec. The leaf discs
then were illuminated (600 µmol m-2 sec-1) for 20 min at
25°C, blotted briefly with blotting paper, rinsed by vacuum infiltration with 25 mL of water,
blotted again, and vacuum infiltrated with Murashige and Skoog 1962 solution, pH 7.2,
containing 10 mM NaHCO3 and 10 mM Met. After further illumination for various chase
periods, duplicate leaf discs were frozen in liquid N2 and extracted with 0.4 mL of leaf
extraction buffer. For analysis of total protein, 0.4 mL of Tris-glycine-SDS sample buffer (Novex) was
added to one of the extracts. His-tagged Rubisco hexadecamers were isolated from the other extract by
affinity purification as described above and eluted in a final volume of 50 µL, to which 50 µL
of Tris-glycine-SDS buffer was added. Proteins in both samples were separated by SDS-PAGE, and the
resulting gels were dried and exposed to a storage phosphor screen for 14
days.
N-Terminal Protein Sequencing
N-terminal amino acid sequencing was
performed on the small subunit bands obtained by SDS-PAGE from Rubisco preparations that had been
purified by Ni-NTA agarose affinity chromatography from transformants tpSSuH4 and SSuH2. The separate
bands corresponding to the two types of small subunits (His tagged and untagged) were excised from the
gels, washed extensively with water, and freeze dried. The gel fragments were allowed to reswell in 10
mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 11.0, containing 0.2% SDS before 10 volumes of 0.1 M
3-(cyclohexylamino)-1-propanesulfonic acid was added. The polypeptides were allowed to adsorb
passively overnight onto pieces of polyvinylidene difluoride membrane. Half of the membrane pieces
were treated overnight with 70% formic acid to cleave any N-terminal formyl groups that might be
present. Polypeptides on all membrane pieces were sequenced using an Applied Biosystems Procise-cLC
sequencer equipped with a 0.8 x 250-mm phenylthiohydantoin column.
Mass
Spectrometric Analyses
Total Rubisco, purified from the wild type and tpSSuH and C3
transformants as described by Whitney et al. 1999 , was denatured by dilution with 4
volumes of 0.1 M Tris-HCl buffer, pH 7.2, containing 6 M guanidine-HCl. The large and small subunits
were separated by gel filtration on a Beckman (San Ramon, CA) Ultraspherogel SEC 4000 column (7.5
x 300 mm) equilibrated with the same buffer. Samples from the small subunit peaks were prepared
for electrospray ionization mass spectrometry (ESI-MS) as described by Neylon et al. 2000 .
Mass spectra were obtained with a VG Biotech (Altrincham, Cheshire, UK) Quattro II mass
spectrometer.
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ACKNOWLEDGMENTS |
|---|
We
thank G. Hudson for contributing to the construction of plasmid pC3, P. Maliga for plasmids and advice
about the biolistic technique, and H. Kane for reading the manuscript.
Received July 21, 2000; accepted November 10, 2000.
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ABSTRACT
INTRODUCTION