PSRP-3 is the higher plant orthologue of a hypothetical protein ycf65 gene product, first reported in the chloroplast genome of a red alga.. Fax: + 1 520 325 7957, Tel.: + 1 520 325 7957,
Trang 1Proteomic identification of all plastid-specific ribosomal proteins
in higher plant chloroplast 30S ribosomal subunit
PSRP-2 (U1A-type domains), PSRP-3a/b (ycf65 homologue)
and PSRP-4 (Thx homologue)
Kenichi Yamaguchi* and Alap R Subramanian
Max-Planck-Institut fuer molekulare Genetik, Berlin-Dahlem, Germany and Department of Biochemistry, University of Arizona, Tucson, USA
Six ribosomal proteins are specific to higher plant
chloro-plast ribosomes [Subramanian, A.R (1993) Trends Biochem
Sci 18, 177–180] Three of them have been fully
character-ized [Yamaguchi, K., von Knoblauch, K & Subramanian,
A R (2000) J Biol Chem 275, 28455–28465; Yamaguchi,
K & Subramanian, A R (2000) J Biol Chem 275, 28466–
28482] The remaining three plastid-specific ribosomal
pro-teins (PSRPs), all on the small subunit, have now been
characterized (2D PAGE, HPLC, N-terminal/internal
pep-tide sequencing, electrospray ionization MS, cloning/
sequencing of precursor cDNAs) PSRP-3 exists in two
forms (a/b, N-terminus free and blocked by
post-transla-tional modification), whereas PSRP-2 and PSRP-4 appear,
from MS data, to be unmodified PSRP-2 contains two
RNA-binding domains which occur in mRNA processing/
stabilizing proteins (e.g U1A snRNP, poly(A)-binding
proteins), suggesting a possible role for it in the recruiting of
stored chloroplast mRNAs for active protein synthesis
PSRP-3 is the higher plant orthologue of a hypothetical
protein (ycf65 gene product), first reported in the chloroplast
genome of a red alga The ycf65 gene is absent from the
chloroplast genomes of higher plants Therefore, we suggest
that Psrp-3/ycf65, encoding an evolutionarily conserved
chloroplast ribosomal protein, represents an example of organelle-to-nucleus gene transfer in chloroplast evolution PSRP-4 shows strong homology with Thx, a small basic ribosomal protein of Thermus thermophilus 30S subunit (with a specific structural role in the subunit crystallographic structure), but its orthologues are absent from Escherichia coli and the photosynthetic bacterium Synechocystis We would therefore suggest that PSRP-4 is an example of gene capture (via horizontal gene transfer) during chloro-ribo-some emergence Orthologues of all six PSRPs are identifi-able in the complete genome sequence of Arabidopsis thaliana and in the higher plant expressed sequence tag database All six PSRPs are nucleus-encoded The cytosolic precursors of PSRP-2, PSRP-3, and PSRP-4 have average targeting peptides (62, 58, and 54 residues long), and the mature proteins are of 196, 121, and 47 residues length (molar masses, 21.7, 13.8 and 5.2 kDa), respectively Func-tions of the PSRPs as active participants in translational regulation, the key feature of chloroplast protein synthesis, are discussed and a model is proposed
Keywords: chloroplast-specific ribosomal protein; proteo-mics
We have recently completed a comprehensive proteome
analysis and protein identification of the chloroplast
ribosome (chloro-ribosome) of a higher plant [1,2] The
results showed that the chloro-ribosomal 30S subunit
contains four chloroplast/plastid-specific ribosomal
pro-teins (PSRPs) in addition to the orthologues of the full
complement of Escherichia coli 30S subunit ribosomal
proteins [1] The specific proteins were designated plastid-specific ribosomal proteins (gene designation, Psrp), PSRP-1 to PSRP-4 The chloro-ribosomal 50S subunit comprised the orthologues of 31 E coli 50S subunit ribosomal proteins (only two E coli ribosomal proteins were unrepresented, L25 and L30), and two additional PSRPs, namely, PSRP-5 and PSRP-6 [2] The intact
Correspondence to A R Subramanian, 5110 East Woodgate Ln., Tucson, AZ 85712, USA Fax: + 1 520 325 7957, Tel.: + 1 520 325 7957, E-mail: alapsubraman@aol.com
Abbreviations: PSRP, chloroplast/plastid-specific ribosomal protein; pRRF, plastid ribosome recycling factor; RBD, RNA-binding domain in RNA-binding protein ( 80 amino-acid residues long); RNP1 and RNP2, conserved hexapeptide and octapeptide sequences in RBD; cpRNP, chloroplast RNA-binding protein; EST, expressed sequence tag; ycf, hypothetical chloroplast frame.
*Present address: Department of Cell Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA, E-mail: yamaken@scripps.edu
Note: The spinach peptide sequences reported in this paper have been deposited in SWISS-PROT under accession numbers, P82277 (PSRP-2), P82412 (PSRP-3) and P47910 (PSRP-4) The cDNA nucleotide sequences (spinach and Arabidopsis) have been submitted to the GenBank/EBI Data Bank with accession numbers AF240462 (PSRP-2), AF239218 (PSRP-3), AF236825 (PSRP-4), spinach, and AF236826 (Arabidopsis PSRP-4) (Received 29 July 2002, revised 30 October 2002, accepted 8 November 2002)
Trang 2chloro-ribosome (70S) revealed an additional protein in
stoichiometric amount, the plastid ribosome recycling
factor (pRRF), which is released on the dissociation of
chloro-ribosome into subunits [2] Thus the
chloro-ribo-some proteome is composed of 59 distinct proteins: six
PSRPs, a bacterial-type pRRF (in E coli pRRF is not a
component of the ribosome), and 52 orthologues of
eubacterial ribosomal proteins [2] These results thus
confirmed the close kinship of the chloro-ribosome with
the eubacterial ribosome [3–5] and also revealed a distinct
departure, i.e recruitment of several large proteins during
the chloro-ribosome evolution The results also
re-con-firmed the great dissimilarities among the three (cyto-,
mito-, and chloro-) major types of ribosome [6]
The rate of protein synthesis in chloroplasts increases
dramatically on illumination, whereas the mRNA levels
remain relatively unchanged through light/dark transitions
(reviewed in [7,8]) Other significant differences between
chloroplasts and bacteria in both gene expression and
regulation of protein synthesis have been recognized
Nuclear factors regulate chloroplast protein synthesis at
several key post-transcriptional steps, e.g mRNA
process-ing, mRNA editprocess-ing, mRNA stability [9–15], and the
translation initiation step plays a major role in the
expression of several plastid genes, e.g light-induced
translation of psbA mRNA is regulated by cis-elements
including ribosome-binding sites and 5¢-UTR-binding
proteins [16–18] Thus, while maintaining an overall
resem-blance to the eubacterial system, chloroplast
transcrip-tion-translation has evolved numerous additional control
elements to achieve its highly effective co-ordination
between photosynthetic protein requirements and the
ribosome function The PSRPs, located on the ribosome
itself, are conceivably one set of control elements that have
permitted the observed translational co-ordination
Plastids (general name for the organelle, of which
chloroplast is one of the differentiated forms) have their
own genome, and employ for gene expression a
transcrip-tion-translation system composed of both plastid-encoded
and nucleus-encoded proteins Plastid ribosomes are
responsible for the synthesis of fewer than a hundred
polypeptides encoded in the plastid DNA, but these include
some of the most abundant proteins in the biosphere, e.g
the large subunit of ribulose-1,5-bisphosphate carboxylase/
oxygenase Moreover, certain key proteins of the
photosys-tems have high rates of protein turnover required during
their function (reviewed in [19]) Thus chloro-ribosomes
have to maintain a high rate of protein synthesis, but, being
dependent on photosynthetic chemical energy for function,
have had to evolve mechanisms dealing with the diurnal and
other variations in light intensity It would be interesting if
PSRPs, either ribosome-bound or free, play a role in these
global regulations of chloroplast protein synthesis
Here, we report the protein isolation and
characteriza-tion, and cloning of the cDNAs of three nuclear-encoded
PSRPs; PSRP-2, PSRP-3a/b, and PSRP-4 Together with
the previously reported results on PSRP-1, PSRP-5a/b/c
and PSRP-6 [1,2,20–22], characterization of all six PSRPs in
spinach (Spinacia oleracea) chloro-ribosome is now
com-plete Homologues of all six PSRPs are identifiable in the
complete genome sequence of Arabidopsis thaliana and in
the expressed sequence tag database of other land plants
We discuss light-dependent chloro-translational regulation, other possible functions, and evolution of the PSRPs
Materials and methods
Spinach chloroplast ribosome, 30S subunits, TP30 Spinach (S oleracea, cv Alwaro) chloroplast ribosomes were prepared as previously described [23] First, 5000 A260 units of ribosomes were run on a zonal sucrose gradient to obtain purified 70S ribosomes, and 3000 A260 units of purified 70S ribosomes were run on a dissociating zonal gradient to obtain 30S and 50S subunits (details in [21]) TP30 was prepared as described previously [1]
Protein/peptide electrophoresis, electroblotting SDS/PAGE was performed by the method of Laemmli [24] 2D PAGE was performed as described previously [25] Tricine SDS/PAGE of peptides was performed by the method of Scha¨gger & von Jagow [26] Molecular mass markers used were ovalbumin (43 kDa), carbonic anhyd-rase (29 kDa), b-lactoglobulin (18.4 kDa), lysozyme (14.3 kDa), bovine trypsin inhibitor (6.2 kDa) and insulin b-chain (3.4 kDa) Electroblotting was carried out as described previously [2]
Protein/peptide purification with RP-HPLC Protein or peptide was resolved with a Vydac C4 column (4.6· 150 or 250 mm) using the HPLC system described previously [1] The solvent systems and gradient conditions are described in the figure legends
Internal peptide preparation from PSRP-2 and PSRP-3a/b
After electrophoresis, 2D gels were stained for 30 min in 0.1% Coomassie Brilliant Blue R-250 (CBB)/45% ethanol/ 10% acetic acid (w/v/v) and destained for 1–2 h in 25% ethanol/8% acetic acid (v/v) In-gel digestion of PSRP-2 using endoproteinase Lys-C was carried out basically by the method of Hellman et al [27] with the slight modifi-cation described in our previous paper [2] For Asp-N digestion or CNBr cleavage, proteins from 2D gel spots corresponding to PSRP-2 and PSRP-3a were extracted as follows Five spots containing 10 lg protein/spot were placed in a 1.5-mL microtube and homogenized in 400 lL extraction buffer (1% SDS, 20 mM Tris/HCl, pH 8.0) using a small fitting pestle A further 400 lL of extraction buffer was added and the tube was shaken for 16 h at room temperature Peptide extract was separated from gel fragments by centrifugation (0.45 lm filter unit, ULTRA-FREE-MC; Millipore), concentrated to 250 lL in a Speed-Vac, precipitated with acetone (1 mL ice-cold acetone;
16 h at)20 C), and collected as a pellet by centrifugation (20 000 g, 15 min) Endoproteinase Asp-N (Sigma) diges-tion was performed [ 50 lg protein in 80 lL 50 mMTris/ HCl (pH 8.0)/2M urea] for 16 h at 37C (enzyme/ substrate, 1 : 100) The reaction was stopped by adding
20 lL 5% (v/v) trifluoroacetic acid, and the digest was subjected to HPLC CNBr cleavage was performed in
Trang 3100 lL 0.15M CNBr/75% (v/v) trifluoroacetic acid (4 lg
protein, 16 h, room temperature in the dark) After the
reaction, CNBr and trifluoroacetic acid were evaporated
under N2 gas and dried in a Speed-Vac The peptides
obtained were kept at)20 C until used
Protein/peptide sequencing and MS
Protein sequencing was carried out at the Laboratory for
Protein Sequencing and Analyses, University of Arizona,
using Applied Biosystem 477A Protein/Peptide sequencer
interfaced with a 120A HPLC analyzer MS analysis was
carried out at the Mass Spectrometry Facility, Department
of Chemistry, University of Arizona, using a Finnigan LCQ
electrospray ionization mass spectrometer (ESI MS) About
50 pmol protein in 10 lL 4% acetic acid was subjected to
ESI MS
Cloning and sequencing of PSRP-2, PSRP-3, PSRP-4
cDNAs
A kgt11 spinach cDNA library prepared previously in our
laboratory [28] was screened by thermal gradient PCR
using a Mastercycler gradient PCR apparatus (Eppendorf
Scientific, Inc.) PCR was performed (3 min at 94C, 35
cycles of 1 min at 94C, 1 min at 43–60 C for degenerate
PCR or 1 min at 55C, 1.5 min at 72 C, and 1 cycle of
10 min at 72C) with 1.25 U Taq DNA polymerase
(Gibco-BRL) in a 50-lL reaction volume containing 1 lL
kgt11 library ( 108 plaque-forming units), 1 lM
gene-specific primer or 2 lM degenerate primer, 1 lM lambda
arm primer (PF or PR), 200 lM each dNTP, 1.5 mM
MgCl2, and 50 mMKCl in 20 mMTris/HCl (pH 8.4) PF
(forward primer) and PR (reverse primer) are
comple-mentary to the cloning site of kgt11 Degenerate
oligonu-cleotide primers, P2-1, P3-1, and P4-1 were designed from
the internal peptide of PSRP-2 (peptide 3, -MDIATTQA-,
including CNBr-cleaved Met, see Fig 4A) and N-terminal
sequence portions of PSRP-3 (-MGNEVDID-) and
PSRP-4 (-PKNKNKG-), respectively Optimal annealing
temperatures for the degenerate RCR were observed to be
57–60C for amplifications of 3¢-portions of Psrp-2 (P2-1/
PR) and Psrp-3 (P3-1/PR), and 50–57C for amplification
of 3¢-portion of Psrp-4 (P4-1/PR) Here, for example,
P2-1/PR stands for PCR amplified DNA using lambda
library and primer sets P2-1 and PR Gene-specific PCR
primers for Psrp-2, Psrp-3 and Psrp-4 (P2-2, P3-2 and
P4-2) were designed from the nucleotide sequence of PCR
products, P2-1/PR, P3-1/PR and P4-1/PR
Tag-sequence-containing primers complementary to 5¢-termini and
3¢-termini of PSRP-2 and PSRP-4 cDNAs (P2-3, P2-4,
P4-3, and P4-4) were designed after sequencing the sets of
PCR products
The full cDNAs encoding PSRP-2 and PSRP-4 were
amplified from the lambda library using the tagged primers
The nucleotide sequences of Psrp-2 and Psrp-4 were
obtained by sequencing P2-3/P2-4 and P4-3/P4-4 using
sequencing primers, TAG1 and TAG2, and the two strands
were completely sequenced by primer walking The phage
clone of PSRP-3 cDNA was obtained by the following
method The PCR product encoding 5¢-PSRP-3 cDNA
portion (PF/P3-2) was labeled with32P as described by the
supplier of Random Primed DNA Labeling Kit (Boehringer Mannheim) The lambda library (150 000 pfu) was plated
on four 132-mm plates, and the plaques were lifted on to ICN BIOTRANS Nylon membrane Prehybridization was performed in 500 mMsodium phosphate, pH 7.0, at 50C for 2 h and hybridization in 500 mM sodium phosphate (pH 7.0)/7% SDS at 50C for 16 h The membrane was washed twice in 100 mM sodium phosphate (pH 7.0)/1% SDS at 37C for 10 min followed by a 10-min wash in
40 mMsodium phosphate (pH 7.0)/1% SDS at 37C and autoradiographed Plaques giving positive signals were purified and preserved by standard procedures [29] Insert DNA in the phage clone (PSRP-3-D1) was amplified by PCR using primer sets PF and PR, then cleaved by EcoRI digestion and subcloned into the plasmid vector pBluescript
SK–(Stratagene) The insert DNA in Psrp-3 plasmid clone was sequenced Nucleotide sequencing was carried out at the DNA Sequencing Facility, University of Arizona, using
an Applied Biosystems model 377 sequencer PCR products were analyzed by agarose gel electrophoresis using 1% (w/v) agarose gel and visualized by ethidium bromide staining Oligonucleotides used in this study were: PF, 5¢-CGGGATC CGGTGGCGACGACTCCTGGAGCCC-3¢; PR, 5¢-CG GGATCCCAACTGGTAATGGTAGCGACCGGC-3¢; P2-1, 5¢-ATGGAYATHGCIACIACICARGC-3¢; P2-2, 5¢-TAGCAACTCATTCGTCACTGTC-3¢; P2-3, 5¢-GGA ATTCTAGATATCGTCGACAATTTGTGTTACTACC AAAATC-3¢; P2-4, 5¢-GGAATTCGTCGACGCGTTAA AAAAGATAGCAGCATTGACAC-3¢; P3-1, 5¢-ATGG GIAAYGARGTIGAYATHG-3¢; P3-2, 5¢-CTAGACC TATGTTTTTCTCCATCC-3¢; P4-1, 5¢-CCIAARAAYA ARAAYAARGG-3¢; P4-2, 5¢-CAGATAGGAAGAGGG GCAAGGA-3¢; P4-3, 5¢-GGAATTCTAGATATCGTC GACTTATCTTCAGAACTTGTTGC-3¢; P4-4, 5¢-GGA ATTCGTCGACGCGTTTTTCAACAAATCATCATAT A-3¢; TAG1, 5¢-GGAATTCTAGATATCGTCG-3¢; TAG2, 5¢-GGAATTCGTCGACGCG-3¢
Computer analysis
A homology search was performed using the BLAST
program ORFs from cDNA sequences were analyzed using the map program from the GCG software package [30] Sequence alignments and comparisons were performed usingPILEUP and GAP programs in the same package or
CLUSTAL W[31] The results were displayed usingBOXSHADE
(version 3.21 written by K Hofmann and M D Baron) or manually modified Secondary structure prediction was by the methods of Chou & Fasman [32] and Garnier et al [33] using GCG software
Protein and gene nomenclature The protein and gene nomenclature in this paper are in accordance with the Commission on Plant Gene Nomen-clature rules [34], and follows our previous paper [1,2]
E coli orthologues of ribosomal proteins S1–S21 were designated PRP S1 to PRP S21 (P, for plastid; C was not used for chloroplast to avoid confusion with C, cytosolic) The six PSRPs are designated PSRP-1 to PSRP-6, and their genes, Psrp-1 to Psrp-6 (see Table 1 and our previous papers [1,2])
Trang 4Results and discussion
Identification, isolation, N-terminal/internal peptide
sequencing and MS of PSRP-2, PSRP-3 and PSRP-4
Spinach chloroplast ribosome was first purified on a zonal
sucrose gradient, and the ribosomal 70S peak collected was
then run on a dissociating zonal gradient to obtain pure 30S
and 50S subunits free of adhering stromal proteins (see [21]
for details and gradient profiles) As described [21], efficient
dissociation of chloroplast ribosome required a
phosphate-containing buffer The total protein from the 30S subunit
(TP30, 200 pmol) was subjected to 2D PAGE (Fig 1), and
the resolved proteins were electroblotted on to
poly(viny-lidene difluoride) membrane for N-terminal sequence
ana-lysis All of the 30S protein spots were excised from the blot
and subjected to N-terminal protein sequencing analysis
We identified in the chloroplast 30S subunit the orthologues
of all E coli 30S ribosomal proteins (S1–S21) and the details
are reported in a previous paper [1] We designated the four
additional proteins present in the chloroplast 30S subunit
PSRP-1, PSRP-2, PSRP-3 and PSRP-4 (see Fig 1)
PSRP-1 has been previously characterized [20,21], and its
cDNA cloned and expressed in E coli [35] PSRP-4 has also
been previously identified but only partially sequenced and
had been designated S31 [36] We therefore proceeded with
the characterization of PSRP-2, PSRP-3, and PSRP-4
The N-terminal sequence and the yield/recovery of
phenylthiohydantoin (PTH)-amino acids from Edman
degradation were: PSRP-2, NH2-VVTEETSSSSTASSSS
DGEGA- (21 amino acids, 41 pmol per spot); PSRP-3,
NH2-VAPETISDVAIMGNEVDIDDDLLVNKEKLK
VLVKPMDKXXLVL- (43 amino acids, 7 pmol per spot);
PSRP-4, NH2-GRGDRKTAKGKRFNHSFGNARPKN
KNKGRGPPKAPIFPKGDPS- (43 amino acids, 37 pmol
per spot) The yield of 37–41 pmol PTH-amino acids per
spot for PSRP-2 and PSRP-4 corresponded to that for the
other 30S ribosomal proteins (average, over 30 pmol) The
results supported the view that PSRP-2 and PSRP-4 are unit
proteins on the chloroplast 30S subunit With respect to
PSRP-3, the yield of PTH-amino acids was significantly lower (7 pmol per spot); however, the Coomassie Blue staining intensity of its spot was similar to that of the other spots in that region of the gel Therefore, a partially blocked N-terminus for PSRP-3 was indicated
To confirm whether PSRP-3 is N-terminally blocked, electrophoretic separation of the two forms (blocked and unblocked) was attempted by running the first dimension gel of the 2D PAGE for twice as long The PSRP-3 spot was resolved into two spots, a slower migrating spot marked a, and a faster migrating spot marked b (Fig 1 inset) The two
Fig 1 Two-dimensional gel pattern of spinach chloroplast 30S subunit proteins:resolving PSRP-2, PSRP-3a/b, and PSRP-4 TP30 (200 pmol) electropherogram stained with Coomassie Blue (as land-marks, S1a/b, S4, S6a-e and S10a/b are shown) First dimension:
pH 5.0 in 4% (w/v) acrylamide gel containing 8 M urea; second dimension: pH 6.7 in 10% (w/v) acrylamide gel containing 0.2% SDS Inset shows a poly(vinylidene difluoride) blot of the acidic proteins, stained with Amido Black For better resolution, the gel was run twice
as long in the first dimension PSRP-3 spots a and b (circled) indicate the N-terminal blocked and unblocked forms, respectively Note: small acidic proteins are stained weakly by Amido Black Molecular sizes and isoelectric points (pI) shown are based on the characterization in our two previous papers [1,2].
Table 1 Characteristics of chloroplast-specific ribosomal proteins (spinach).
Protein
name
Subunit
location
Chain length
Molecular mass
Isoelectric pointa Ref.c
Other name and ref Similar protein and ref.
PSRP-1 30S 236 26 805b 6.2 [21] CS-S5 [20]
S22 [92]
S30 [21]
Synechococcus lrtA light-repressed transcript A product [86]
PSRP-2 30S 198 21 665b 5.0 New Chloroplast ribonucleoproteins [44,45] PSRP-3 30S 121 > 13 794 a (a)
13 794 a (b)
< 4.9 (a) 4.9 (b)
New P purpurea ycf65 hypothetical chloroplast
reading frame product [48]
PSRP-4 30S 47 5 174b 11.8 New S31 [36]
SCS23 [93]
T thermophilus 30S ribosomal protein Thx [52] PSRP-5 50S 80 (a)
58 (b)
54 (c)
9 255 b (a)
7 066b(b)
6 638b(c)
11.5 (a) 12.2 (b) 12.2 (c)
[2] L40 [94]
PsCL18 [22]
PSRP-6 50S 69 7 387 a 10.6 [2] PsCL25 [22]
a
Calculated from mature protein sequence.bObtained from mass spectrometry.cFor cloning and sequencing of cDNA encoding full precursor sequence.
Trang 5spots were excised and subjected to N-terminal analysis.
Edman degradation gave a clear N-terminal sequence for
the weaker staining b spot (9 pmol per spot), but an unclear
sequence for the stronger staining a spot (insignificant yield,
less than 2 pmol per spot) From its slower electrophoretic
migration in the first dimension gel, the a form is expected
to be more acidic than the b form (i.e loss of a positive
charge/net gain of a negative charge, making the protein
more acidic)
To confirm that the more acidic a form is N-blocked
PSRP-3, a spots were excised from several gels, and the
extracted protein was cleaved with CNBr The CNBr
fragments were separated by Tricine
SDS/PAGE/electro-blotting, and two of the CNBr fragments (Peptide 1 and
Peptide 2, Fig 2D) were sequenced: Peptide 1, GNEVDI-;
Peptide 2, EKNIGLALDQTIPG- Peptide 1 has the same
sequence as part of the N-terminal sequence of PSRP-3
(Gly13–Ile18) above Peptide 2 is a new sequence, and it
was considered to be an internal peptide sequence from
PSRP-3 (subsequently confirmed by DNA sequencing of
PSRP-3 clone) The experiments thus demonstrated that
PSRP-3 exists in two forms (designated a and b), the
alpha form being N-blocked and the b form having a free
N-terminus The approximate ratio of the two forms is
3 : 1
For cloning PSRP-2 (by PCR screening a spinach
cDNA library using degenerate oligonucleotide
pri-mers), the predominantly serine/threonine-rich N-terminal
sequence obtained above was unsuitable Therefore,
inter-nal peptides of PSRP-2 were prepared HPLC resolution
of the peptides from two protease digests (endoproteinase
Lys-C and endoproteinase Asp-N) are shown in Fig 2A,B
Long peptides containing aromatic (high UV absorption)
and/or acidic amino acids are eluted late in RP-HPLC;
these amino acids (W, Y, F, D, E) have less than average
degeneracy Therefore, four of the late-eluted tall peaks
were taken for sequence analysis In addition, PSRP-2
spots were excised from several 2D gels and the extracted
protein was subjected to CNBr cleavage (CNBr generates
long fragments generally; also the cleavage occurs at the
carboxyl of Met which has zero degeneracy) Peptide
frag-ments were separated by Tricine SDS/PAGE (Fig 2C),
and two were taken for sequencing The five internal
sequences obtained were: Peptide 1, YDKYSGRSR
RFGFVTM-; Peptide 2, VNITEKPLEGM-; Peptide 3,
DIATTQAEDSQFVESPYKVY-; Peptide 3a,
DSQFV-(contained in Peptide 3 sequence); and Peptide 4,
DFFSEKGKVLGAKVQRTPG-
Being a very basic protein, PSRP-4 could be readily
purified by RP-HPLC of 1 mg TP30 on a Vydac C18
column (Fig 3A); the purified protein showed a single
band, corresponding to the fastest migrating band of TP30
on the 1D gel, and a single spot on the 2D gel (Fig 3A,
insets) The purified PSRP-4 was subjected to ESI MS The
spectrum (Fig 3B) showed multicharged (+ 4 to + 11)
ions in the 400–1400 m/z (mass to charge ratio) region The
deconvoluted mass spectrum (Fig 3C) showed a single
peak with a molecular mass of 5174 Da This observed mass
and the sequence mass calculated from the PSRP-4
amino-acid sequence (see next section, Fig 4C) are in excellent
agreement Thus, PSRP-4 is not post-translationally
modi-fied to any significant degree
Isolation of PSRP-2 and PSRP-3 directly from TP30 on
an HPLC C18column was not effective, because they were coeluted with a few other proteins [1] However, LC/MS analysis of an HPLC fraction (pool 18 in [1]), which contained PSRP-2, PRP S4 and PRP S8 resolved the molecular masses, and yielded an observed mass of
Fig 2 Isolation of internal peptides from PSRP-2 and PSRP-3 (A) HPLC separation of endoproteinase Lys-C in-gel digest of PSRP-2 (10 lg, extracted from 2D gel spots) Peptide 2 and the N-terminal peptide were sequenced CBB, peak of Coomassie Blue (B) HPLC separation of endoproteinase Asp-N digest of PSRP-2 (40 lg, extracted from 2D gel spots) Peptides 3a and 4 were sequenced RP-HPLC was carried out on a Vydac C4 column (150 · 4.6 mm) using a step linear gradient of acetonitrile (MeCN) in 0.1% (v/v) trifluoro-acetic acid (0% MeCN up to 5 min, 40% MeCN at 65 min, 80% MeCN at 75 min), at a constant flow rate of 0.5 mLÆmin)1 (C) Poly(vinylidene difluoride) blot of CNBr-cleaved fragments of PSRP-2 (4 lg, CNBr fr.) and intact PSRP-2 (2 lg) Peptides 1 and 3 were sequenced (D) Poly(vinylidene difluoride) blot of CNBr-cleaved fragments of PSRP-3a (4 lg) and intact PSRP-3a (2 lg) Peptides 1 and 2 were sequenced CNBr fragments were separated by Tricine SDS/PAGE and electroblotted on to a poly(vinylidene difluoride) membrane, and stained with Amido Black Peptide peaks/bands indicated were analyzed in an automated protein sequencer.
Trang 621 665 Da for PSRP-2 This value exactly corresponds to
the sequence-calculated mass of PSRP-2 (see next section,
Fig 4A), suggesting almost no post-translational
modifica-tion in the mature protein
PSRP-3 could not be observed using ESI-LC/MS or
MALDI-TOF MS This may be due to the poor ionization
of this protein A few other ribosomal proteins, e.g PRP L2,
could also not be observed in our ESI MS and
MALDI-TOF MS experiments [2] Thus, we can offer no suggestions
on the nature of the N-terminal blocking in PSRP-3a or on
the possibility of additional post-translational modifications
in either form
cDNA cloning and nucleotide sequencing of the cytoplasmic mRNAs for PSRP-2, PSRP-3 and PSRP-4 PSRP-2, PSRP-3, and PSRP-4 were considered to be nuclear-encoded proteins because the N-terminal sequences and internal sequences, reported above, are not encoded in the plastid genome sequence of spinach [37] or other higher plants [38] Therefore, inosine-containing degenerate prim-ers were designed from the peptide sequence information, and used to screen a previously described kgt11 cDNA library [28] First, partial cDNA was specifically amplified using sets of degenerate primer/lambda arm primer, and further PCR amplifications were carried out using sets of gene-specific primer (based on the cDNA sequence ob-tained) and lambda arm primer (PF or PR) The cDNA clones were finally obtained by a third PCR amplification using tagged primers complementary to the 5¢ and 3¢ ends of cDNA inserts (see Materials and methods for further information), and both strands of the cDNAs obtained were completely sequenced The nucleotide sequences of the cDNAs encoding the precursors of PSRP-2, PSRP-3, and PSRP-4 are shown in Fig 4
The PSRP-2 precursor cDNA comprises 1242 bp [exclu-ding poly(A) tail], the ORF (nucleotides138–920) enco[exclu-ding
a putative 220-residue protein The N-terminal sequence of mature PSRP-2 begins at residue 63, suggesting a 62-residue transit peptide, and a 198-residue mature protein of sequence mass 21 665.02 Da and theoretical pI 4.99 The nucleotide sequence of PSRP-3 precursor cDNA comprises
751 bp, with an ORF (nucleotides 41–580) encoding a putative 179-residue protein The mature PSRP-3 begins at residue 59, suggesting a 58-residue transit peptide and a 121-residue mature protein of sequence mass 13 794.02 Da and theoretical pI 4.93 [The molecular mass of PSRP-3, estimated from Tricine SDS/PAGE (Fig 3D) or SDS/ PAGE [2], is 14.0 kDa, close to the sequence mass, indicating no heavy post-translational modifications.] The nucleotide sequence of PSRP-4 precursor cDNA comprises
521 bp, with an ORF (nucleotides 22–327) encoding a putative 101-residue protein The mature PSRP-4 begins at residue 55, indicating a 54-residue transit peptide and a 47-residue mature protein of sequence mass, 5173.80 Da and theoretical pI 11.80 The 87, 57, and 43 amino acids of PSRP-2, PSRP-3, and PSRP-4 sequences, determined from protein work (underlined in Fig 4, corresponds to 44%, 47%, and 91%, respectively, of the mature protein chain lengths), showed 100% match to the cDNA-derived sequences As noted above, the MS molar masses of mature PSRP-2 and PSRP-4 suggest an absence of post-transla-tional modifications
Sequence homology of PSRP-2 to ribonucleoproteins containing two U1A-type RNA-binding domains
A homology search using the BLASTPprogram revealed a significant sequence similarity of PSRP-2 to a large number of proteins that carry one or more conserved RNA-binding domains These domains (called RBD, but also RRM for RNA recognition motif) are well charac-terized in human U1A small nuclear ribonucleoprotein (U1A snRNP) An RBD is defined as having an 80-residue sequence, containing a conserved octapeptide
Fig 3 Purification and MS of PSRP-4 (A) RP-HPLC profile of TP30
(1 mg) resolved on a Vydac C 18 column (250 · 4.6 mm) using a step
linear gradient of isopropanol (IPA) in 0.1% (v/v) trifluoroacetic acid
(10% IPA from 0 to 10 min, 25% IPA at 80 min, 45% IPA at
250 min) at a constant flow rate of 0.5 mLÆmin)1 Every peak in the
0–100 min retention time was subjected to SDS/PAGE, MS, and
protein sequencing (asterisk, nonprotein peak) S17frg is a truncated
form of S17, see [1] Inset (1D and 2D) shows that the smallest protein
band of TP30, a 7.5-kDa protein, corresponds to PSRP-4 in 2D PAGE.
(B) ESI MS of PSRP-4 Each peak represents an individual charged
ion The m/z ratio and the number of positive charges on the ion are
shown above each peak (C) Deconvoluted mass spectrum of the m/z
series in (B) indicates a single protein of molecular mass 5174.30 Da.
Trang 7(RNP1) and a hexapeptide (RNP2) separated by about 30 amino acids [39] The RBD folds into a compact structure
of four antiparallel b-sheets and two a-helices (b1-a1-b2-b3-a2-b4) with the conserved RNP1 and RNP2 located in the b1 and b3 antiparallel strands [39] Both RNP1 and RNP2 are important elements for recognizing the target RNA
The highest alignment score of PSRP-2 was actually to a small group of RNA-binding proteins (cpRNPs) present in the chloroplast stroma Other high-alignment hits were: polyadenylate-binding proteins, glycine-rich RNA-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), and small nuclear ribonucleoprotein (snRNP) [40–43] The three tobacco cpRNPs, cp29A, cp31, and cp33 [44,45], are shown aligned with the PSRP-2 sequence in Fig 5A, with sequence identities (similarities) of 36.5% (48.7%), 36.2% (48.9%), and 39.1% (48.7%), respectively The primary structure of PSRP-2 appears to be related
to that of the cpRNPs, with a similar arrangement of the two RBDs, but with a shorter, less negatively charged N-terminal domain and a truncated (by 8–30 residues) C-terminal domain (Fig 5A) A comparison of PSRP-2 with several other RBD-containing proteins (spinach 28 RNP, Chlamydomonas reinhardtii RB47 poly(A)-binding protein, barley cold-inducible glycine-rich RNA-binding protein, Anabaena variabilis RNA-binding protein, human hnRNP, and human U1A snRNP) shows high conservation
of the RNP1 and RNP2 sequences between these proteins and PSRP-2 (Fig 5B)
Convergent evolution has been suggested for the sequence relationship between bacterial RNA-binding pro-teins and eukaryotic glycine-rich propro-teins, and a phylo-genetic analysis has indicated that cpRNPs are likely to have diverged from eukaryotic glycine-rich proteins, rather than from cyanobacteria [46] As it appears structurally closely related to cpRNPs, PSRP-2 may also have a eukaryotic, evolutionary origin
The RBD-containing proteins are generally associated with RNA processing: splicing, localization, and stabil-izing In the U1A protein, the aromatic residues of RNP1 and RNP2 are critical for RNA-base stacking [39] Because such aromatic residues in the putative RBDs of PSRP-2 are conserved (Fig 5A,C), PSRP-2 probably performs a similar RNA-binding activity As protein synthesis in chloroplasts is remarkable for its mRNA storage in the dark and light-induced translational spurt,
we would like to suggest a role for PSRP-2 in this process However, as PSRP-2 could bind mRNA, rRNA
or both, and if it binds mRNA, it could just as well be involved in translation initiation as in light-regulated translation
Fig 4 Cytoplasmic precursors and complete mature forms:the nuc-leotide sequences of PSRP-2, PSRP-3 and PSRP-4 cDNAs and corre-lating experimentally determined peptide sequences Initiation contexts conforming to the Kozak rule [91] are boxed Chloroplast-targeting signal sequences (transit peptides) are shown in italics Arrows indicate the cleavage site of the transit peptide The experimentally determined N-terminal sequences and internal peptides are underlined The stop codon is indicated by an asterisk (A)n, polyadenylation.
Trang 8Fig 5 Sequence alignment of PSRP-2 with three chloroplast RNA-binding proteins:domain arrangement/comparison of RBDs between PSRP-2 and RBD-containing proteins (A) PSRP-2 is aligned with three chloroplast RNA-binding proteins (cp29, cp31 and cp33, from wood tobacco, Nicotiana sylvestris [44,45]), to which it is closely related in structure, having two RBDs in similar arrangement The octapeptide RNP1 and the hexapeptide RNP2 motifs in each of the two RBDs are boxed Negatively charged amino acids (D/E) in the acidic N-terminal region of cpRNPs are shown in bold letters Identical amino acids or conserved replacements in the four proteins are shown shaded Chain lengths of the mature proteins and percentage identity (I) and similarity (S) are indicated after the C-terminus (B) Schematic diagram of RBD domain arrangement (filled boxes) in PSRP-2 and six other RBD-containing proteins Abbreviations and accession numbers: cpRNP, chloroplast ribonucleoprotein 28RNP from spinach (P28644); GRP, cold-inducible glycine-rich RNA-binding protein from barley (U49482); cyRBP, RNA-binding protein from a cyano-bacterium, A variabilis (I39621); PABP, poly(A)-binding protein from an alga, C reinhardtii (T07933); hnRNP, heterogeneous nuclear ribonu-creoprotein A1 from human (P09651); snRNP, small nuclear ribonucleoprotein U1A spliceosomal protein from human (P09012) (C) Alignment of the 50-amino-acid sequence portion of RBD-1 and RBD-2 from PSRP-2 and the six other RBD-containing proteins in (B) The positions of conserved amino-acid residues are highlighted: black, identical, and grey, similar.
Trang 9PSRP-3 and the hypothetical chloroplast reading frame
(ycf65) protein
ABLASTsearch of the PSRP-3 sequence against database
sequences showed several close homologues They were all
unidentified hypothetical proteins, designated YCF65
(hypothetical chloroplast frame 65 product [47]), first
reported in the chloroplast genome of Porphyra purpurea,
a red alga [48] Homologous sequences are present in the
prasinophycean alga, Mesostigma viride, and the
crypto-phyte alga, Guillardia theta The ycf65 gene is absent from
the chloroplast genome of the green algae Chlorella vulgaris
[49] and C reinhardtii (J Maul, J W Lilly and D B Stern,
unpublished results, www.biology.duke.edu/chlamy_
genome/chloro.html) However, a PSRP-3 homologue can
be identified in the Chlamydomonas expressed sequence tag
(EST) sequences (Table 2), suggesting a relocation of the
ycf65gene to the nuclear genome of C reinhardtii
A homologue of the Psrp-3/ycf65 gene is absent from
the database of eubacterial (nonphotosynthetic) and
archaebacterial genomes However, a Psrp-3 homologue
is present in the two (photosynthetic) cyanobacterial
genomes (Synechocystis and Synechococcus) in the database
Although not yet experimentally shown, it is likely that the
PSRP-3 protein is a component of cyanobacterial
ribo-somes The overall sequence identities among higher plant
PSRP-3 homologues are high (71–80%), whereas those
between higher plant and algal/cyanobacterial PSRP-3
homologoues are lower (41–52%) The PSRP-3 sequences
contain five invariant residues which are the relatively rare
tryptophan, and, moreover, a highly conserved decapeptide
motif, -Y(Y/F)FWPRXDAW-, containing five conserved
aromatic amino acids, is present in the central region
(Fig 6) Compared with the algal and cyanobacterial
sequences, the mature spinach PSRP-3 carries a negatively
charged N-terminal extension and a 20-residue truncation
at the C-terminus
It is likely that the Psrp-3 gene, having had its origins in
the cyanobacterial genome, was retained during the
emer-gence of algal/higher plant chloroplasts As PSRP-3 is
specifically associated with ribosomes that have to perform
protein synthesis in a photosynthetic environment, it may
have been evolved for a role that does not exist in E coli,
e.g linking protein synthesis and light Spinach PSRP-3
coexists in two forms, a form post-translationally modified
at the N-terminus and an unmodified form It is not known
if the ratio of these two forms is constant under all
conditions The ratio could be variable, depending on the intensity and duration of light, like the growth rate-dependent N-terminal modification in certain E coli ribo-somal proteins [50] It would be interesting to identify the plant gene for the enzyme that catalyzes PSRP-3 post-translational modification, and to check whether the cyanobacterial genome carries a homologous enzyme gene
Sequence homology of PSRP-4 withThermus thermophilus 30S subunit protein Thx and a plant mitochondrial protein
From the incomplete sequence data then available [36,51], the T thermophilus 30S ribosomal protein Thx was previously identified as a homologue of PSRP-4 protein (it was then designated S31) Recently the complete sequence of Thx, having only 26 residues, has been confirmed by nucleotide sequencing [52] Figure 7 shows the sequence alignment
A search usingBLASTrevealed two PSRP-4 homologues
in the complete genome of Arabidopsis One of them was very closely related to the PSRP-4 sequence (chromosomal locus number AT2g38140), and so we obtained the corresponding EST (clone 122F4T7) from the Arabidopsis EST Stock Center and sequenced it completely (see accession number AF236826 for sequence data) The data revealed the cDNA of the PSRP-4 precursor protein of Arabidopsischloroplast ribosome (see Fig 7A) The other homologue was a hypothetical protein, named here Ath-4h (chromosomal locus AT2g2129), with less
PSRP-4 homology A transcript of this hypothetical protein was not identifiable in the Arabidopsis EST database However, sequences closely related to it are found in the ESTs of other plants, the closest one being a maize EST sequence The PSRP-4 precursor sequences from spinach (Sol-PSRP-4), Arabidopsis (AthPSRP-4) and tomato (LesPSRP-4) are aligned in Fig 7A with the T themophilus Thx, and included there are the PSRP-4h hypothetical proteins from Arabidopsisand maize The PSRP-4 precursors carry plastid transit peptides of 50 amino-acid residues The mature PSRP-4 proteins have (a) lysine/arginie-rich Thx-like regions, (b) hydrophobic proline-rich motifs, and (c) nonconserved C-terminal regions The PSRP-4h hypothet-ical proteins have shorter putative transit peptides, but contain lysine/arginine-rich Thx-like regions, and con-served, hydrophobic, proline-rich C-terminal regions (-PWPLPFKLI-COOH)
Table 2 Homologues of spinach PSRPs in other angiosperms, a bryophyte, green alga, and photosynthetic bacterium Accession numbers are in parentheses Chromosomal locus of Arabidopsis genes are shown in square brackets EST, in italic ?, Significant homologues not found in public database, NI, not identified in the complete genome sequence.
Spinach Arabidopsis Barley Moss C reinhardtii Synechocystis PSRP-1 (M55322) AF370148 [AT5g24490] BF262651 AW561215 AV390148 P74518 PSRP-2 (AF240462) AY039568 [AT3g52150] BF267982 ? ? NI
PSRP-3 (AF239218) AV530526 [AT1g68590]
? [AT5g15760]
AW201242 Z98114 BG847093 Q55385 PSRP-4 (AF236825) AF236826 [AT2g38140] BE558713 AW598994 BE452645 NI
PSRP-5 (AF261940) BE037671 [AT3g56910] BF621665 AW509867 ? NI
PSRP-6 (AF245292) AV532737 [AT5g17870] BG300387 AW126633 AV622927 NI
Trang 10The hypothetical AthPSRP-4h and ZmaPSRP-4h
sequences were predicted to be chloroplast proteins by
the ChloroP program [53], but on the other hand, the
PSORT program [54] predicted them as mitochondrial
matrix proteins or nuclear proteins It has been suggested
that an N-terminal amphiphilic helix is a critical
deter-minant for mitochondrial sorting [55], and recent NMR
structures of mitochondrial and plastid transit peptides
[56,57] have shown a significant difference in their
secondary structures, i.e mitochondrial transit peptides
have an N-terminal a-helix region, but plastid transit
peptides have a nonhelical N-terminal region
Secondary-structure prediction of the N-terminal regions of
Ath-PSRP-4h and ZmaAth-PSRP-4h showed arginine-containing
amphiphilic helices like those of known mitochondrial
transit peptides, whereas the N-termini of PSRP-4
precursors were predicted to be nonhelical structures
(Fig 7A) Therefore, although the possibility of
N-terminal extended PSRP-4 homologues in the cytoplasm
or in the nuclear matrix cannot be ruled out, we suggest
that the PSRP-4h hypothetical proteins are probably
mitochondrial proteins
Thx has been visualized in the crystal structure of
T thermophilus30S ribosomal subunit [58] It fits into the
cavity found between the 16S rRNA helices H30, H41,
H41a, H42, and H43 at the top of the 30S head, the high
positive charge of Thx stabilizing the organization of the
RNA elements [58] These rRNA helices are also conserved
in the plastid 16S rRNA Therefore, we suggest that PSRP-4
is most likely located at the top of the chloroplast 30S
subunit head, with the Thx-like N-terminal sequence anchoring it to the ribosome
The sequence homology between a Thermus ribosomal protein and the higher plant PSRP-4 requires a comment A Thx homologue is not identifiable in the genome sequences
of not only E coli and other mesophilic bacteria, but also in the genomes of other thermophilic bacteria such as Aquifex aelolicus and Thermotoga maritima Thus, the unusual sequence resemblance between Thx and PSRP-4 may be due
to a convergent evolution, i.e PSRP-4 and Thx genes may have arisen independently acquiring a similar function (stabilization of the rRNA helices) Another possibility is that PSRP-4 emerged during chloro-ribosome evolution by
a process of gene capture (by horizontal gene transfer) from the progenitors of T thermophilus Whatever its origin, experiments transforming E coli with Thx or PSRP-4 gene would probably address the questions of thermal stability and function
The two PSRPs on the chloroplast large ribosomal subunit, PSRP-5 and PSRP-6, are also highly basic, positively charged, small proteins (Table 1) Although these proteins do not share significant sequence similarity, they share certain structural characteristics with PSRP-4 Figure 7B shows the sequences of PSRP-4, PSRP-5c (the shortest form of PSRP-5 [2]), PSRP-6 and Thx (manually aligned) and the schematic diagram of a discernible consensus secondary structure, composed of an N-terminal lysine/arginine-rich domain, a hydrophobic proline-rich motif, and a nonconserved C-terminal region The basic N-terminal domains of PSRP-5 and PSRP-6 may also serve
Fig 6 Sequence alignment of spinach PSRP-3 with algal ycf65 gene product and with homologues from higher plants and cyanobacteria Aligned sequences (and accession numbers) are: Arabidopsis (1: AT1g8590 and 2: AT5g15760, see also Table 2 and text), barley (AW201241); YCF65 gene products from P purpurea (P51351); Guillardia theta (O78422); and Mesostigma viride (AAF43868); YCF65-like hypothetical proteins from Synechococcus PCC7942 (O05161) and Synechocystis PCC6803 (Q55385) The positions of conserved residues are highlighted: black, identical, and grey, similar.