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J. Biol. Chem., Vol. 275, Issue 37, 28466-28482, September 15, 2000
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From the Department of Biochemistry, The University of Arizona,
Tucson, Arizona 85712 and the Max-Planck-Institut für Molekulare
Genetik, Berlin-Dahlem, Germany 14195
We have completed identification of
all the ribosomal proteins (RPs) in spinach plastid (chloroplast)
ribosomal 50 S subunit via a proteomic approach using two-dimensional
electrophoresis, electroblotting/protein sequencing, high
performance liquid chromatography purification, polymerase chain
reaction-based screening of cDNA library/nucleotide sequencing, and
mass spectrometry (reversed-phase HPLC coupled to electrospray
ionization mass spectrometry and electrospray ionization mass
spectrometry). Spinach plastid 50 S subunit comprises 33 proteins, of
which 31 are orthologues of Escherichia coli RPs and two
are plastid-specific RPs (PSRP-5 and PSRP-6) having no homologues in
other types of ribosomes. Orthologues of E. coli L25 and
L30 are absent in spinach plastid ribosome. 25 of the plastid 50 S RPs
are encoded in the nuclear genome and synthesized on cytosolic
ribosomes, whereas eight of the plastid RPs are encoded in the plastid
organelle genome and synthesized on plastid ribosomes. Sites for
transit peptide cleavages in the cytosolic RP precursors and formyl Met
processing in the plastid-synthesized RPs were established.
Post-translational modifications were observed in several mature
plastid RPs, including multiple forms of L10, L18, L31, and PSRP-5 and
N-terminal/internal modifications in L2, L11 and L16. Comparison of the
RPs in gradient-purified 70 S ribosome with those in the 30 and 50 S
subunits revealed an additional protein, in approximately
stoichiometric amount, specific to the 70 S ribosome. It was identified
to be plastid ribosome recycling factor. Combining with our recent
study of the proteins in plastid 30 S subunit (Yamaguchi, K., von
Knoblauch, K., and Subramanian, A. R. (2000) J. Biol.
Chem. 275, 28455-28465), we show that spinach plastid
ribosome comprises 59 proteins (33 in 50 S subunit and 25 in 30 S
subunit and ribosome recycling factor in 70 S), of which 53 are
E. coli orthologues and 6 are plastid-specific proteins
(PSRP-1 to PSRP-6). We propose the hypothesis that PSRPs were evolved
to perform functions unique to plastid translation and its regulation,
including protein targeting/translocation to thylakoid membrane via
plastid 50 S subunit.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF238221, AF245292, AF250384, AF250923, and
AF261940.
The Plastid Ribosomal Proteins
IDENTIFICATION OF ALL THE PROTEINS IN THE 50 S SUBUNIT OF AN
ORGANELLE RIBOSOME (CHLOROPLAST)*
*
This work was supported by Max-Planck-Gesellschaft through a
Sponsored Project Grant (protein synthesis and regulation).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: 5110 East Woodgate
Ln., Tucson, AZ 85712. Tel./Fax: 520-325-7957; E-mail:
alapsubraman@cs.com.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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