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PDBsum entry 1trj
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Signaling protein
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PDB id
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1trj
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References listed in PDB file
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Key reference
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Title
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Identification of the versatile scaffold protein rack1 on the eukaryotic ribosome by cryo-Em.
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Authors
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J.Sengupta,
J.Nilsson,
R.Gursky,
C.M.Spahn,
P.Nissen,
J.Frank.
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Ref.
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Nat Struct Mol Biol, 2004,
11,
957-962.
[DOI no: ]
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PubMed id
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Abstract
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RACK1 serves as a scaffold protein for a wide range of kinases and
membrane-bound receptors. It is a WD-repeat family protein and is predicted to
have a beta-propeller architecture with seven blades like a Gbeta protein. Mass
spectrometry studies have identified its association with the small subunit of
eukaryotic ribosomes and, most recently, it has been shown to regulate
initiation by recruiting protein kinase C to the 40S subunit. Here we present
the results of a cryo-EM study of the 80S ribosome that positively locate RACK1
on the head region of the 40S subunit, in the immediate vicinity of the mRNA
exit channel. One face of RACK1 exposes the WD-repeats as a platform for
interactions with kinases and receptors. Using this platform, RACK1 can recruit
other proteins to the ribosome.
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Figure 3.
Figure 3. Ribosomal binding site for RACK1. (a) View of the
anchoring region of RACK1 (red) showing interactions with the
18S rRNA helices 39 and 40. (b) View of RACK1 and protein
neighbors, rpS3 (S3p), rpS5 (S7p), rpS16 (S9p) and rpS20 (S10p)
from the 40S subunit head region (corresponding ribosomal
proteins in E. coli are indicated in parentheses). Thumbnails to
the right of each panel represent 40S subunit structure in the
corresponding views to aid orientation. Atomic coordinates for
small subunit proteins and 18S rRNA were taken from PDB entry
1S1H23, and all representations were made using Ribbons44.
Landmarks of the 40S subunit: bk, beak; pt, platform.
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Figure 5.
Figure 5. Src interaction site on the ribosome. (a) The
approximate position of Src-interacting residues on the 80S
ribosome relative to the mRNA path (red) and P-site tRNA
(green). (b) Ribbon representation of the RACK1 homology model
in the same orientation as in a with the two Src-interacting
residues indicated.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Struct Mol Biol
(2004,
11,
957-962)
copyright 2004.
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Secondary reference #1
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Title
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Release of eif6 (p27bbp) from the 60s subunit allows 80s ribosome assembly.
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Authors
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M.Ceci,
C.Gaviraghi,
C.Gorrini,
L.A.Sala,
N.Offenhäuser,
P.C.Marchisio,
S.Biffo.
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Ref.
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Nature, 2003,
426,
579-584.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1: Endogenous RACK1 is found on ribosomes and binds to
eIF6 in the cytoplasm. a, Interaction between endogenous
proteins: A431 lysates immunoprecipitated with anti-eIF6 or
preimmune sera were immunoblotted with anti-RACK1 (top panel)
and anti-eIF6 (bottom panel). b, MBP -RACK1 pulls down
endogenous eIF6. MBP -RACK1 and MBP affinity purification
followed by immunoblotting for eIF6. c, Direct interaction
between RACK1 and eIF6 by far-western blotting. Immobilized
RACK1 revealed with anti-RACK1 (positive control) or
biotinylated eIF6 or streptavidin (negative control). *,
degradation product. d, e, Endogenous RACK1 is present in the
cytoplasm by immunofluorescence (d) and on ribosomes when
detected by immunoblotting of fractions collected by sucrose
density gradient (e). 1,2, soluble; 4, 40S peak; 9 -13,
polysomes. Lower panel, eIF6 (transfected + endogenous). Bar, 5
µm.
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Figure 5.
Figure 5: eIF6 anti-association activity in vitro is modulated
by RACK1 -PKC beta- II.
0.7 pmol 80S was used per experiment. Four fractions were
assayed: soluble, 40S, 60S and 80S. a, b, Salt-washed ribosomal
subunits remain dissociated in 1 mM Mg2+ (a) but associate after
raising the concentration to 5 mM Mg2+, in the absence of eIF6
(b, left panel). The right panel of b shows endogenous proteins
in salt-washed ribosomes. c, Association of ribosomal subunits
in 5 mM Mg2+ is inhibited by eIF6 (top), which associates with
the free 60S fraction (lower panel). d, e, Addition of activated
PKC  II
(d) or RACK1 -PKC II
(e) results in release of eIF6 from the 60S subunit (lower
panel, soluble fraction) and 80S joining (top). f, g, S235A eIF6
inhibits ribosomal joining in the presence of RACK1 -PKC II.
The blots also show RACK1 (see main text for details).
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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Secondary reference #2
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Title
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Cpc2/rack1 is a ribosome-Associated protein that promotes efficient translation in schizosaccharomyces pombe.
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Authors
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B.Shor,
J.Calaycay,
J.Rushbrook,
M.Mcleod.
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Ref.
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J Biol Chem, 2003,
278,
49119-49128.
[DOI no: ]
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PubMed id
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Figure 2.
FIG. 2. Distribution of Cpc2H6F in sucrose gradient
fractionations of wild type cell extracts. A, cells containing
the Cpc2H6F fusion protein were grown at 30 °C to 1 x 10^7
cells/ml prior to the preparation of cell lysates. Cycloheximide
(100 µg/ml) was included in the extraction buffers and
sucrose gradients. Supernatants from 14,000 rpm centrifugations
of the cell lysates were fractionated on 7-47% sucrose
gradients, and absorbance at 260 nm was recorded to locate
ribosome subunits, 80 S couples, and polysomes. Western blots of
Cpc2H6F and ribosomal protein L7 are aligned below the traces to
indicate their distribution in the sucrose gradient. B, wild
type cell extracts containing the Cpc2H6F fusion protein were
prepared as described above. In addition to cycloheximide, EDTA
(40 mM), NaCl (0.7 M), or RNase A (0.5 mg/ml) were added as
indicated. Supernatants were fractionated on 5-25% sucrose
gradients.
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Figure 3.
FIG. 3. Cpc2 is localized in the cytoplasm and excluded
from the nucleus. A montage of cells containing the Cpc2-EGFP
fusion protein during various stages of the yeast life cycle.
The arrows indicate conjugating cell pairs. Images were captured
using fluorescence microscopy (A) or confocal laser imaging (B).
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #3
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Title
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Asc1p, A wd40-Domain containing adaptor protein, Is required for the interaction of the RNA-Binding protein scp160p with polysomes.
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Authors
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S.Baum,
M.Bittins,
S.Frey,
M.Seedorf.
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Ref.
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Biochem J, 2004,
380,
823-830.
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PubMed id
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Headers
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