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PDBsum entry 2z4d
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Nuclear protein
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PDB id
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2z4d
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References listed in PDB file
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Key reference
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Title
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Proteasome subunit rpn13 is a novel ubiquitin receptor.
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Authors
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K.Husnjak,
S.Elsasser,
N.Zhang,
X.Chen,
L.Randles,
Y.Shi,
K.Hofmann,
K.J.Walters,
D.Finley,
I.Dikic.
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Ref.
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Nature, 2008,
453,
481-488.
[DOI no: ]
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PubMed id
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Abstract
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Proteasomal receptors that recognize ubiquitin chains attached to substrates are
key mediators of selective protein degradation in eukaryotes. Here we report the
identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the
proteasome. Rpn13 binds ubiquitin through a conserved amino-terminal region
termed the pleckstrin-like receptor for ubiquitin (Pru) domain, which binds
K48-linked diubiquitin with an affinity of approximately 90 nM. Like proteasomal
ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like (UBL) domains of
UBL-ubiquitin-associated (UBA) proteins. In yeast, a synthetic phenotype results
when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are
combined, indicating functional linkage between these ubiquitin receptors.
Because Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating
enzyme, our findings suggest a coupling of chain recognition and disassembly at
the proteasome.
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Figure 1.
Figure 1: Murine Rpn13 binds ubiquitin chains. a, mRpn13 cDNA
fragments were cloned into pYTH9 vector in frame with the Gal4
DNA-binding domain. The resulting bait vectors were transformed
into yeast strain Y190 with prey pACT2 vectors containing
wild-type ubiquitin, I44A ubiquitin or hRpn2 (positive binding
control) cDNA in frame with Gal4 DNA-activating domain. b,
Architecture of Rpn13 from various species. The N-terminal
domain is generally conserved (black box) whereas the C-terminal
region (grey box) is absent in S. cerevisiae and has diverged
beyond recognition in one of the two Saccharomyces pombe
proteins (S. pombe (1)). S. pombe (1) = SPCC16A11.16; S. pombe
(2) = SPBC342.04. The percentage identity to the conserved
hRpn13 Pru domain is provided at right. c, Alignment of Rpn13
N-terminal sequences. Residues that are invariant or conserved
in at least 50% of sequences are shaded in black or grey,
respectively. d, To identify the minimal region required for
ubiquitin binding, mRpn13 deletion mutants were expressed as
GST-fused proteins, purified and tested for their binding to
linear tetraubiquitin by immunoblotting with anti-ubiquitin
antibodies. Tetraubiquitin was obtained by thrombin cleavage of
GST-fused tetraubiquitin (GST 4  Ub)
and equivalent amounts of GST-fused deletion mutants were used
in GST pull-down assay.
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Figure 6.
Figure 6: Phenotypic effects of the loss of ubiquitin receptor
function by Rpn13. a, Canavanine sensitivity of single and
double mutants in ubiquitin receptor genes. Cells in late log
phase (top: SY998b, SY980f, SY1004c and SY920b; middle: SY1076,
SY1073a, SY1012a and SY1080a; bottom: SY1076, SY1074a, SY1012a
and SY1082a) were serially diluted and stamped on plates using a
pin array. Plates were incubated at 30 °C for either 2
(left) or 3 (right) days. b, Endogenous ubiquitin conjugate
levels in proteasomal ubiquitin receptor mutants. Cells (SY998a,
SY980a, SY1004a and SY920a) were grown to log phase, and
whole-cell extracts prepared. Proteins were resolved by 4–12%
gradient SDS–PAGE, transferred to polyvinylidene fluoride, and
probed with antibody against ubiquitin. The membrane was
stripped and probed with antibody against eIF5a. c, Substrate
stabilization in rpn13-KKD mutants. Cells (SY992b, SY1004b)
expressing Ub^V76–Val-e^  K–
 -gal
from a GAL promoter were grown to mid-log phase under inducing
conditions. Protein synthesis was quenched at time zero by
adding cycloheximide. Aliquots were withdrawn at the time points
indicated, and lysates prepared. Proteins were visualized by
SDS–PAGE/immunoblot analysis, using an antibody to -galactosidase,
and quantified with imaging software (Kodak EDAS 290). The rate
of degradation of Ub^V76–Val-e^ K–
-gal
was reduced approximately twofold in the rpn13-KKD mutant
compared with wild type. Asterisks indicate distinct -galactosidase-derived
partial breakdown products, whose relative intensities differ
between wild type and mutant. d, rpn13-KKD mutants are not
deficient in proteasome levels. Cells (SY992a, SY1004a) were
grown and lysed as in e. Extract (150  g)
was resolved by native PAGE, and proteasome complexes visualized
using suc–LLVY–AMC (top) and Coomassie blue as a loading
control (middle). The asterisk indicates the expected position
of the proteasome core particle, which is not visualized owing
to low levels. Extracts were also subject to a quantitative
proteasome assay, using suc–LLVY–AMC (bottom). e,
Proteasomes from rpn13-KKD mutants are loaded with Rpn13-KKD
protein. Cells (SY933, SY936, SY950 and SY952) were grown to
late log phase at 30 °C in yeast extract (10 g l^-1),
peptone (20 g l^-1) and dextrose (20 g l^-1) (YPD), harvested
and lysed as described (see Supplementary Information). Extract
(100 g)
was incubated with 1 g
of either GST–Rpn13 (+ lanes) or GST only (samples where
GST–Rpn13 is absent) on ice. Proteasome complexes were
resolved by native PAGE and visualized by suc–LLVY–AMC
overlay assay.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2008,
453,
481-488)
copyright 2008.
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