PDBsum entry 2z4d

Nuclear protein

PDB id: 2z4d

Contents

Protein chain

96 a.a. *

* Residue conservation analysis

PDB id:

2z4d

Name:

Nuclear protein

Title:

Nmr structures of yeast proteasome component rpn13

Structure:

26s proteasome regulatory subunit rpn13. Chain: a. Fragment: residues 6-101. Synonym: proteasome component rpn13, proteasome non-atpase subunit 13. Engineered: yes

Source:

Saccharomyces cerevisiae. Yeast. Organism_taxid: 4932. Gene: rpn13. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

8 models

Authors:

N.Zhang,K.J.Walters

Key ref:

K.Husnjak et al. (2008). Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature, 453, 481-488. PubMed id: 18497817 DOI: 10.1038/nature06926

Date:

15-Jun-07 Release date: 20-May-08

Protein chain

O13563  (RPN13_YEAST) -  26S proteasome regulatory subunit RPN13 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 156 a.a.

Struc: 96 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1038/nature06926

Nature 453:481-488 (2008)

PubMed id: 18497817

Proteasome subunit Rpn13 is a novel ubiquitin receptor.

K.Husnjak, S.Elsasser, N.Zhang, X.Chen, L.Randles, Y.Shi, K.Hofmann, K.J.Walters, D.Finley, I.Dikic.

ABSTRACT

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.

Selected figure(s)

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.

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.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 453, 481-488) copyright 2008.

Figures were selected by an automated process.