Biomol NMR Assign
DOI 10.1007/s12104-013-9506-2
ARTICLE
1
H, 13C and 15N resonance assignments of Rpn9, a regulatory
subunit of 26S proteasome from Saccharomyces cerevisiae
Yujie Wu • Yunfei Hu • Changwen Jin
Received: 8 May 2013 / Accepted: 29 June 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract The 26S proteasome is an essential molecular
machine for specific protein degradation in eukaryotic
cells. The 26S proteasome is formed by a central 20S core
particle capped by two 19S regulatory particle (RP) at both
ends. The Rpn9 protein is a non-ATPase subunit located in
the lid complex of the 19S RP, and is identified to be
essential for efficient assembly of yeast 26S proteasome.
Bioinformatics analysis of Saccharomyces cerevisiae Rpn9
suggested it contains a PCI domain at the C-terminal
region. However, high-resolution structures of either the
PCI domain or the full-length Rpn9 still remain elusive.
Herein, we report the chemical shift assignments of 1H, 13C
and 15N atoms of the individual N- and C-domains, as well
as full-length S. cerevisiae Rpn9, which provide the basis
for further structural and functional studies of Rpn9 using
solution NMR technique.
Keywords Proteasome
Rpn9
PCI
NMR

Assignments
Y. Wu
Y. Hu
C. Jin
Beijing Nuclear Magnetic Resonance Center, Peking University,
Beijing 100871, China
Y. Wu
C. Jin
College of Life Sciences, Peking University,
Beijing 100871, China
Y. Hu (&)
C. Jin (&)
College of Chemistry and Molecular Engineering, Peking
University, Beijing 100871, China
e-mail: yunfei@pku.edu.cn
C. Jin
e-mail: changwen@pku.edu.cn
Biological context
The eukaryotic 26S proteasome is a multi-subunit protein
complex which plays an important role in selected protein
degradation in the ubiquitin–proteasome pathway (Glick-
man and Ciechanover 2002). The 26S proteasome holo-
complex contains two types of particles: the cylindrical
core particle (CP) harboring the protease activity (Heine-
meyer et al. 1997), and the regulatory particles (RPs)
binding to both ends of the CP (Peters et al. 1994; Murata
et al. 2009). The central cavity inside the CP is the place
where targeted protein degradation takes place; while the
RPs participate in ubiquitinated proteins recognition,
deubiquitylation, unfolding, and assisting substrates trans-
location into the CP (Glickman et al. 1998).
The RP is composed of two complexes, the base com-
plex and the lid complex (Glickman et al. 1998). Rpn9 is an
important subunit of the lid complex, and is necessary for
the integrity and efficiency of the 26S proteasome
(Takeuchi et al. 1999; Inai and Nishikimi 2002). Rpn9
interacts with the ubiquitin receptor Rpn10 (Takeuchi et al.
1999), and the Drpn9 strain was found to accumulate multi-
ubiquitinated proteins at restrictive temperatures in Sac-
charomyces cerevisiae (Takeuchi et al. 1999). Bioinfor-
matic and biochemical analyses of S. cerevisiae Rpn9
suggested it contains an N-terminal domain (NTD) and a
C-terminal PCI domain. A structure model of the 26S
proteasome has been derived from cryo-EM maps, sug-
gesting that the NTD and PCI domains of Rpn9 may sep-
arately contribute to interactions with the Rpn10 and Rpn5
subunits in the 19S RP (Beck et al. 2012). However, high-
resolution structures of the Rpn9 subunit are still lacking.
Herein, we report the 1H, 13C and 15N resonance assign-
ments of S. cerevisiae Rpn9 NTD and PCI domains sepa-
rately, as well as the full-length protein. Our results provide
123

the basis for further investigations of Rpn9 structures and
interactions using NMR technique.
Methods and experiments
The S. cerevisiae rpn9 gene encoding the Rpn9 protein was
cloned into pET-21a (?) vector with or without a C-ter-
minal His6-tag. The NTD (residues 1–160) of S. cerevisiae
Rpn9 (abbreviated as Rpn9-NTD later on) was cloned into
pET-28a (?) vector. All recombinant proteins were
expressed in E. coli BL21(DE3) strain (Novagen). The
E. coli cells were cultured in 1 L Luria–Bertani (LB) liquid
medium with ampicillin (100 mg/L) or kanamycin (50 mg/
L) at 35 °C. When OD600 reached 0.8, cells were centri-
fuged at 4,000g and re-suspended in 500 ml M9 medium.
Protein expression was induced by adding isopropyl-b-D-
Thiogalactoside (IPTG) to a final concentration of 0.4 mM,
and cells were harvested after 16 h induction at 25 °C.
13 15
C/ N-labeled or 15N-labeled protein was produced using
M9 media containing 15N–NH4Cl (1 g/L) with or without
13
C6 glucose (4 g/L) (Marley et al. 2001). Uniformly 2H,
13
C, 15N-labeled Rpn9 protein was expressed in minimal
M9 culture prepared in D2O with 15N–NH4Cl and 13C6
glucose.
The full-length Rpn9 (with C-terminal His6-tag) and the
Rpn9-NTD (with N-terminal His6-tag) were purified by
affinity chromatography Ni–NTA (nickel nitrilotriacetic
acid agarose) followed by gel filtration (Superdex-75)
using ÄKTA FPLC system (GE Healthcare Life Sciences).
The N-terminal His6-tag of Rpn9-NTD was removed by
thrombin cleavage. Full-length Rpn9 protein without any
fusion tags was purified by ion exchange chromatography
(Q- Sepharose) and gel filtration (Superdex-75). The puri-
ties of the proteins were greater than 95 % identified by
SDS-PAGE.
For preparation of PCI domain of Rpn9 (abbreviated as
Rpn9-PCI later on), full-length Rpn9 protein (with or
without C-terminal His6-tag) was digested by trypsin (at
Rpn9:trypsin mass ratio 100:1) at 25 °C for 2 h. PMSF was
added at a final concentration of 1 mM to quench the
protease activity. Rpn9-PCI was subsequently purified by
gel filtration (Superdex-75). Analysis by mass spectroscopy
and amino acid N-terminal sequencing suggested that the
Rpn9-PCI thus obtained is comprised of residues 181–356.
Purity of the Rpn9-PCI sample was higher than 95 %
identified by SDS-PAGE.
For NMR studies, both the Rpn9-NTD and the full-
length Rpn9 with C-terminal His6-tag samples were pre-
pared in 50 mM sodium phosphate buffer (pH 7.0) with
100 mM NaCl. The Rpn9-PCI samples were prepared in
20 mM Tris buffer (pH 7.5) with 50 mM NaCl, and the
full-length Rpn9 samples without His6-tag were prepared
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Y. Wu et al.
in 50 mM MES buffer (pH 6.7). D2O (10 %) was added
into the NMR samples, and 2,2-dimethyl-2-silapentane-
sulfonic acid (DSS) was added as the internal chemical
shift reference.
The NMR experiments were performed on Bruker
Avance 500, 600 and 800 MHz spectrometers equipped
with cyroprobes. For Rpn9-NTD and Rpn9-PCI, two
dimensional 1H–15N and 1H–13C heteronuclear single-
quantum coherence (HSQC) spectra and three-dimensional
(3D) HNCA, HN(CO)CA, HNCO, HN(CA)CO, HNCACB,
CBCA(CO)NH, 15N-edited TOCSY–HSQC (mixing time
30 ms), HBHA(CO)NH, HCCH–TOCSY, (H)CCH–TOC-
SY, HCCH–COSY, and (H)CCH–COSY (Sattler et al.
1999) were collected for chemical shift assignments. 3D
15
N and 13C edited NOESYHSQC spectra (mixing time
100 ms) were collected to confirm the chemical shift
assignments. The above NMR experiments were performed
at 25 °C.
For full-length Rpn9 backbone chemical shift assign-
ments, TROSY-based 1H–15N HSQC (Pervushin et al.
1997) and triple resonance experiments were collected with
2
H/13C/15N-labeled samples, including TROSY-based
HNCA, HN(CO)CA, HN(CO)CACB, HNCACB, HNCO,
and HN(CA)CO (Salzmann et al. 1998). HCCH–TOCSY,
(H)CCH–TOCSY, HCCH–COSY, and (H)CCH–COSY
were collected with 13C/15N-labeled protein samples for
side-chain assignments. Since the C-terminal tail of Rpn9
was prone to degradation, a 13C/15N-labeled Rpn9 sample
with C-terminal His6-tag was prepared and conventional
HNCACB and CBCA(CO)NH experiments were collected
to confirm the chemical shift assignments for this region
(residues 367–393). The above NMR experiments were
performed at 30 °C.
All spectra were processed by NMRPipe (Delaglio et al.
1995) and analyzed using CARA (Keller 2004) and
NMRView (Johnson and Blevins 1994).
Assignments and data deposition
The 2D 1H–15N HSQC spectrum of S. cerevisiae Rpn9-
NTD is shown in Fig. 1. Rpn9-NTD contains 160 amino
acid residues and has a molecular weight of 18.9 kDa. The
backbone chemical shift assignments were obtained for
154 out of 160 residues. Besides three prolines, the unas-
signed residues include Asn116, His122 and Glu154. More
than 90 % of the backbone and side-chain chemical shift
assignments were obtained.
The 2D 1H–15N HSQC spectrum of Rpn9-PCI is shown
in Fig. 2. Rpn9-PCI contains 176 amino acid residues and
has a molecular weight of 20.2 kDa. The backbone
chemical shift assignments were obtained for 166 out of
176 residues. Besides five prolines, the unassigned residues
1 13 15
H, C and N resonance assignments of Rpn9

Fig. 1 2D 1H–15N HSQC

spectrum of Rpn9-NTD
annotated with the backbone
assignments. The assignments
are labeled with the one-letter
amino acid code and residue
number. The side chain NH2
peaks of Asn and Gln are
connected by horizontal lines.
Folded peaks are colored by red

Fig. 2 2D 1H–15N HSQC

spectrum of Rpn9-PCI
annotated with the backbone
assignments. The assignments
are labeled with the one-letter
amino acid code and residue
number. The side chain NH2
peaks of Asn and Gln are
connected by horizontal lines.
Folded peaks are colored by red

123

Fig. 3 2D TROSY-based 1H–15N HSQC spectrum of full-length Rpn9 (with C-terminal His6-tag) annotated with the backbone assignments. The
assignments are labeled with the one-letter amino acid code and residue number. Folded peaks are colored by red
include Phe181–Asp184 and Ser246. More than 90 % of
the backbone and side-chain chemical shift assignments
were obtained.
The 2D TROSY based 1H–15N HSQC spectrum of full-
length Rpn9 with C-terminal His6-tag is shown in Fig. 3.
Rpn9 contains 393 amino acid residues and has a molecular
weight of 45.8 kDa. The backbone chemical shift assign-
ments were obtained for 370 out of 393 residues. Besides
nine prolines, the unassigned residues include Met1, Phe2,
Asn116, Asp121, His122, Asp148 and Thr364–Val371.
About 70 % of the backbone and side-chain chemical shift
assignments were obtained. The chemical shifts observed
for residues in the isolated domains did not show much
difference from the full-length protein, suggesting that the
two domains do not interact with each other and adopt
similar conformations in either state. The only regions that
showed relatively large chemical shift difference are resi-
dues N117–K160 at the C-terminal of Rpn9-NTD and
residues F185–S204 at the N-terminal of Rpn9-PCI. These
123
Y. Wu et al.
segments correspond to the region linking NTD and PCI
domains, and may have different conformations in the
isolated domains and the full-length protein.
The chemical shift assignments of the full-length Rpn9
and the Rpn9-NTD, Rpn9-PCI domains and have been
deposited in the BioMagResBank (http://www.bmrb.wisc.
edu/) under the accession numbers 19219, 19220 and
19221, respectively.
Acknowledgments All NMR experiments were carried out at the
Beijing NMR Center. This research was supported by Grant
2006AA02A323 from the Ministry of Science and Technology of
China to C. J.
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