Báo cáo khoa học: Analysis of the RNA degradosome complex in Vibrio angustum S14 potx

Analysis of the RNA degradosome complex in
Vibrio angustum S14
Melissa A. Erce, Jason K. K. Low and Marc R. Wilkins
Systems Biology Laboratory, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
Introduction
Post-transcriptional control of gene expression is an
important regulatory mechanism, as the length of time
that a transcript is available for translation limits the
expression of its protein product. Messenger RNA
(mRNA) half-lives can differ by as much as two orders
of magnitude within a single cell. In Escherichia coli,
where the average message has a half-life of about
5 min, individual mRNA half-lives can be as short as
several seconds or as long as 1 h [1]. Through the reg-
ulation of mRNA stability, patterns of protein synthe-
sis in the cell can be modulated in response to changes
in growth conditions [2]. In addition, selective mRNA
decay results in the differential expression of gene
products in some polycistronic mRNAs [3]. The multi-
Keywords
degradosome; microdomains; RhlB;
RNase E; two-dimensional Blue
Native-SDS ⁄ PAGE
Correspondence
M. Wilkins, School of Biotechnology and
Biomolecular Sciences, University of New
South Wales, Sydney, NSW 2052, Australia
Fax: +61 2 9385 1483
Tel: +61 2 9385 3633
E-mail:
(Received 28 June 2010, revised 28

September 2010, accepted 22 October
2010)
doi:10.1111/j.1742-4658.2010.07934.x
The RNA degradosome is built on the C-terminal half of ribonuclease E
(RNase E) which shows high sequence variation, even amongst closely
related species. This is intriguing given its central role in RNA processing
and mRNA decay. Previously, we have identified RhlB (ATP-dependent
DEAD-box RNA helicase)-binding, PNPase (polynucleotide phosphory-
lase)-binding and enolase-binding microdomains in the C-terminal half of
Vibrio angustum S14 RNase E, and have shown through two-hybrid analy-
sis that the PNPase and enolase-binding microdomains have protein-bind-
ing function. We suggest that the RhlB-binding, enolase-binding and
PNPase-binding microdomains may be interchangeable between Escherichi-
a coli and V. angustum S14 RNase E. In this study, we used two-hybrid
techniques to show that the putative RhlB-binding microdomain can bind
RhlB. We then used Blue Native-PAGE, a technique commonly employed
in the separation of membrane protein complexes, in a study of the first of
its kind to purify and analyse the RNA degradosome. We showed that the
V. angustum S14 RNA degradosome comprises at least RNase E, RhlB,
enolase and PNPase. Based on the results obtained from sequence analyses,
two-hybrid assays, immunoprecipitation experiments and Blue Native-
PAGE separation, we present a model for the V. angustum S14 RNA
degradosome. We discuss the benefits of using Blue Native-PAGE as a tool
to analyse the RNA degradosome, and the implications of microdomain-
mediated RNase E interaction specificity.
Structured digital abstract
l
A list of the large number of protein–protein interactions described in this article is available
via the MINT article ID
MINT-8049250

Abbreviations
BACTH, bacterial adenylate cyclase two-hybrid; CTH, C-terminal half; DMP, dimethyl pimelimidate.2HCl; NTH, N-terminal half;
PNPase, polynucleotide phosphorylase; PVDF, poly(vinylidene difluoride); RhlB, ATP-dependent DEAD-box RNA helicase; RNase E,
ribonuclease E.
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5161
enzyme RNA degradosome is involved in the steady-
state regulation of transcripts in E. coli. Apart from its
role in mRNA degradation, the degradosome is also
responsible for the processing of 5S ribosomal RNA
and tRNAs, as well as the degradation of tmRNAs [4–8].
The principal proteins comprising the degradosome
include the 3¢ to 5¢ exoribonuclease polynucleotide
phosphorylase (PNPase), the ATP-dependent DEAD-
box RNA helicase (RhlB) and the glycolytic enzyme
enolase [9–12]. The scaffolding for the degradosome is
provided by RNase E. One of the largest proteins
found in E. coli, RNase E, is 1061 amino acids in
length [10,13,14]. It is defined by two functionally
distinct domains of approximately equal size – the
N-terminal half (NTH, residues 1–498) and the
C-terminal half (CTH, residues 499–1061) [15,16].
The catalytic activity of E. coli RNase E resides within
its globular NTH. The sequences within this essential
region are highly conserved amongst eubacteria [17].
In contrast, the CTH lacks sequence conservation, has
little structural character, has no known catalytic func-
tion [17,18] but provides the scaffolding for the recruit-
ment of the degradosome through short recognition
motifs [19]. The RNase E CTH contains two regions
which can bind RNA and interact with substrates such

as the 9S precursor for 5S ribosomal RNA [18].
Apart from the canonical degradosome components,
polyphosphate kinase, poly(A) polymerase, ribosomal
proteins and chaperone proteins GroEL and DnaK
have been found to be present in the degradosome,
but in substoichiometric amounts [11,20,21].
The molecular interactions of RNase E with other
proteins of the RNA degradosome are proposed to be
mediated by short microdomains of 15–40 amino acids
on RNase E [22]. These microdomains exhibit higher
amino acid sequence conservation than the rest of the
RNase E CTH. It is through these regions that specific
molecular interactions are believed to occur to direct
RNase E function, and hence confer adaptive reorgani-
zation of protein complex formation [17,22–25].
In order to study RNase E and its interaction part-
ners, it would be useful if it could be isolated. The
purification and characterization of RNase E have pro-
ven challenging [15], and obtaining an amount suffi-
cient for analysis is even more difficult. Methods for
its purification as part of the RNA degradosome, as
well as the reconstitution of an active degradosome
through the purification of its individual components,
have been developed [11,26–29]. Large-scale prepara-
tions have involved purification under denaturing con-
ditions; others have involved the overexpression of
individual components and subsequent reconstitution
of the degradosome. Recently, a method for the
preparation of a recombinant degradosome has been
described [30]. Co-immunoprecipitation methods have

also been used to study the RNA degradosome. This
technique is especially useful when studying bacteria in
which the introduction of genetic tags is difficult [31].
These methods, however, require a large amount of
starting material. Furthermore, as they often require
the overexpression and ⁄ or tagging of the RNA
degradosome components, the complexes formed may
not truly represent what is occurring in the cell. In
view of these limitations, we decided to use Blue-
Native PAGE (BN-PAGE), a ‘charge shift’ technique
employed in the separation of mitochondrial mem-
brane proteins and complexes [32,33]. This technique
has been successfully employed recently to study the
E. coli complexome [34]. It requires far less starting
material and, when used in conjunction with immuno-
blotting and ⁄ or mass spectrometric analysis, may
provide a better view of the dynamic nature of the
RNA degradosome.
In this study, we characterized the RNA degradosome
from an environmental species: the marine heterotro-
phic, Gram-negative bacterium, Vibrio angustum S14
[35]. V. angustum S14 is a model organism for the
study of starvation, as it exhibits remarkable physio-
logical changes and increased mRNA stability during
carbon starvation [36,37]. We have demonstrated pre-
viously, using two-hybrid screening, that RNase E
from V. angustum S14 contains sites for interaction
with enolase and PNPase [38]. Here, we use a combi-
nation of proteomic techniques, such as BN-PAGE,
co-immunoprecipitation and tandem MS to identify

the components of the RNA degradosome in this
organism. We also show, using two-hybrid analysis,
that the CTH of RNase E from V. angustum S14 inter-
acts with RhlB.
Results
RhlB binds to V. angustum S14 RNase E and
PNPase
Previously, we predicted that the RNase E CTH from
V. angustum S14 possessed interaction sites for PNPase
and enolase, and demonstrated these interactions
through two-hybrid analysis [38]. We also predicted
that RhlB should bind to RNase E at residues 719–
753. Here, we undertook two-hybrid analysis to test
this; the results of this analysis and our previous analy-
sis [38] are shown in Table 1. This two-hybrid system
introduces two proteins of interest fused to the T18
and T25 domains of Bordetella pertussis adenylate
cyclase into E. coli cya
)
on plasmids. When physically
RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al.
5162 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS
Table 1. Escherichia coli two-hybrid analysis results. Incubations were at 25 °C. The horizontal axis indicates the protein fused with the T18 domain and the vertical axis indicates the pro-
tein fused with the T25 domain. Empty cells indicate crosses not carried out in this study. ‘++’ denotes strong interactions, ‘+’ denotes moderate interactions and ‘)’ denotes weak ⁄ no
interactions.
pKT25
pUT18 ⁄ pUT18C
1 2 3 4 5 6 7 8 9 10111213
E. coli
RhlBsite

684–784
V. S14
RhlBsite
714–758
V. S14
CTH
526–1094
E. coli
PNPsite
844–1061
V. S14
PNPsite
1015–1094
E. coli
Enosite
833–851
V. S14
Enosite
885–909
E. coli
PNPase
V. S14
PNPase
E. coli
RhlB
V. S14
RhlB
E. coli
enolase
V. S14

enolase
T18
a
T18C
b
T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C T18 T18C
A E. coli
RhlBsite
)) )) ++ ++ ++ + )) ))
B V. S14
RhlBsite
)) )) ++ + ++ ++ )) ))
C V. S14
CTH
+
c
++
c
)
c
)
c
)
c
+
c
++ ++ ++ ++ )
c
+
c

++
c
++
c
D E. coli
PNPsite
)
c
+
c
)
c
)
c
)) ))
E V. S14
PNPsite
)
c
)
c
)
c
+
c
)) )) )
c
)
c
)

c
)
c
F E. coli
Enosite
)
c
)
c
)
c
)
c
)) )) +
c
+
c
+
c
+
c
G V. S14
Enosite
)
c
)
c
)
c
)

c
)) )) +
c
++
c
+
c
+
c
H E. coli
PNPase
)) )) +
c
)
c
++
c
)
c
+
c
)
c
)
c
)
c
)
c
)

c
)
c
++
c
)
c
++
c
++++)
I V. S14
PNPase
)) )) ++
c
)
c
+
c
)
c
+
c
)
c
)
c
)
c
)
c

)
c
)
c
++
c
)
c
++
c
+ ) + )
J E. coli
RhlB
++ ++ ) ++ ++ ++ )) )) )) )) +++)) ++ ++ ++ +
K V. S14
RhlB
+ ))++ ++ ++ ) ) )) )) )) )) )) )+ ))
L E. coli
enolase
)) )) ++
c
)
c
)) )
c
)
c
)
c
)

c
+
c
+
c
++
c
++
c
++
c
+
c
M V. S14
enolase
)) )) ++
c
++
c
)) )
c
)
c
+
c
+
c
++
c
+

c
++
c
++
c
++
c
++
c
a
Two-hybrid result between bait fused with T25 on its N-terminus and prey fused with T18 on its C-terminus (pUT18).
b
Two-hybrid result between bait fused with T25 on its N-terminus
and prey fused with T18 on its N-terminus (pUT18C).
c
Two-hybrid result that has been published previously [38].
M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5163
separated, the T18 and T25 domains are inactive, but
the interaction of the hybrid proteins results in the
functional complementation of adenylate cyclase in
E. coli cya
)
and the subsequent expression of the lac
operon [39].
RhlB demonstrated positive interactions with the
CTH of V. angustum S14 RNase E (grids 3K and
11C). In order to better define the region of interac-
tion of RNase E with RhlB, we tested the putative
RhlB-binding domain of V. angustum S14 RNase E

(residues 719–753, plus five amino acid residues flank-
ing on either side); it was found to interact with RhlB
(grid 2K and its reciprocal cross in 11B). Interest-
ingly, this region was also capable of interacting with
E. coli RhlB (grid 2J and its reciprocal cross in 10B).
It should be noted, however, that, when the
T18 domain was fused to the C-terminus of the
V. angustum S14 RNase E RhlB-binding site (residues
714–758), the interaction with RhlB was negative,
probably because of a difference in conformation
(grid 2K) or because the T18 domain occluded the
RhlB-binding site. Previous reports have suggested
that RhlB and PNPase can interact independently of
RNase E in E. coli [16]. Our two-hybrid analysis fur-
ther confirmed this (grid 8J and its reciprocal cross in
10H). We also observed an interaction between
V. angustum S14 PNPase and RhlB; however, this was
weaker than that seen in E. coli (grid 11I, C-terminal
fusion of the T18 domain). We did not expect PNPase
to interact with the RhlB-binding site, and we
observed negative interactions for this (grids 1H, 1I,
2H, 2I, 8A, 8B, 9A and 9B). Negative interactions
were also seen between RhlB and the PNPase-binding
site of RNase E (grids 4J, 4K, 5J, 5K, 10D, 10E, 11D
and 11E), which was expected. These, together with
the negative interaction of PNPase with the RhlB-
binding site, served as negative controls for the exper-
iment (grids 1H, 1I, 2H, 2I, 8A, 8B, 9A and 9B).
A series of cross-species’ interactions was also tested
here. We observed strong interactions for E. coli RhlB

and the CTH of V. angustum S14 RNase E (grids 3J
and 10C). Weaker interactions were seen for other
cross-species’ crosses involving RhlB and the RhlB-
binding site (grids 1K, 2J, 10B and 11A). Further, we
observed the self-interaction of E. coli RhlB (grid 10J),
suggesting that this protein can self-interact [40].
PNPase and RhlB copurify with V. angustum S14
RNase E
Having shown that PNPase and RhlB can interact
with RNase E microdomains as well as the RNase E
CTH and with each other in a two-hybrid assay, we
investigated whether they interacted in vivo. First, we
determined whether V. angustum S14 proteins can be
detected by antisera against E. coli RNase E, PNPase
and RhlB (Fig. 1A). Following that, we then carried
out several immunoprecipitation experiments to deter-
mine whether V. angustum S14 RNase E forms a
complex with V. angustum S14 PNPase and RhlB,
and to determine whether other possible interaction
partners were present. V. angustum S14 PNPase and
RhlB were found to coprecipitate with RNase E when
antiserum against RNase E was used for immuno-
precipitation (Fig. 1B). Similarly, when antiserum
against PNPase was used for immunoprecipitation,
V. angustum S14 RhlB and RNase E were found to
associate with V. angustum S14 PNPase (Fig. 1C). As
enolase comigrated in the same region in the gel as
RNase E
PNPase
RhlB

188
98
62
49
MW
(kDa)
RhlB
RNase E
PNPase
250
148
64
50
MW
(kDa)
RNase E
PNPase
IgG
RhlB
250
148
64
50
Mw
(kDa)
A
B
C
Fig. 1. Immunoprecipitation of RNase E and PNPase in V. angu-
stum S14 by antisera against E. coli RNase E and PNPase. (A)

V. angustum S14 lysate probed with antisera against RNase E,
PNPase and RhlB. (B) Immunoprecipitation using RNase E antise-
rum. The RNase E antiserum was incubated with V. angustum S14
lysate and then isolated by protein G-conjugated Sepharose. The
eluted fraction was separated by SDS ⁄ PAGE and analysed by
immunoblotting using a series of antisera. PNPase and RhlB were
found to copurify with V. angustum S14 RNase E. The antibody
heavy chain is indicated. Composite image obtained when the
PVDF membrane was probed serially with antisera against RNa-
se E, PNPase and RhlB. (C) Immunoprecipitation using PNPase
antiserum. Antiserum against PNPase was incubated with V. angu-
stum S14 lysate and then isolated by protein G-conjugated Sepha-
rose. Following SDS ⁄ PAGE separation, the immunoprecipitates
were analysed by western blot and probed in a serial fashion with
antisera against RNase E, PNPase and RhlB. RNase E and RhlB
were found to copurify with PNPase.
RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al.
5164 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS
the IgG heavy chain, any signal from probing with
enolase antiserum was masked by IgG at that posi-
tion in the gel.
Identification of the RNA degradosome in
V. angustum S14 using BN-PAGE
Previously, we predicted that V. angustum S14 RNa-
se E contains interaction sites for RhlB, PNPase and
enolase [38]. Using two-hybrid analysis here and in a
previous study, we demonstrated the interactions of
V. angustum S14 RNase E microdomains and the
CTH of RNase E with RhlB, enolase and PNPase.
From the results of our immunoprecipitation experi-

ments, we found that RhlB and PNPase associate with
RNase E in V. angustum S14. Together, these results
strongly suggest, but do not prove, that all of these
proteins form a degradosome complex. To ascertain
that these proteins all co-associate into a degrado-
some-like complex in V. angustum S14, we separated
the cell lysate using BN-PAGE (Fig . 2A). This
revealed a range of protein complexes of various sizes
up to 1.2 MDa. To identify the RNA degradosome
amongst all these complexes, the proteins were
transferred onto a poly(vinylidene difluoride) (PVDF)
membrane and probed with antisera against RNase E,
PNPase and RhlB (Fig. 2B). On probing with
RNase E-specific antibodies, RNase E was detected in
two high-molecular-mass bands above 1 MDa and in
bands corresponding to approximately 480 and
650 kDa. The latter is consistent with RNase E’s ho-
motetrameric state (Fig. 2B). It should be noted that
the true molecular mass of RNase E is 116 kDa, but
its apparent molecular mass is approximately 180 kDa
[41]. PNPase was observed in the same two high-
molecular-mass bands (above 1 MDa) as detected by
antiserum against E. coli RNase E. PNPase was also
observed at approximately 750 kDa (a potential trimer
of trimers), as well as in a band running at approxi-
mately 240 kDa, which is consistent with its trimeric
form (Fig. 2B). The presence of RhlB was detected in
the same two high-molecular-mass complexes above
1 MDa, which also contained RNase E and PNPase
(Fig. 2B), but was not seen below 1 MDa. The detec-

tion of lower mass bands containing RhlB may have
been reduced as the proteins were in their native rather
than denatured state, making the epitopes more
difficult to recognize by the antiserum. It is interesting
to note that RNase E, PNPase and RhlB are part of
the high-molecular-mass complexes, but did not
co-associate in other lower mass heteromultimers.
Probing with antiserum against enolase proved to be
uninformative as enolase is present in high abundance
in the cell and comigrates with other protein complexes.
Antibodies were not available for the identification
of other possible interactor proteins; we therefore used
MS to analyse Bands A and B (Fig. 2A). This verified
the presence of enolase and PNPase, but not RNase E
(Table 2). This was not unexpected because of the low
abundance of RNase E in the cell. The amino acid
sequence of RNase E is arginine-rich, giving rise to
peptides that are either too short or too long for frag-
mentation when subjected to digestion by trypsin. This
may have occluded the identification of RNase E in
the mass spectrometric analysis of Bands A and B.
However, RNase E was detected by MS of immuno-
precipitates. Ribosomal protein L4 was detected in
Bands A and B, together with GroEL, a large number
of ribosomal proteins and a high abundance of meta-
bolic proteins (data not shown). It is unclear whether
these ribosomal proteins are truly complexed with the
RNA degradosome in V. angustum S14 or whether
parts of the ribosome or, indeed, other protein com-
plexes co-electrophorese with the degradosome; this is

likely when one considers the abundance of these pro-
teins within the cell and the large complexes which
comprise the ribosome. The proteins identified, which
have been described to associate with the RNA
MW
(kDa)
1236
1048
1236
MW
(kDa)
RNase E
PNPase
RhlB
Band A
Band B
720
1048
480
720
242
480
146
66
242
A
B
Fig. 2. Separation of protein complexes through BN-PAGE. (A)
Analytical analysis of protein complexes in V. angustum S14.
V. angustum S14 lysate (17 lg) was separated by BN-PAGE and

silver stained. (B) Western blot of V. angustum S14 RNA degrado-
some components. V. angustum S14 lysate (200 lg) was sepa-
rated through BN-PAGE and electroblotted onto a PVDF
membrane. Bands were detected by probing serially with antisera
against RNase E, PNPase and RhlB. Molecular mass markers are
shown in kDa.
M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5165
degradosome in E. coli, are shown in Table 2. Only
one peptide was identified for PNPase and ribosomal
protein L4, but these have been found previously to be
associated with RNase E [42]. Fragmentation spectra
for both of these showed a strong series of y-ions and
highly significant identification scores (Fig. 3).
Two-dimensional BN-SDS
⁄
PAGE analysis
To better understand the subunit composition of the
degradosome protein complex, we used two-dimen-
sional BN-SDS ⁄ PAGE. In this technique, protein com-
plexes can be separated into subunits in an
SDS ⁄ PAGE second dimension, following first dimen-
sion BN-PAGE. We excised six bands from the first
dimension BN-PAGE separation (Fig. 4A), and ran
these bands separately in a second dimension reducing
SDS ⁄ PAGE to separate their constituent proteins
according to size (Fig. 4B). As a control, cleared lysate
was loaded (Fig. 4B, lane CL). It can be seen that the
composition of each of the bands excised from the
BN-PAGE separation varied (Fig. 4B, lanes 1–6) and

was different from the control. It was evident that each
band contained proteins and complexes apart from the
RNA degradosome. This is to be expected as BN-
PAGE was separating a whole-cell lysate. After second
dimension SDS ⁄ PAGE separation, the proteins were
electroblotted onto a PVDF membrane and serially
probed with antisera against RNase E, PNPase, RhlB
and enolase (Fig. 4C). We found that all BN-PAGE
bands excised between 900 kDa and 1.2 MDa
contained RNase E (Fig. 4C, lanes 3–5). However,
RNase E was not present in Band 6. In all instances in
which RNase E was present, PNPase, RhlB and eno-
lase were also present, but in varying quantities.
Although the antisera used have different affinities for
their target protein, Band 4 showed more RNase E
and PNPase than RhlB and enolase. By contrast,
Bands 1, 2, 3 and 5 showed roughly equivalent
amounts of each of these proteins in the four samples.
Interestingly, Band 6, which corresponded to a band
excised in the vicinity of the 380-kDa region of the
first dimension BN-PAGE separation, only contained
PNPase, enolase and RhlB. There was no full-length
RNase E. However, there was evidence of a band
migrating at approximately 80 kDa, which migrated
above PNPase. This corresponds to a fragment of
RNase E which is seen in one-dimensional SDS ⁄ PAGE
analyses and immunoblots of whole-cell lysate of
V. angustum S14 RNase E (Figs 1B and 4D), and
which has been observed previously to migrate in this
region [18]. This suggests that RhlB, enolase and

PNPase may be found together in association with a
fragment of RNase E containing its CTH. This would
be consistent with the known positions of the microdo-
mains in RNase E. Although more unlikely, we cannot
rule out that, if this fragment of RNase E is its NTH,
and not its CTH, RhlB, enolase and PNPase may be
able to form an association independent of RNase E.
Discussion
Through sequence analysis, we have shown previously
that the CTH of V. angustum S14 RNase E contains
binding sites for RhlB (residues 714–758), enolase (resi-
dues 885–909) and PNPase (residues 1015–1094). We
Table 2. Identification of V. angustum S14 RNase E-associated proteins through mass spectrometric analysis.
V. angustum S14
protein
Mass
(Da) Score Peptide Position
Individual
peptide score
a
Band A
Enolase (Q1ZNA5)
b
45920 123 LNQIGSLTETLAAIK 345–359 90
SGETEDATIADLAVGTAAGQIK 374–395 53
GroEL (Q1ZKN3)
b
57302 72 NAGDEESVVANNVK 457–470 20
EVASQANDAAGDGTTTATVLAQAIIAEGLK 76–107 70
50S Ribosomal protein

L4 (Q1ZJA7)
b
21707 63 LIVVDNFALEAPK 117–129 63
Band B
Enolase (Q1ZNA5)
b
45920 106 GITNSILIK 336–344 54
LNQIGSLTETLAAIK 345–359 76
PNPase (Q1ZJW2)
b
76671 31 IAELAEAK 245–251 31
50S Ribosomal protein 21707 116 SILSELVR 106–113 30
L4 (Q1ZJA7)
b
AIDPVSLIAFDK 173–184 75
GADALTVSETTFGR 7–20 61
Immunoprecipitation
RNase E (Q1ZS71)
b
122521 54 AALSTLDLPQGMGLIVRT 153–169 54
a
For this analysis, peptides with scores > 10 are considered to be statistically significant.
b
Swiss-Prot accession number.
RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al.
5166 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS
have also shown, using two-hybrid analysis, that
the enolase- and PNPase-binding sites are capable of
interacting with enolase and PNPase [38]. Here, we
report that the putative V. angustum S14 RNase E

RhlB-binding site (residues 714–758) interacts with
RhlB. Further, results from our BN-PAGE analysis
and immunoprecipitation experiments confirmed that
RNase E complexes with RhlB, enolase and PNPase in
V. angustum S14. V. angustum S14 PNPase exists as a
trimer and, through two-hybrid experiments, we
showed that enolase and PNPase in V. angustum S14
can self-associate. Based on our observations, we
present a model for the RNA degradosome complex in
V. angustum S14 which shows the positions of the
microdomains in the CTH of RNase E, the proteins
that bind to them, as well as their dimerization state
(Fig. 5). We have predicted previously that there
are five regions of increased structural order in the
V. angustum S14 RNase E CTH: the first corresponds
to Segment A, which is involved in membrane binding
(residues 565–584); the second is a putative RNA-bind-
ing domain (residues 604–661); the third has a function
# b Seq y #
1 114.0913 L13
2 227.1754 I 1315.7256 12
3 326.2438 V 1202.6416 11
4 425.3122 V 110.3.5732 10
5 540.3392 D 1004.5047 9
6 654.3821 N 889.4778 8
7 801.4505 F 775.4349 7
8 872.4876 A 628.3664 6
9 985.5717 L 557.3293 5
10 1114.6143 E 444.2453 4
11 1185.6514 A 315.2027 3

12 1282.7042 P 244.1656 2
13 K 147.1128 1
1 114.0913 I8
2 185.1285 A 731.3934 7
3
314.1710
E 660.3563 6
4 427.2551 L 531.3137 5
5 498.2922 A
418.2296
4
6 627.3348 E 347.1925
3
7 698.3719 A 218.1499 2
# b Seq y #
8K147.1128 1
A
B
0 200
L
L
y(1)
a(2)
b(2)
y(2)
y(4)
y(5)
y(6)
y(7)
y*(4)++ ,y*(2)

b(6)++
a(2) , a(4)++
b(2) , y*(2)
b(3)
y(4)
y(6)
y(7)
y(8)
y(9)
y(10)
y(11)
400 600 800 1000 1200
0 200 400 600 800 1000 1200
y*(6)++
Fig. 3. Fragmentation spectra for peptide sequences from V. angustum S14 proteins identified in Bands A and B. (A) Fragmentation spectra
for peptide sequence LIVVDNFALEAPK from ribosomal protein L4 identified in Band A. For this peptide, the theoretical ion mass was
1427.802 Da and the experimental ion mass was 1427.767 Da. (B) Fragmentation spectra for peptide sequence IAELAEAK from PNPase
identified in Band B. For this peptide, the theoretical ion mass was 843.470 Da and the experimental ion mass was 843.454 Da. The boxes
illustrate the fragment ions for these peptide sequences as predicted by Mascot. Fragments that match between our data and the predicted
peptide fragments are shown in red. The fragment ions for both peptide sequences include a near-complete y-ion series and two from the
b-ion series, indicating a good match of the peptide fragments to the predicted sequence.
M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5167
which has yet to be described (residues 792–797); the
fourth corresponds to the enolase-binding microdo-
main (residues 885–909); and the fifth corresponds to
the region which binds PNPase (residues 1015–1094).
The RhlB-binding site, which has been shown here to
be capable of binding RhlB, did not correspond to a
region of increased structural order (residues 714–758).

From the results obtained in all of our analyses, it
would be reasonable to conclude that the core of
the V. angustum S14 RNA degradosome comprises
RNase E, PNPase, RhlB and enolase, all of which
are also components of the so-called canonical
degradosome in E. coli. This is not surprising as
V. angustum S14 belongs to the same subset of
c-proteobacteria as E. coli. Organisms belonging to
this group have previously been classified to have
Type A RNase E homologues [22].
The interactions observed in the immunoprecipita-
tion experiments and two-hybrid analyses are pairwise
interactions; therefore, we sought to prove that these
proteins actually form a single protein complex using
BN-PAGE. Previous techniques to isolate the RNA
AB C
D
Fig. 4. Analysis of the V. angustum S14 RNA degradosome complex through two-dimensional BN-SDS ⁄ PAGE. (A) Coomassie-stained gel of
the preparative separation of V. angustum S14 protein complexes in the first dimension by BN-PAGE. Bands 1–6 were excised and sepa-
rated in a second dimension SDS ⁄ PAGE. Bands were chosen for excision based on the results obtained from our BN-PAGE blot (Fig. 2B).
We chose to excise Band 1 as we suspected that it consisted mainly of protein aggregation. Band 2 was chosen as we wanted to deter-
mine whether the excision of a band slightly higher than Bands 3 and 4 (where RNA degradosome components were detected previously)
would also contain degradosome proteins. Bands 3 and 4 were excised as they corresponded to the high-molecular-mass bands, Bands A
and B in Fig. 2A, where the degradosome components were detected previously (Fig. 2B). Band 5 was excised to determine whether the
degradosome components seen in Fig. 2B were still present across the mass range. Band 6 was chosen on the basis of the apparent migra-
tion of the PNPase trimer on the BN-PAGE blot. (B) Coomassie-stained 4–12% Bis-Tris gel of the second dimension SDS ⁄ PAGE separation
of the excised bands (Bands 1–6). Cleared lysate (CL) is shown for comparative purposes. There is a clear difference in proteins present in
each band, indicating that the bands excised from the BN-PAGE gel are composed of different proteins. (C) Western blot of the second
dimension SDS ⁄ PAGE separation of the excised bands. The blot was probed simultaneously with a combination of RNase E, PNPase, RhlB
and enolase antisera. Bands 1 and 3 are more enriched in the components of the degradosome than the others. Band 6 does not contain

full-length RNase E. Cleared lysate (CL) is again shown for comparison. The lower half of the blot is not shown as there was no signal
detected below the 40-kDa region. A band migrating at approximately 80 kDa (*) cross-reacts with the RNase E antiserum and may be the
RNase E CTH. (D) Western blot of V. angustum S14 lysate probed with RNase E antiserum. Molecular mass markers are shown in kDa.
CTHNTH
RNase E
RhlB
(714–758)
Putative
RNA-binding
domain
?
(792–797)
“Segment A”
(565–581)
Enolase
(885–909)
PNPase
(1015–1094)
(604–661)
Fig. 5. V. angustum S14 RNA degradosome model. The CTH of V. angustum S14 RNase E contains sites for interaction with RhlB, enolase
and PNPase. In addition, we have identified regions with sequence homology to ‘Segment A’ and the RNA-binding domain in E. coli.A
remaining region (residues 792–797) which may be involved in interactions has yet to be characterized. Figure not drawn to scale.
RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al.
5168 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS
degradosome have required large amounts of starting
material, or have involved the denaturing purification
of individual components followed by their reconstitu-
tion into the RNA degradosome. As emerging evi-
dence points to the RNA degradosome as a dynamic
complex with many possible components, these tech-

niques provide limited avenues for truly assessing
what is occurring in vivo. We report the use of BN-
PAGE, a technique commonly employed to separate
protein complexes, two-dimensional BN-SDS ⁄ PAGE
and immunoprecipitation to analyse the RNA de-
gradosome from V. angustum S14. As little as 17 lg
of protein from whole-cell lysate is sufficient to visu-
alize the separation of protein complexes through
BN-PAGE. Our results from the BN-PAGE separa-
tion and subsequent immunodetection indicate that
PNPase and RhlB associate with RNase E in a com-
plex above 1.2 MDa, but that these proteins also co-
associate in smaller complexes of approximately
900 kDa. This is in agreement with current findings
that the RNA degradosome may be an assembly
which can range from 500 kDa to possibly in excess
of 4 MDa [30]. Mass spectrometric analysis of the
bands at 1.1 and 1.2 MDa identified GroEL and
ribosome proteins as possible interaction partners.
This was not surprising as ribosomal proteins have
been found in association with RNase E and the de-
gradosome, and play a role in its regulation, espe-
cially in times of stress [42]. However, it could also
be that components of the ribosome are comigrating
with the RNA degradosome in the native gels.
Together with ribosomal proteins, we identified eno-
lase and PNPase in these high-molecular-mass com-
plexes, confirming the results obtained in our
previous E. coli two-hybrid analysis and immunopre-
cipitation experiments [38]. It remains to be eluci-

dated whether the multiple locations of degradosome
components observed in BN-PAGE was a result of a
heterogeneous population of RNA degradosomes
in the cell, or whether the degradosome subunits
dissociated in preparation steps preceding BN-PAGE
separation.
We have identified sequence homologues for the
degradosome proteins in V. angustum S14 and showed
that they are capable of interacting with the CTH of
V. angustum S14 RNase E as well as their respective
binding sites ([38] and this work). Previously, we have
shown that the RNase E enolase-binding and PNPase-
binding sites from V. angustum S14 and E. coli may
be interchangeable. Enolase and PNPase from
V. angustum S14 and E. coli can bind to RNase E
microdomains (used in two-hybrid analysis) in V. an-
gustum S14 RNase E and E. coli RNase E. Here, we
have expanded on these results and shown that RhlB
can interact with the CTH of V. angustum S14 RNa-
se E and the predicted RhlB-binding microdomain.
Interestingly, we found that, despite low sequence
identity between the CTH of E. coli and V. angu-
stum S14 RNase E (28% identity) and the high varia-
tion in sequences flanking the microdomains, E. coli
RhlB, enolase and PNPase are able to bind to the
V. angustum S14 RNase E CTH. These results suggest
that specific protein interactions with RNase E can
occur despite the disordered nature and variability of
the sequences flanking the microdomains. This high-
lights the importance of microdomain sequence conser-

vation; the cross-species’ setting of the two-hybrid
experiments lends further strength to this. The fact
that the CTH of RNase E appears to adopt little struc-
ture probably plays a role in providing the flexibility
that is required by many molecular interactions
[18,38]. Further, there is decreased evolutionary con-
straint, allowing the sequences to adapt to the specific
requirements of the organism. Future experiments may
be aimed at the further investigation of how microdo-
mains direct the specificity of RNase E and how they
influence the assembly of different types of degrado-
somes in the cell.
Experimental procedures
Bacterial strains and plasmids
Vibrio angustum S14 was used to prepare protein samples
for BN-PAGE analysis and co-immunoprecipitation of
RNase E. E. coli strain DHM1 was used in E. coli two-
hybrid assays to study protein–protein interactions as
described below.
Media and growth conditions
Vibrio angustum S14 was grown in high-salt Luria–Bertani
broth (LB20) (10 gÆL
)1
tryptone, 5 gÆL
)1
yeast extract,
20 gÆL
)1
NaCl). E. coli strains were grown in Luria–Bertani
broth (LB10) (10 gÆL

)1
tryptone, 5 gÆL
)1
yeast extract,
10 gÆL
)1
NaCl). Solid medium was made by the addition of
15 gÆL
)1
of agar. Where appropriate, isopropyl thio-b-d-
galactoside (IPTG), the lac promoter inducer, was added to
a final concentration of 0.5 mm, and 5-bromo-4-chloro-
3-indolyl-b-d-galactopyranoside (X-gal), a substrate of
b-galactosidase, was added to a final concentration of
40 mgÆL
)1
.
Liquid bacterial cultures were inoculated with day-old
single colonies and grown in the appropriate medium on a
rotary shaker at 180 r.p.m. V. angustum S14 was grown at
25 °C and E. coli cultures were grown at 37 °C, unless
M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5169
otherwise specified. Where appropriate, the medium was
supplemented with antibiotics (ampicillin, kanamycin and
nalidixic acid at 100, 50 and 30 mgÆL
)1
, respectively).
E. coli two-hybrid analysis of interactions of
RNase E with RhlB

The Bacterial Adenylate Cyclase Two-Hybrid (BACTH)
system [39] was used to test for an interaction between
RhlB and RNase E. All genes and gene fragments were
cloned into the BACTH parental plasmids, whereby the
T25 domain was fused to the N-terminus (pKT25) of the
fragment and the T18 domain was fused to both the N-
(pUT18C) and C-termini (pUT18) (see Table S1 for a com-
plete list of strains and constructs, and Table S2 for the
primers used). The genes used included V. angustum S14
RhlB (Swiss-Prot Accession No. Q1ZNA5) and V. angu-
stum S14 PNPase (Q1ZJW2). Gene fragments included the
V. angustum S14 RNase E CTH (residues 526–1094) and
the V. angustum S14 RNase E putative RhlB-binding micr-
odomain (residues 714–758). Experimental details and posi-
tive and negative controls were the same as in a previous
study [38]. The negative interaction of RhlB with the
RNase E PNPase-binding site also served as a negative
control. Briefly, reciprocal crosses were performed where
appropriate by cotransforming chemically competent E. coli
DHM1 cells and plating on LB–X-gal plates supplemented
with appropriate antibiotics. The transformants were grown
at 30 °C for 26 h before being picked and patched onto
new plates. The patched transformants were then grown
separately at 25 °C for 48 h. The BACTH patch assay
results were scored by a ‘++’ for strong interactions, ‘+’
for moderate interactions and ‘)’ for very weak or no inter-
actions, based on colour intensity.
Analysis of protein complexes
Vibrio angustum S14 cell pellets were resuspended in 1·
Native PAGE buffer (50 mm Bis-Tris, 6 m HCl, 50 mm

NaCl, 10% w ⁄ v glycerol, 0.001% Ponceau S, 0.8% Triton
X-100, pH 7.2) supplemented with Roche Complete
EDTA-free protease inhibitor cocktail (one tablet per
50 mL of solution; Roche Diagnostics, Mannheim,
Germany) and lysed by sonication (Branson sonifier, Bran-
son Ultrasonics Corporation, Danbury, CT, USA). The
resulting lysate was clarified by centrifugation (22 000 g,
4 °C, 30 min). Samples were loaded onto 3–12% Native-
PAGE Novex Bis-Tris gels (Invitrogen Life Techonologies,
Carlsbad, CA, USA). Gels were run at a constant voltage
of 100 V using 1· NativePAGE running buffer (50 mm Bis-
Tris, 50 mm Tricine, pH 6.8) at the anode and a light blue
cathode buffer (50 mm Bis-Tris, 50 mm Tricine, 0.02%
Coomassie G-250, pH 6.8) until the dye front migrated to
the end of the gel. The apparent molecular mass of protein
complexes was estimated by comparison with very high-
molecular-mass markers of range 20–1236 kDa (Invitrogen
Life Techonologies). Protein bands were visualized either
by silver staining for analytical gels or Coomassie blue
staining for preparative gels. Bands of interest were excised
and analysed through mass spectrometric analysis.
For subsequent analysis in a second dimension via
SDS ⁄ PAGE, following first dimension separation, the BN-
PAGE gel was equilibrated in 1· Mes buffer (Invitrogen
Life Technologies) for 10 min. Bands of interest were
excised and placed into the wells of a 4–12% Novex Bis-
Tris gel (Invitrogen Life Technologies). Electrophoresis was
performed at a constant voltage of 150 V until the dye
front migrated to the end of the gel. Protein bands were
visualized with Bio-Safe Coomassie (Bio-Rad Laboratories,

Hercules, CA, USA).
Western blotting and immunodetection
For western blot analysis, proteins were separated on a
3–12% NativePAGE Novex Bis-Tris gel, 4–12% Novex
Bis-Tris gel, or both (Invitrogen Life Technologies), and
transferred onto PVDF membranes (Millipore, Bedford,
MA, USA) using the Invitrogen XCell II blot apparatus in
1· NativePAGE Transfer buffer (25 mm Bicine, 25 mm Bis-
Tris, 1 mm EDTA, pH 7.2) for native gels and 1· Native-
PAGE transfer buffer in 20% methanol (v ⁄ v) for Bis-Tris
gels. Transfer was carried out at a constant 600 mA for
90 min. After blocking with 5% (w ⁄ v) dried skimmed milk
in NaCl ⁄ Pi with 0.1% Tween 20 (v/v) overnight, mem-
branes were probed by incubation with a 1 : 5000 dilution
of primary antisera (against E. coli RNase E, RhlB, enolase
or PNPase, which were generous gifts from Dr A. J. Carp-
ousis, CNRS-Universite
´
Paul Sabatier, Toulouse) in
NaCl ⁄ Pi with 0.1% Tween 20 (v/v), followed by incubation
with a 1 : 5000 dilution of anti-rabbit IgG conjugated to
horseradish peroxidase (GE Healthcare, Little Chalfont,
Buckinghamshire, UK) in NaCl ⁄ Pi with 0.1% Tween 20
(v/v). Protein bands recognized by each specific antibody
were then detected using chemiluminescence (GE Health-
care) and visualized using a Fujifilm LAS 3000 imager
(Fuijifilm, Tokyo, Japan).
Immunoprecipitation
Escherichia coli RNase E antiserum (a generous gift from
Dr A. J. Carpousis) was crosslinked to Dynabeads Protein

A (Invitrogen Dynal, Oslo, Norway) using dimethyl pim-
elimidate.2HCl (DMP, Thermo Scientific, Rockford, IL,
USA) according to the manufacturer’s instructions. Briefly,
the Dynabeads were incubated with antibodies for 4 h on a
rotating wheel at 4 °C. Excess antibodies were washed off
with NaCl ⁄ P
i
(0.16 mm Na
2
HPO
4
.H
2
O, 5.51 mm NaH
2-
PO
4
.2H
2
O, 140 mm NaCl, pH 7.4), followed by 200 mm
triethanolamine, pH 8.0. The antibody-bound beads
were incubated with DMP (20 mm DMP, 200 mm
RNA degradosome complex in Vibrio angustum S14 M. A. Erce et al.
5170 FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS
triethanolamine, pH 8.0) for 45 min. The crosslinking reac-
tion was quenched with 50 mm Tris-Cl, pH 7.4.
Vibrio angustum S14 cells were grown in 500 mL of LB20
medium to an attenuance D at 600 nm of 0.7, and har-
vested by centrifugation at 5000 g for 15 min at 4 °C. Cell
pellets were resuspended in NaCl ⁄ P

i
containing Roche
Complete EDTA-free protease inhibitor cocktail (one tablet
per 50 mL of solution) and lysed through sonication (Bran-
son sonifier). The resulting lysate containing 5 mg of pro-
tein was clarified by centrifugation at 22 000 g for 30 min
at 4 °C and mixed with 100 lL of Dynabeads Protein A
crosslinked to an equal volume of antiserum raised against
E. coli RNase E. The mixture was rotated at 4 °C for 2 h.
The beads were washed three times using NaCl ⁄ P
i
and the
target protein was eluted by incubating the beads in 1·
SDS buffer for 15 min at 55 ° C or boiling the beads in 1·
SDS gel loading buffer for 10 min. Following SDS ⁄ PAGE
separation, the proteins were visualized by silver staining,
or processed for western blot analysis and immunodetec-
tion. The bands of interest were excised and subjected to
mass spectrometric analysis.
In a parallel experiment, V. angustum S14 RNase E was
immunoprecipitated using either E. coli RNase E or
PNPase antisera, employing the Protein G Immunoprecipi-
tation Kit (Sigma-Aldrich, Castle Hill, NSW, Australia) fol-
lowing the manufacturer’s instructions.
Mass spectrometric analysis
The bands of interest were excised, digested with trypsin
and analysed by LC-MS-MS, as described previously [43].
Searches were performed using the Mascot search engine
[44] employing a database of V. angustum S14 proteins.
Acknowledgements

We thank Dr A. J. Carpousis for generously providing
antisera against E. coli RNase E, PNPase, RhlB and
enolase. We thank Dr Mark Raftery for the mass spec-
trometric results which were obtained at the Bioanalyti-
cal Mass Spectrometry Facility within the Analytical
Centre of the University of New South Wales. Subsi-
dized access to this facility is gratefully acknowledged.
We gratefully acknowledge Dr Kathy Takayama and
Dr Paul March for establishing the background for this
research. This research was funded by the University of
New South Wales. M. A. Erce and J. K. K. Low are
recipients of the Australian Postgraduate Award.
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Supporting information
The following supplementary material is available:
Table S1. List of bacterial strains and plasmids used.
Table S2. List of oligonucleotide primers used for
amplification.
This supplementary material can be found in the

online version of this article.
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M. A. Erce et al. RNA degradosome complex in Vibrio angustum S14
FEBS Journal 277 (2010) 5161–5173 ª 2010 The Authors Journal compilation ª 2010 FEBS 5173