A Novel miRNA Processing Pathway
Independent of Dicer Requires
Argonaute2 Catalytic Activity
Daniel Cifuentes,1Huiling Xue,1David W. Taylor,2Heather Patnode,1Yuichiro Mishima,1,3
Sihem Cheloufi,4,5Enbo Ma,6Shrikant Mane,7Gregory J. Hannon,4Nathan D. Lawson,8
Scot A. Wolfe,8,9Antonio J. Giraldez1,10*
Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent
miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to
other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in
MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2
catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued
by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the
secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA
function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA
secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated
cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs
independently of Dicer.
their target mRNAs (1, 2). In animals, most
miRNAs are processed from a primary transcript
(termed pri-miRNA) by two ribonuclease III
(nt) small RNAs that regulate dead-
enylation, translation, and decay of
(RNase III) enzymes, Drosha and Dicer. Recent
studies have identified several miRNA classes
that bypass Drosha-mediated processing, namely
miRtrons, tRNAZ, and small nucleolar RNA
been viewed as a central processing enzyme in
the maturation of small RNAs (2). But are there
pathways that might process miRNAs in a Dicer-
independent manner, we sequenced small RNAs
(19 to 36 nt) in wild-type and maternal-zygotic
dicer mutants (MZdicer) (7). We analyzed 48-
hour-old embryos in two wild-type replicates
and two dicer mutant alleles (8), dicerhu715and
dicerhu896(fig. S1). Of the ~2 million reads per
sample, 69 to 82% mapped to known 5′- or 3′-
derived miRNAs in the wild type, whereas 4 to
9% mapped to miRNAs in the MZdicer mutants
1Department of Genetics, Yale University School of Medicine,
New Haven, CT 06510, USA.2Department of Molecular Bio-
physics and Biochemistry, Yale University School of Medicine,
School of Science, Kobe University, 1-1 Rokkodaicho Nadaku,
Kobe 657-8501, Japan.4Howard Hughes Medical Institute,
Watson School of Biological Sciences, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY 11724, USA.5Program in
6Department of Molecular and Cell Biology, University of
California, Berkeley, CA 94720, USA.7Yale Center for Genome
Analysis,Yale West Campus, Orange,CT06477,USA.8Program
in Gene Function and Expression, University of Massachusetts
Medical School, Worcester, MA 01605, USA.9Department of
Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, Worcester, MA 01605, USA.
10Yale Stem Cell Center, Yale University School of Medicine,
New Haven, CT 06520, USA.
*To whom correspondence should be addressed. E-mail:
Fig. 1. MicroRNA anal-
ysis in wild type (wt) and
in MZdicer and MZago2
mutants. (A and B) Nor-
malized reads from wild
MZago2 (B) libraries for
all annotated zebrafish
miRNAs. Some miRNAs
are shown as a reference
miRNAs (solid circles);
miR-144-5′ (green) and
miR-451-5′ (red) are
expressed in the same
pri-miRNA. (C) Scheme
of miR-144/miR-451 ge-
nomic loci and predicted
secondary structure of
miRNA in red). (D) Total
number of reads that
MZdicer, and MZago2.
Nontemplated uridines are
shown in red. (E) Domain
organization of Ago2. The
90-nt deletion (D90) re-
sults in a predicted trun-
cated protein lacking two
of three catalytic residues.
Amino acid positions are based on the mammalian Ago2. (F) Northern blot of embryos to detect slicer cleavage of an injected GFP target mRNA with three
complementary targets to miR-1 (3xPT-miR-1) in the presence(+) or absence (–) of miR-1 (7). Slicer activity isindicated by the higher-mobility product (fig.
25 JUNE 2010 VOL 328
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123456789 10 11 12
miR-451 hairpin miR-451 mm10-11
in vitro processing Ago2 processing+RNaseI protection IP + northern blot to detect miR-451
(10%) (10%) (100%)
(10%) (10%) (100%)
(10%) (10%) (100%)
(added after 3h)
miR-451 northern blot miR-451 northern blotendogenous miRNAs @48hpf
Fig. 2. Ago2 binds and processes pre-miR-451. (A) Immunoprecipitation of
FLAG-mAgo2 in wild-type and mutant embryos injected with pre-miR-451
followed by Northern blot analysis to detect bound miR-451. Input (I),
supernatant (S), and immunoprecipitate (IP) are indicated. (B and C) In vitro
cleavage assay using hAgo2 or hDicer protein and 5′-radiolabeled pre-miR-
430orpre-miR-451. (C)Ago2processingreactionsweretreated with(+)or
without (–) RNase I to assay protection of the processed hairpin by Ago2.
(D to F) Northern blot analyses to detect mature miR-451 after injection
with pre-miR-451 (+) [(D) and (E)] or endogenous miR-451 and miR-430
(F). Injection of wild-type mAgo2 but not a catalytically dead mAgo2D669A
rescues pre-miR-451 processing in vivo (E). The processing of miR-
451mm10-11is strongly reduced. Endogenous pre-miR-451 at 48 hpf is
processed in wild type and MZdicer but not in MZago2 mutants. Diagrams
forpredicted hairpins,cleavageintermediates,maturemiR-451 (red),miR-
430 (green), and miRNA* (blue) are shown. P32* indicates that injected
hairpins were radiolabeled (18).
Fig. 3. MZago2mutantsshowreducederythropoiesis.
but are reduced in MZago2 mutants [group II (mild)
MZago2 mutants (n = 61) compared to wild-type
embryos (n = 200), showing strongly reduced (group
III; light gray) and partially reduced (group II; gray)
numbers of o-das (+) cells (c2test, P < 0.001). (C)
Whole-mount in situ hybridization of ago2 expression
at 24 hpf. (D) May-Grünwald/Giemsa stain of eryth-
rocytes from wild-type, MZago2 mutants, and MZago2
injected at one-cell stage with various RNAs as shown
(+). Erythrocytes are representative of the mean for
each group. (E) Scatterplot of the nuclear cytoplasmic
ratio (N:C) for each genotype in (D) as a readout of
erythrocyte maturation (17). Distributions of the N:C
ratios in wild-type compared to MZago2 differed
correction, P < 10−15). Erythrocyte maturation is
rescued bymiR-451-duplex(MZago2and MZago2+
and MZago2+mAgo2, P < 10−15) but not
catalytically dead mAgo2D669A(MZago2 and
MZago2+mAgo2D669A, P > 0.1).
group II group III
wild type MZago2
Wt # of o-das (+) cells
Reduced # of o-das (+)
Loss of most o-das (+)
Percentage of embryos with
normal number of o-das+ cells
erythrocyte N:C ratio
VOL 32825 JUNE 2010
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(fig.S2). Several miRNAs appeared refractory to
dicer loss of function, notably miR-451-5′, miR-
2190-5′, miR-2190-3′, and miR-735-5′ (Fig. 1A
cy, reproducibility, and evolutionary conservation,
we focused subsequent analysis on miR-451.
for several reasons: (i) It is encoded in a con-
served 42-nt hairpin (fig. S5) with a 17-nt stem,
processing (9); (ii) miR-451 has a defined 5′ end
but a variable 3′ end that extends over the loop
and D); and (iii) reads stopped at nucleotide 30,
uridines, with nucleotide 31 mostly being a non-
templated U (Fig. 1D). The final templated base
pairs with nucleotide 10 of the mature miRNA
(Fig. 1C and fig. S1), a site where slicer activity
cleaves the passenger strand in siRNAs (10–12).
These observations lead us to hypothesize that
Ago2 slicer activity could participate in miRNA
maturation (fig. S1).
To determine whether Ago2 participates in
miRNA maturation, we generated a deletion
in the Piwi domain of the ago2 gene (ago2D90)
with the use of zinc finger nucleases (13–15)
(Fig. 1E and fig. S1). Because argonaute genes
are maternally expressed (fig. S6), we generated
maternal-zygotic ago2 mutants (MZago2). In-
deed, slicer cleavage of an mRNAwith perfectly
complementary targets to miR-1 was severely
reduced in MZago2 but not Zago2 relative to
wild-type embryos (Fig. 1F and fig. S1).
To investigate the role of Ago2 in miRNA
processing, we sequenced small RNAs (19 to 36
nt) from 48-hour-old MZago2 mutant embryos.
Comparing the normalized read frequency for
each 5′- and 3′-mature miRNA between wild-
type and MZago2 mutants revealed a reduction
in the number of reads that mapped to miR-451
(Fig. 1, B and D). In contrast, other miRNAs re-
mained largely unchanged. miR-451 and miR-144
arecoexpressed from a common primary transcript
miR-451 accumulated in the absence of Dicer
(factor of ~3 increase), miR-144 reads were re-
duced by a factor of >200 in MZdicer mutants
(18) (Fig. 1A). Conversely,ago2 loss of function
did not affect the read frequency of miR-144
of >8000. Taken together, these results indicate
that Ago2 regulates miR-451 levels posttran-
scriptionally by affecting either its processing
Recent studies suggest that Ago2 binds pre-
where miRNA* denotes the complementary
strand. Ago2 interacted with radiolabeled syn-
of Flag-mouse-Ago2 (mAgo2) with pre-miR-
451 or a mutant pre-miR-451mm10-11(with two
mismatches in the predicted slicer cleavage
site) followed by Ago2 immunoprecipitation
showed that Ago2 bound to both mature miR-
451 and pre-miR-451mm10-11(Fig. 2A). Incuba-
tion of human Ago2 (hAgo2) with pre-miR-451
but not pre-miR-430 resulted in a sharp 30-nt
band corresponding with the predicted slicer
Fig. 4. ADicer-independentmiRNA.(A)Zebrafishpre-miRNAsandduplexesas
indicated. pre-miR-430ago2-hairpinis a miR-430c hairpin that has been mutated
and shortened to form a 42-nt hairpin mimicking pre-miR-451 (ago2-hairpin).
dsRed mRNA (red). The GFP reporter contains three complementary target sites
tomiR-430inits 3′-untranslated region.(C)Northern blottodetect miR-430in
wild-type embryos injected with hairpins as indicated. a-Amanitin was co-
injected to inhibit transcription of endogenous pri-miR-430. (D) Northern blot
to detect 5′-radiolabeled pre-miR-430ago2-hairpinafter in vitro processing by
recombinant hAgo2 and hDicer. (E) In vivo assay to rescue miR-430 function in
MZdicer mutants. Bright-field and fluorescent images of the dorsal view of the
Brain outline (dashed line), mid-hindbrain boundary (green asterisk), and
ventricles(red,whiteasterisk)are shown.Morphogenesisdefectsarerescued by
injection of a Dicer-independent pre-miR-430ago2-hairpinor a miR-430-duplex
but not a Dicer-dependent pre-miR-430.
25 JUNE 2010VOL 328
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cleavage product of miR-451 (Fig. 2B). Converse-
ly, recombinant Dicer bound both pre-miRNAs
(fig. S7) but could only process pre-miR-430
(Fig. 2B). To investigate whether Ago2 pro-
cesses miR-451, we injected pre-miRNAs into
one-cell-stage embryos. Synthetic and endog-
enous pre-miR-451 hairpins were processed into
~30-nt intermediates and a ~22- to 26-nt ma-
ture miR-451in wild-type andMZdicermutant
but not in MZago2 mutant embryos (Fig. 2, D
and F). In contrast, a canonical mature miR-430
was processed in both wild-type and MZago2
mutant embryos but not in MZdicer (Fig. 2F).
On the basis of the sequencing results, we hy-
pothesized that Ago2-processed hairpin might
undergo nucleolytic trimming at the 3′ end (Fig.
slicer-cleaved intermediate from RNase I in
vitro, resulting in a ~20- to 26-nt 3′-end trimmed
product (Fig. 2C), similar to the mature miRNAs
observed in vivo (Fig. 2, D to F). Ago2 slicer
activity depends on its catalytic triad (DDH) and
the pairing between the guide and the target
mRNA (23–25). Expressing wild-type but not
catalytically dead (D669A) mAgo2 in MZago2
mutants rescued pre-miR-451 processing in
vivo (Fig. 2E). Furthermore, a hairpin with
slicer cleavage was bound by Ago2 (fig. S7)
but was inefficiently processed into mature
miR-451 (Fig. 2E). These results indicate that
Ago2 binds and cleaves pre-miR-451 in a pro-
cess that requires the slicer catalytic activity
and is independent of Dicer.
MZago2 mutant embryos displayed nor-
mal morphogenesis during gastrulation, brain
development, and heart development (fig. S8).
Ago2 is maternally expressed, and later in de-
velopment it acquires tissue-specific expres-
sion in the brain and intermediate cell mass
(ICM) (Fig. 3C and fig. S6). The ICM corre-
sponds to the hematopoietic precursors and
overlaps with the expression domain of miR-
451 (16), which plays an important role in eryth-
rocyte maturation in zebrafish (16, 17).
Consistent with the Ago2-dependent processing
of miR-451, MZago2 but not MZdicer mutants
showed a reduction in the number of hemo-
globinized erythrocytes (Fig. 3, A and B, and
fig. S8). In zebrafish, erythrocyte maturation
can be monitored by changes in erythrocyte
morphology and reduced nuclear/cytoplasmic
(N:C) ratio (17, 26, 27). Erythrocyte matura-
tion was delayed in MZago2 mutants, as man-
ifested by a significant increase in N:C ratio at
60 hours post-fertilization (hpf) (P < 10−15)
(Fig. 3, D and E). Providing back wild-type
mAgo2 or mature miR-451-duplex but not cat-
alytically dead mAgo2D669Arescued erythrocyte
maturation in MZago2 mutants (Fig. 3, D and
E). Thus, Ago2 catalytic function plays an
important role during erythrocyte maturation.
Whereas miR-451 is a 42-nt miRNA hairpin,
canonical vertebrate miRNAs are ~60 nt, and
unlike most miRNAs, mature miR-451 extends
into the loop of the hairpin where it overlaps
with the miRNA* (Fig. 4A and fig. S5). We hy-
pothesized that selection of the processing
pathway may be determined by structural dif-
ferences or by specific sequence motifs. To
distinguish between these two scenarios, we
modified the sequence of pre-miR-451 to en-
code a Dicer-dependent miRNA (miR-430c or
miR-1) mimicking pre-miR-451 structure and
length (pre-miRNAago2-hairpin) (Fig. 4A and fig.
S10). miR-430c is a member of a zygotically
expressed miRNA family that regulates ma-
ternal mRNA clearance, gastrulation, and brain
morphogenesis (7, 28). These processes are dis-
rupted in MZdicer mutants but can be rescued
by injection of a Dicer-independent miR-430-
duplex (7, 28). Three lines of evidence indi-
cate that pre-miR-430ago2-hairpinis processed
and functional independently of Dicer: (i)
Synthetic pre-miRNAago2-hairpinwas processed
into a ~23-nt mature miRNA in vivo (Fig. 4C
and fig. S10) and processed by recombinant
hAgo2 but not hDicer in vitro (Fig. 4D); (ii)
injection of miR-430cago2-hairpininto MZdicer
embryos repressed translation of a green flu-
orescent protein miR-430 reporter (GFP-miR-
430) relative to a dsRed control (Fig. 4B); and
(iii) injection of pre-miR-430cago2-hairpininto
MZdicer mutants rescued the gastrulation and
brain morphogenesis defects similarly to a
miR-430-duplex (Fig. 4E). In contrast, equi-
molar levels of the annotated Dicer-dependent
pre-miR-430 did not rescue the MZdicer phe-
notype (Fig. 4E). A second engineered miRNA
(miR-1ago2-hairpin) was also processed indepen-
dently of Dicer and down-regulated a GFP-
miR-1 reporter in vivo (fig. S10). These results
support a model in which the secondary
structure of the hairpin determines whether a
pre-miRNA is processed by Ago2 to form a
physiologically functional Dicer-independent
Our study defines a Dicer-independent path-
way for miRNA processing that is dependent
on Ago2 catalytic activity. We propose a mod-
el whereby Ago2 binds the pre-miRNA and
cleaves the paired miRNA* passenger strand
10 nucleotides away from the 5′ end of the
Ago2-bound miRNA guide strand (18). On
the basis of our small RNA sequencing, this in-
termediate would undergo polyuridylation and
nuclease-mediated removal of uridines and
templated nucleotides not protected by Ago2
to generate the mature miRNA (fig. S11). Pre-
vious studies have shown that the terminal
uridylyl transferase (TUT4) is recruited by
lin-28 to uridylate pre-let7 (29), which blocks
miRNA maturation and accelerates its degrada-
tion. Although we cannot exclude the possi-
bility that miR-451–uridylated intermediates are
targeted for complete degradation, our model
favors a scenario where uridylated Ago2-
cleaved pre-miRNAs are trimmed by a cellular
nuclease to generate mature miRNA sequences
protected by Ago2.
Ago2 has been reported to cleave siRNAs
and pre-miRNAs (21). Ago2-cleaved precur-
sors (ac-pre-miRNAs) can serve as Dicer sub-
strates, but their physiological functions remain
unclear (21). Here, we show that Ago2 cleavage
is necessary for the generation of a functional
miRNA (Figs. 1, 2, and 4). The identification
of a miRNA-processing pathway that bypasses
Dicer function might have wide implications
for the processing of canonical miRNAs. Our
study provides a biological context in which
Ago2 slicer activity is needed to process a
blood-specific miRNA, miR-451 (30). Al-
though it is likely that Ago2 has additional
roles in the cell by cleaving perfectly com-
plementary targets (1), the strong conserva-
tion of the sequence and secondary structure of
miR-451 across vertebrates suggests that con-
straints are in place to maintain this Ago2-
mediated miRNA processing pathway through
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31. We thank J. Doudna, D. O’Carroll, L. Zon, S. Lacadie,
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VOL 32825 JUNE 2010
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Scholar program, and the Pew Scholars Program in the Download full-text
Biomedical Sciences (A.J.G.). Contributions: D.C. and
A.J.G. designed and performed experiments; H.X.
performed computational analysis; D.C. and D.W.T.
performed in vitro assays; H.P. performed in situ
hybridizations; Y.M., G.J.H., and S.C. helped with initial
small RNA library sequencing and discussion; E.M.
provided recombinant Ago2; S.M. provided small RNA
sequencing; S.A.W. and N.L. designed the zinc finger
nucleases; and A.J.G. wrote the manuscript. Sequencing
data are deposited in Gene Expression Omnibus
(accession number GSE21503).
Supporting Online Material
Materials and Methods
Figs. S1 to S11
12 April 2010; accepted 27 April 2010
Published online 6 May 2010;
Include this information when citing this paper.
Control of Membrane Protein Topology
by a Single C-Terminal Residue
Susanna Seppälä,1Joanna S. Slusky,1Pilar Lloris-Garcerá,1Mikaela Rapp,1* Gunnar von Heijne1,2†
The mechanism by which multispanning helix-bundle membrane proteins are inserted into
their target membrane remains unclear. In both prokaryotic and eukaryotic cells, membrane
proteins are inserted cotranslationally into the lipid bilayer. Positively charged residues flanking
the transmembrane helices are important topological determinants, but it is not known whether
they act strictly locally, affecting only the nearest transmembrane helices, or can act globally, affecting
the topology of the entire protein. Here we found that the topology of an Escherichia coli inner
membrane protein with four or five transmembrane helices could be controlled by a single positively
charged residue placed in different locations throughout the protein, including the very C terminus.
This observation points to an unanticipated plasticity in membrane protein insertion mechanisms.
ntegral a-helical membrane proteins carry
out a wide range of central biological func-
tions. They have two conspicuous structural
features: hydrophobic transmembrane a helices
and a strong bias inthe distribution ofpositively
charged arginine (Arg) and lysine (Lys)residues
between cytoplasmic and extracytoplasmic loops,
with up to three times the frequency of Arg and
charged residues exert local control over the ori-
entation of transmembrane helices in their im-
mediate neighborhood (2, 3), but whether they
can also affect the global topology of a protein is
unknown. Multispanning membrane proteins in-
sert into their target membrane cotranslationally;
therefore, positively charged residues in a more
C-terminal region of the protein might be ex-
distant N-terminal transmembrane helices. How-
0 1 2 3 4 5 6
02468 10 12
Growth density (au)
Normalized growth = growthmutant
Dilution factor (log10)
Fig. 1. (A) The dual-topology protein EmrE and the EmrE(Nin) and EmrE(Nout) constructs
EtBrper ml.Thenormalized growth of a particularconstructiscalculated as the ratioof the
area under its growth-dilution curve relative to that obtained for wild-type EmrE, after
subtraction of thearea fortheempty vectorcontrol.(C) Normalizedgrowthvaluesfor EmrE
constructs discussed in thetext. Error bars indicate T1 SEM.
and Biophysics, Stockholm University, SE-106 91 Stockholm,
Sweden.2Science for Life Laboratory, Stockholm University, Box
1031, SE-171 21 Solna, Sweden.
*Present address: Department of Medical Biochemistry and
†To whom correspondence should be addressed. E-mail:
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