multiple roles for the twin arginine leader sequence of dimethyl

Multiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide

Reductase of Escherichia coli

Damaraju Sambasivarao, Raymond J. Turner1, Joanne L. Simala-Grant2, Gillian

Shaw, Jing Hu, and Joel H. Weiner #

MRC Group in Molecular Biology of Membrane Proteins, Department of

Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

1Present address: Structural Biology Group, Department of Biological Sciences,

University of Calgary, Calgary, Alberta, Canada, T2N 1N4

2 Present address: Dept. of Medical Microbiology and Immunology, University of

Alberta, Edmonton, Alberta, Canada T6G 2H7

Running Title: Twin Arginine Leader of DMSO Reductase

# Author to whom correspondence should be addressed

Phone: 780-492-2761; Fax: 780-492-0886

email: [email protected]

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 8, 2000 as Manuscript M909289199 by guest on February 11, 2018

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Summary

Dimethyl Sulfoxide (DMSO) Reductase of Escherichia coli is a terminal electron

transport chain enzyme that is expressed under anaerobic growth conditions and

is required for anaerobic growth with DMSO as the terminal electron acceptor.

The trimeric enzyme is composed of a membrane extrinsic catalytic dimer

(DmsAB), and a membrane intrinsic anchor (DmsC). The amino terminus of

DmsA has a leader sequence with a twin arginine motif that targets DmsAB to

the membrane via a novel Sec-independent mechanism termed MTT for

Membrane Targeting and Translocation. We demonstrate that the Met1 present

upstream of the twin arginine motif serves as the correct translational start site.

The leader is essential for the expression of DmsA, stability of the DmsAB dimer

as well as membrane targeting of the reductase holoenzyme. Mutation of

arginine17 to aspartate abolished membrane targeting. The reductase was labile

in the leader sequence mutants. These mutants failed to support growth on

glycerol-DMSO minimal medium. Replacing the DmsA leader with the TorA

leader of trimethylamine N-oxide reductase produced a membrane-bound

DmsABC with greatly reduced enzyme activity and inefficient anaerobic

respiration indicating that the twin arginine leaders may play specific roles in the

assembly of redox enzymes.

Introduction

Recently, we and others have reported the discovery of a novel system which

targets and translocates folded, cofactor-containing proteins to and across the

cytoplasmic membrane of bacteria (1-4). A similar system was previously

identified in chloroplasts and some mitochondria (5). This system, named MTT

(for Membrane Targeting and Translocation) or TAT (for Twin Arginine

Translocation) recognizes an amino-terminal leader sequence that is distinct from

the one recognized by the well-characterized Sec system (1-4). The MTT leader

sequence is typically 30-60 amino acids long, contains a conserved twin arginine

motif (S/T R R X F X/L K), and has been found to be associated with a large

number of periplasmic redox proteins(6,7). Although the role of this signal

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sequence in protein export was originally proposed for the periplasmic

hydrogenase of Desulfovibrio vulgaris (8), understanding of this unique leader had

to await the discovery of the MTT genes.

One of the twin Arg leader proteins identified in a database search was the

catalytic subunit of DMSO reductase(6). This enzyme is a heterotrimer,

composed of a molybdenum molybdopterin guanine dinucleotide containing

catalytic subunit (DmsA, 85.8 kD), an iron sulfur subunit (DmsB, 23.1 kD) and a

membrane intrinsic subunit (DmsC, 30.8 kD) (9,10). The DmsAB subunits

function as a catalytic dimer while DmsC is the membrane anchor subunit and

binds menaquinone (11). It was argued by Berks that DmsA together with DmsB

could be targeted for export via the MTT export pathway (6). However, extensive

biochemical, immunological, electron microscopic and electron paramagnetic

resonance studies have been carried out on the topological organization of the

DMSO reductase (12,13). The two membrane extrinsic subunits, DmsAB were

shown to face the cytoplasmic side of the membrane, contrary to the periplasmic

localization of most other twin Arg leader containing proteins.

The twin Arg motif of DmsA was not recognized in the initial cloning and

sequencing of the dms operon (9). N-terminal analysis of the purified DmsA

subunit, revealed that the mature, purified protein began with the sequence Val-

Asp-Ser… (Fig. 1) and the first upstream Met (16 residues upstream, M30 in Fig.

1) was proposed as the initiating Met (9). However, it now appears that DmsA

may actually initiate at a Met, 46 residues upstream of the V46 (Fig. 1). This 45

amino acid leader encodes a twin Arg motif and has Ala-His-Ala immediately

upstream of the cleavage site, in accord with other proteins containing this type

of leader (6,14).

The electron transfer properties of the DMSO reductase in E. coli are well

documented (15,16). However, very little is known about the role of the leader

peptide. In the present study, we confirm that DmsA does indeed initiate at the

more upstream Met (which we have now designated as M1, Fig. 1) We also

examined the influence of the DmsA leader sequence on the assembly and

stability of the enzyme, together with the membrane targeting functions.


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Experimental Procedures

Materials. Oligonucleotides were purchased from the DNA core facility,

Department of Biochemistry, University of Alberta, Edmonton, Canada or

GIBCO BRL, Canada. All molecular biology reagents were purchased from Life

Technologies Inc. The Sculptor in vitro mutagenesis kit was obtained from

Amersham Corp. ECL western blotting detection reagents were purchased from

Amersham Life Sciences. All other reagents were of analytical grade.

Bacterial Strains and plasmids. The following bacterial strains were used from our

laboratory collection. TG1 (∆(lac-pro) supE thi hsdD5/F’ traD36 proAB lacIq lacZ

DM15); DSS301 (as TG1; Kmr ∆dmsABC) and K38 (HfrC, λ−). The plasmids

carrying mutations in the DmsA leader sequence were generated using the

Sculptor site directed mutagenesis kit (15). The following primers were used to

construct the plasmids containing the native anaerobic promoter: pDMSM1 Stop

(GTGAGCCATTtaaAAAACGAAAATCC), pDMSR17D (GGTGAGTCGCgaT

GGTT TGGTAAAAAC) pDMSA43N:A 45N (TTAGTCGGATTaatCACaaTGTC

GATAGCG) and pDMSA43N (TTAGTCGGATTaatCACAATGTCGATAGCG).

The mutated residues were indicated in lower case. Plasmid pDMS160 (derived

from pBR322 vector) served as the template DNA. The wild-type and mutant dms

plasmids under the control of the T7 promoter were constructed as described

earlier (17-19). Briefly, the DNA fragments carrying the dms operon (EcoRI/Sal I

digest) from pDMS160, pDMSR17D and pDMSM1 Stop were cloned in to

pTZ18R as EcoRI/Sal I inserts to yield the T7 promoter constructs pDMS223,

pT7R17D and pT7M1 Stop, respectively. E. coli strain K38 carrying the plasmid

pGP1-2 (20) was transformed with the T7 promoter driven dms constructs to

study the expression of Dms polypeptides. Molecular biological procedures such

as restriction enzyme digestion, gel elution of DNA, ligation, filling in the

recessed ends of DNA after restriction digestion and transformation of DNA into

bacterial strains were carried out as described (21).

Plasmid pTDMS10, has a deletion of the dms leader sequence up to and including

the putative cleavage site at position 45 of the leader (Fig 1). This corresponds to

a 133 base pair (bp) deletion (nucleotide 808 to 940 of the dms operon map (9,18)),

and was constructed using the polymerase chain reaction (PCR). Two PCR


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products (A, 868bp and B, 640 bp ) were generated using the plasmid pDMS160

as the template and the primer pairs, BR322-R1 (CACGAGGCCCTTTCG) /

TDMS3 (cccggggctagccagctgtctagacatatgccccatggATAATGGCTCACTCAAGC)

for product A and TDMS2 (ccatggggcatatgtctagacagctggctagccccggg

GTCGATAGCGCCATTCC)/ TDMS1 (CGCGTTTCGCCAGGG) for product B,

respectively. The primer pairs TDMS3/TDMS2 encompass a 38 bp

complimentary overhang (shown in lower case) coding for the multicloning site:

NcoI, NdeI, XbaI, PvuII, NheI, SmaI. Except for PvuII, all other sites are unique.

Introduction of the multicloning site codes for the following amino acid sequence

and includes the initiating methionine: MGHMSRQLASPG. Following these 11

engineered amino acids is V46 of the mature protein (Fig 1).

PCR products A and B were gel purified, mixed and subjected to a second round

of PCR using primers BR322-R1 and TDMS1. The resulting composite PCR

product (1471 bp) has the multicloning site replacing the leader sequence. The

DNA fragment was gel purified and subjected to EcoRI and EcoRV restriction

digestion to yield a 1394 bp fragment. This DNA now lacking the entire leader

was used to replace the native DNA sequence between the nucleotide positions 1

and 1491 of the plasmid pDMS160, at the EcoRI and EcoRV restriction sites

respectively, to generate pTDMS10 (Fig 1). Plasmids pDMS160 and pTDMS10

harbor the native anaerobic promoter and the rest of the dms operon.

Construction of plasmids carrying the dms genes under control of the lac promoter.

Plasmid pTDMS10 was digested with NcoI and EcoRI and the fragments were

separated on a 1% agarose gel in a TAE buffer system. The larger fragment (7762

bp) containing the entire dms operon (less the leader) was gel eluted. The smaller

NcoI/EcoRI fragment (805 bp) containing the native anaerobic promoter was

discarded. The 205 bp fragment containing the lac promoter was obtained by a

PCR reaction using the DS-18 (atatgaattcGAATTAATTCTCACTCATTAGG)/DS-

19 (CTAGAAGAAGCTTGGGATC) primer pair and pYZ4 DNA (pBR322 derived

vector) as a template (22). The PCR product was digested with EcoRI and NcoI

and ligated to the gel eluted 7762 bp fragment, to generate pDMS189.

Plasmid pDMS190 and pDMS191 were derived from pDMS189 and harbor the

DmsA and TorA leader sequences, respectively, upstream of mature DmsA. The


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DNA sequence for the dms and torA leaders were obtained by PCR, using the

primers DS-11 (atatccatggtgAAAACGAAAATCCCTGATG)/DS-12(atggatcca

gctgGACAGCGTGCGCAATCCG) and SL-S2 (atatccatggtgAACAATAACG

ATCTCTTTC)/SL-A2(atggatcccagctgCGAGATGACAGCGTCAGT),

respectively. The plasmid pDMS160 served as a template DNA for DS-11/DS-12

PCR reaction and chromosomal DNA was used as a template for amplifying the

torA leader. The PCR DNA products have NcoI/PvuII flanking ends and were

cloned in to NcoI/SmaI cut pDMS189 to generate plasmids pDMS190 and

pDMS191.

All constructs were verified by restriction digestion analysis of the mini DNA

preparations and DNA sequencing reactions to confirm the mutations or

deletions in the dms operon.

Media and Growth Conditions. Aerobic overnight bacterial cultures were grown at

370C unless indicated otherwise, in Luria-Bertani (LB) medium supplemented

with the required antibiotics (11). A 1% innoculum was used from these

overnight cultures for all experiments, unless indicated otherwise. Anaerobic

growth experiments in glycerol minimal media were carried out in 160 ml screw

capped conical flasks with a Klett tube attachment (Klett flasks) for direct

monitoring of the bacterial growth as a function of time. The minimal medium

was supplemented with casamino acids, vitamin B1, terminal electron acceptor,

ammonium molybdate and antibiotics, as described (11). DMSO served as the

terminal electron acceptor for the growth experiments in Klett flasks.

For the measurement of DMSO reductase activity, the cells were grown

anaerobically in 1000 ml conical flasks at 370C for 48 hours on glycerol-fumarate

minimal medium (G-F medium); for 24 hours in peptone–fumarate medium or

for 8 hours in Terrific broth (TB) medium (21). DMSO reductase was expressed

constitutively under the above anaerobic growth conditions. Cells were

harvested and treated as described earlier for the preparation of the membrane

and supernatant (periplasm plus cytoplasm) fractions (11).

Gel electrophoresis and western blotting. Proteins were fractionated on a


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7.5 % or a 12% resolving polyacrylamide gel using a Bio-Rad mini slab gel

apparatus with sodium dodecyl sulfate (SDS) buffer system (23). Proteins from

SDS-PAGE gels were transferred to nitro-cellulose filters and probed with the

anti-DmsA and anti-DmsB antibodies as described (11). Western blot analysis of

whole cells was performed essentially as above, except the cell pellets were

directly lysed in the SDS-solubilization buffer and heated in a boiling water bath

for 2 minutes before loading on SDS-PAGE gels.

T7 expression studies. Strain K38/pGP1-2 carrying pDMS223 encoding dmsABC or

various mutants was grown overnight in LB medium at 300C. A 5% innoculum of

this culture was transferred into fresh LB medium (6.0 ml), grown for 2 hours,

and a 1.0 ml aliquot was drawn for the zero time sample. To the remaining 5.0 ml

of bacterial culture, was added fresh LB medium (5.0 ml) at 540C, to facilitate

rapid temperature equilibration of the culture to 420C. The incubation was

continued for another 15 minutes in a water bath set at 420C to induce T7 RNA

polymerase. Rifampicin (at 250 µg/ml of culture) an inhibitor of chromosomally

encoded RNA polymerase, was added and the incubation was continued at 420C

for 15 min. All subsequent incubations were at 370C (20). Aliquots (1.0 ml) were

drawn at various time points, spun in an Eppendorf centrifuge, and the bacterial

pellets resuspended in 50 µL of the SDS-solubilization buffer. The solubilized

pellets were heated in a boiling water bath for 2 minutes; spun to separate the

insoluble material and a 5 µL aliquot of the clear supernatant was loaded per

lane on a SDS-PAGE gel.

Enzyme Assays. DmsAB dimer enzyme assays were carried out by following the

substrate (TMAO) dependent oxidation of reduced benzyl viologen (BV) (11,24).

DmsABC holoenzyme assays were carried out by monitoring the oxidation of the

menaquinol analogue, lapachol as described earlier (25).

Analytical Techniques. DNA sequencing was carried out using an Applied

Biosystems model 373A DNA sequencer at the DNA core facility in the

Department of Biochemistry, University of Alberta, Edmonton, Canada.

All the data reported here are the result of at least two independent experiments

and the results from a representative experiment is presented.


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Results

Investigation of the translational start site of the DmsA leader sequence and its role on

the expression of the DmsAB polypeptides. When the dmsABC sequence was

reported (9,10), we proposed that DmsA initiated at a Met, 16 residues upstream

of a Val, the first amino acid of the purified DmsA subunit (M30 in Fig. 1).

Subsequent bioinformatic analysis of the dms sequence by Berks (6) suggested

that DmsA actually initiated at a Met which is 45 residues upstream of the Val

(M1, Fig. 1) and contained a consensus twin Arginine motif. To determine if M1

was the initiating Met, we mutated this start codon to the stop codon TAA, and

expressed the construct (pT7M1 Stop) using a T7 promoter system. Following

expression, the proteins were probed by western blotting with anti-DmsA and

anti-DmsB antibodies. As shown in Fig 2, vector pTZ18R did not express

DmsAB, whereas pDMS223 (wild-type dmsABC) showed good expression of the

DmsAB subunits. The DmsAB polypeptides were observed as early as 30 min

and peaked at 2 hours. The expression from the pT7M1 Stop plasmid showed

that the intensity of the DmsA polypeptide was drastically reduced with limited

expression at 30 and 60 min followed by rapid degradation. Expression of the

DmsB polypeptide was relatively unaffected, even up to 21 hours. These results

indicated that DmsA had a long 45 amino acid leader which contained a

consensus twin Arginine motif, and that an internal Met30 could not serve as an

efficient initiating Met (Fig. 1). Under these experimental conditions, DmsB could

not protect the truncated form of DmsA originating at the putative second

initiating Met30 in the pT7M1 Stop (Fig 1) from degradation.

Role of the conserved arginine in the DmsA leader sequence. The two arginines of the

twin Arg consensus sequence has been shown to be highly conserved within the

family of polypeptides exhibiting the motif (Fig. 1, R16 and R17). These residues

have been shown to be important for the expression and or the function of the

polypeptides that rely on the MTT system for protein targeting and transport

(14,26,27). We mutated Arg17 in the DmsA leader to an Asp (R17D) and

examined the effect on the expression and stability of DmsA in the T7 system as

described above (Fig. 2). Western blot analysis revealed that DmsAB were

expressed to levels comparable to the wild-type and the amount of DmsA was


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only slightly reduced at 21 hrs. compared to the wild-type (Fig 2; pT7R17D).

These results indicate that the R17D mutation has no adverse affects on the

expression or stability of the DmsAB polypeptides under the conditions of the T7

expression and analysis of the whole cell lysates by western blotting. The

expression of the DmsC polypeptide could not be analysed in these experiments

due to the lack of the corresponding antibody (12).

The DmsA leader sequence is essential for anaerobic growth on DMSO. Anaerobic

growth with glycerol as carbon and energy source and DMSO as the terminal

electron acceptor (G-D medium) provides a simple means of monitoring the

physiological function of DmsABC (11). E. coli DSS301, deleted for the

chromosomal dms operon is unable to support growth on G-D medium (11). The

wild-type plasmid, pDMS160 carrying the entire dms operon restored the

anaerobic growth of this strain (Fig. 3A). As expected, pDMSM1 Stop could not

complement growth. Similarly, the construct lacking the entire leader

(pTDMS10) could not complement growth. Although pDMSR17D produced the

DmsAB (Fig. 2, and presumably the DmsC) polypeptides, this construct was

unable to complement growth of DSS301. A mutant at the putative cleavage site

in which alanine 43 was changed to aspargine (pDMSA43N) and a double

mutant in which both the alanines at 43 and 45 were changed to aspargine

(pDMSA43N:A45N) showed normal growth profiles (Fig. 1 and Fig. 3A).

We investigated replacement of the dms leader with the TorA leader of inducible

TMAO reductase. We used lac promoter constructs, pDMS189, pDMS190 and

pDMS191 to facilitate the interpretation of the leader substitution (or deletions)

while keeping the promoter background constant. The TorA leader has a twin

Arg motif and translocation of TMAO reductase to the periplasm is dependent

on the MTT system (2,3). Plasmid, pDMS191 with a torA leader in place of the

dmsA leader showed poor growth (Fig. 3B). These studies indicate that an intact

dmsA leader was essential for the production of functional enzyme. Examination

of the control lac promoter constructs (Fig. 3B) indicated that pDMS189, lacking a

dmsA leader, could not support growth, similar to its parent pTDMS10 (Fig. 3A).

The wild-type lac promoter-dmsA leader construct (pDMS190) supported good

growth even in the absence of added inducer.


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The role of the DmsA leader on the targeting, assembly and the activity of DMSO

reductase. To probe the enzymatic activity of the leader mutants described above

we measured the DMSO-dependent oxidation of benzyl viologen (BV) as a

measure of DmsAB function and lapachol catalyzed reduction of DMSO as a

measure of the DmsABC in the membrane and soluble fractions from E. coli

DSS301 cells grown on minimal glycerol-fumarate medium (Table 1). DMSO

reductase activity was localized predominantly to the membrane fraction in cells

harboring pDMS160, pDMS190, and the cleavage site mutants pDMSA43N and

pDMSA43N:A45N. The activity data correlated well with the anaerobic growth

experiments (Fig. 3).

Membrane vesicles from E. coli strain DSS301/pDMSM1 Stop, expressed very

low levels of BV and lapachol activities. The BV activity observed was

predominantly in the soluble fraction (Table 1), in agreement with the growth

measurements. Similarly, there was no detectable BV or lapachol enzyme activity

when the leader was deleted (pTDMS10). In this mutant, the leader was replaced

with a multicloning site that encodes 11 amino acids, including an initiating Met

at the N-terminus of DmsA unrelated to the DmsA leader (Fig. 1). These studies

indicate that the leader sequence is an absolute requirement to sustain the

anaerobic growth, enzyme activity, stability and localization of the DMSO

reductase holoenzyme (Fig. 3 and Table 1).

Strain DSS301/pDMSR17D with a mutation in the second Arg of the twin Arg

motif expressed DmsA (Fig. 4B, below) but this enzyme did not associate with

the membrane and only very minimal levels of enzyme activity were detected in

the soluble fraction. Although nearly normal levels of DmsA appear to

accumulate following T7 expression (Fig. 2), we have found that under the

standard growth conditions, (peptone-fumarate medium for 24 hours or on

glycerol-fumarate medium for 48 hours (data not shown), the precursor form of

DmsA is degraded. As will be shown below (Fig. 4B) mutant precursor enzyme

from pDMSR17D was poorly processed to the mature form.

We have examined the expression of dmsABC using the lac promoter construct

(pDMS190). In minimal medium pDMS190 expressed similar total activity units


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in the absence of an inducer (IPTG) compared to pDMS160. The membrane

distribution of the BV and the lapachol activities were also similar to pDMS160

(Table 1). We also compared the activity profiles of the various constructs grown

on rich medium (TB) for 8 hours (Table 2). For the wild-type pDMS190, 97% of

the activity was found in the membrane fraction.

E. coli/pDMS189 lacking a leader had only 6% of the wild-type activity and 76%

of this activity was soluble. DSS301/pDMS191 with a TorA leader in place of

DmsA leader, had about 20% of the wild-type activity and 90% was membrane-

bound, corroborating the slow anaerobic growth seen on glycerol-DMSO

medium. These results indicate the twin arginine leader is required for stability

and for the association of DmsAB with the membrane.

Western blot analysis of the DmsA subunit from the wild-type and signal sequence

mutants. To confirm the role of the leader in membrane targeting and stability of

DmsA we used western blot analysis of membrane and soluble fractions from

cells harboring various plasmids encoding DMSO reductase. The distribution of

DmsA with the wild-type and mutant leaders was examined from cells grown

anaerobically on G-F medium for 48 hours to corroborate the activity

measurements summarized in Table 1. DmsA expressed by DSS301/pDMS160,

pDMS190, pDMSA43N and the double mutant pDMSA43N:A45N was

predominantly in the membrane fraction (Fig. 4A). The distribution of DmsA

reflected the distribution of specific activity (Table 1). Cells harboring plasmids

pDMSM1 Stop (Fig. 4A), pTDMS10 (data not shown) showed no immunoreactive

material in either the membrane or soluble fractions.

Analysis of the DmsA from DSS301/pDMSR17D mutant was carried out from

cells grown on TB medium for 8 hours (Fig 4B). Cells grown on G-F or peptone-

fumarate medium did not show any immunoreactive DmsA presumably due to

degradation (data not shown). However, analysis of the cell fractions derived

from cells grown in rich medium revealed three immunoreactive bands

corresponding to the pre-DmsA (P-form, 90.4 kD), the mature (M-form, 85.8 kD)

and a proteolytically degraded (D-form) in the soluble fraction. The membrane

fraction had only a very small amount of the M-form and D-form. These results


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are in agreement with the hypothesis that the DmsA leader is required for the

correct targeting and stability of the holoenzyme.

In Fig. 4B we also analyzed the membrane and soluble fractions for lac promoter-

controlled expression of DmsABC. The DmsA immunoreactive bands were

observed predominantly in the membrane fraction for E. coli DSS301/pDMS190

(wild-type leader) and pDMS191 (TorA leader). A very small amount of the D-

form of DmsA was observed in the soluble fraction of pDMS191. There was no

detectable immunoreactive material in the membrane or the soluble fraction

from the leaderless construct, pDMS189, consistent with the distribution of the

enzyme activity data (Table 2).

Discussion

The MTT system plays an essential role in the translocation of several folded,

cofactor-containing, redox proteins to the periplasm (1-3). These proteins have

long amino-terminal leader sequences with a twin Arg motif and are usually

soluble or bound to the periplasmic side of the membrane. The DmsABC and

FdoGHI enzymes are membrane-bound, multi-subunit proteins and both the

DmsA subunit and the FdoG subunit have similar twin Arg signal peptides.

However, a great deal of experimental evidence has shown that DmsAB and

FdoGH subunits face the cytoplasmic side of the membrane and are not

translocated across the membrane (12,28). We have proposed that the MTT

system is needed to associate the DmsAB subunits with the membrane anchor

subunit, DmsC (1,11).

In this study we provide experimental evidence for the presence of a 45-residue

twin Arg leader on the DmsA subunit. We also show that the leader is essential

for the stability and function of the holoenzyme. This contrasts with the

periplasmic TMAO and DMSO reductases from E. coli and R. sphaeroides (2,29).

In these enzymes, deletion of the twin Arg leader resulted in cytoplasmic

localization of the enzyme, consistent with the predicted role of the leader

peptide (2,3,29), however the leaderless enzymes were stable and incorporated

the molybdopterin cofactor.


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The DmsA leader has two methionine residues, Met1 and Met30 (Fig. 1). Met30

was found to be a poor initiation site and led to minimal synthesis of the

reductase (Fig. 2). This Met is not conserved in DmsA homologues (6). The

confirmation of a 45-residue leader necessitates re-numbering of amino acid

residues in DmsA. Previous publications have assumed that DmsA initiated at

Met30 based on the original sequence paper (9) and the first residue of the

mature protein, Val46 was previously numbered Val16.

Mutagenesis studies of the conserved Arg, within the twin Arg motif, were

performed on the [Ni-Fe] hydrogenase from Desulfovibrio vulgaris, the nitrous

oxide reductase from the Pseudomonas stutzeri and the glucose-fructose

oxidoreductase from Zymomonas (14,26,30). In all cases the mutant enzymes were

not translocated to the periplasm but were stable. The R17D mutant of DMSO

reductase was highly unstable, was not targeted to the membrane and did not

support anaerobic growth (Table 1 and Fig. 3). These studies suggest that the

twin Arg leader could have two distinct functions: targeting and stability. The

twin Arg motif in DmsA differs from the consensus twin Arg leaders by having a

Leu in place of a Phe in the fourth position however it is unlikely that the

targeting versus translocation role is related to this variation. When we replaced

the DmsA leader with the TorA leader, which has a Phe in place of Leu, DmsA

was still targeted to the membrane. The TorA leader-DmsA construct appeared

to be far less stable than the DmsA leader DmsA construct suggesting that there

are unique interactions between the leader and mature protein.

The twin Arg leader is cleaved by a peptidase (leader peptidase I?) and we have

examined the processing of DmsA by mutating the conserved amino acids

adjacent to the putative cleavage site. Nearly all of the twin arginine leaders have

a conserved A-X-A motif amino-terminal to the cleavage site and cleavage at this

site was confirmed for a number of enzymes (6,14). The DmsA leader also has the

A-X-A motif (Fig. 1), and the N-terminal analysis of the purified DmsA

polypeptide showed cleavage at this site (9). Both the cleavage site mutants

(pDMSA43N and pDMSA43N:A45N) exhibited enzyme activity, were processed

to the M-form, supported anaerobic growth on G-D medium and had the

expected membrane localization of activity. These mutations were chosen based


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on algorithms of von Heijne which suggested that Asn residues were least

preferred by leader peptidase I (31). Data in Figure 4A show that the

DmsA43N:A45N protein migrates as mature DmsA suggesting that cleavage is

catalyzed by a protease which is not inhibited by Asn or a nearby alternate

cleavage site is being utilized. These results lead to a conundrum. If DmsA is not

translocated to the periplasm, when and where is it cleaved? Although in earlier

papers we suggested that cleavage may be an artifact of cell lysis and protein

purification (9,10) it now appears that cleavage is an essential step to maturation

of functional enzyme (Sambasivarao, Dawson, Jing and Weiner, in preparation).

The twin Arg leader is able to export both homologous and heterologous

proteins. Reporter proteins such as dihydrofolate reductase, β-lactamase, alkaline

phosphatase and the 23 kD protein of the plant photosystem II oxygen-evolving

complex were exported and processed to the mature forms when expressed with

a TorA signal peptide (3,14,32,33). We have replaced the DmsA leader with the

TorA leader (pDMS191) and investigated the membrane assembly and function

of the TorA-DMSO reductase holoenzyme (Table 2, Figs 3 and 4). While the

TorA leader behaved similarly to the DmsA leader in its ability to target the

holoenzyme to the membrane, nearly a five-fold decrease in specific activity was

noted for the mutant enzyme, compared to the wild-type enzyme. The mutant

enzyme did not support efficient anaerobic growth on Glycerol DMSO minimal

medium. The incompatibility of the TorA leader supports our contention that the

DmsA leader has important stabilizing properties during maturation of

DmsABC holoenzyme. Interestingly, when the TorA leader was fused to the

soluble DMSO reductase from Rhodobacter sphaeroides the enzyme also failed to

show optimal enzyme activities implying the role of the leader in the overall

folding and function of the enzyme complex (29). These studies show that twin

Arg leaders may play specific roles in the assembly of redox enzymes and may

not be interchangeable.

Acknowledgments- This work was funded by the Medical Research Council of

Canada. We thank Alberta Heritage Foundation for Medical Research for a

summer student fellowship to Jing Hu.


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13. Rothery, R. A., and Weiner, J. H. (1993) Biochemistry 32(22), 5855-61.

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20. Latour, D. J., and Weiner, J. H. (1989) Biochem Cell Biol 67(6), 251-9.

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180(24), 6625-34.

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31. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Int J

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Figure Legends

Figure 1. Signal sequence of the DmsA subunit. The wild-type DmsA signal

sequence is shown for plasmid pDMS160 and for the mutants listed in

experimental procedures. The twin Arg motif is boxed and the consensus

sequence is shown at the bottom. Amino acid changes, or additions resulting

from the cloning manipulations, are shown in lower case. Gaps in the leader

sequences are introduced to facilitate alignment of the constructs. The start of

mature DmsA at position 46 is shown by an arrow. The AXA motif which is

conserved among the twin arginine leader sequences and may be the recognition

site for the protease involved in the processing of the signal sequence is

underlined. Amino acids deleted following the M1 in pTDMS10 and pDMS189

are represented by - -. The stop codon in pDMSM1 Stop is shown by the symbol

(*). The lac promoter construct pDMS190 carries the entire dms operon, similar to

pDMS160, except for the amino acids introduced as a result of engineering the

restriction sites. The TorA leader replaces the Dms leader in pDMS191 and

contains several amino acids derived from the mature TorA identified by italics.

Construction of the plasmids is described in the experimental procedures.

Figure 2. The twin arginine signal sequence is essential for expression and

stability of DmsA. The expression studies were carried out in the T7-promoter

vector pTZ18R, wild-type (pDMS223) and the mutant DmsA leader sequences

(pT7M1 Stop and pT7R17D) are described in the experimental procedures. The

DmsA and DmsB subunits are indicated.

Figure 3. The twin arginine signal sequence of DmsA is essential for

anaerobic growth of E. coli on G-D medium. Bacterial growth measured as Klett

units are plotted as a function of time (hours). The recombinant plasmids in E.

coli DSS301 were tested for growth with either the native anaerobic promoter (A)

or the lac promoter (B). The plasmids are identified in the figure. Individual data

points for the experiments using the plasmids pTDMS10, pDMSR17D, pDMSM1

Stop and DSS301 are omitted for clarity as these plasmids did not show any

growth over 36 hours. Individual growth profiles for the mutants pDMSA43N


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18

and pDMSA43N: A45N were identical and representative data for pDMSA43N:

A45N is shown.

Figure 4. Western blot analysis of the DmsA subunit from strains expressing

the wild-type and mutant enzymes. Recombinant plasmids in E. coli DSS301 are

identified above each lane. See Materials and Methods for full description of the

plasmids. The membrane (m) and soluble (s) fractions were separated on 7.5%

SDS-PAGE gels, transferred to nitrocellulose and blotted for the DmsA subunit.

The precursor (P), mature (M) and the degraded (D) forms of the DmsA subunit

are identified. DMSO STD is purified DMSO reductase. Data shown in A are

from the cells grown on glycerol-fumarate medium for 48 hours. Data shown in

B are from the cells grown on TB medium for 8 hours.


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Table Legends

Table 1: Comparison of the DMSO reductase activity from E. coli DSS301 carrying

wild-type and mutant DmsA leader sequences. Bacterial cultures (1L) were

grown anaerobically in glycerol-fumarate minimal medium for 48 hours at 370C.

Enzyme activities were measured using either reduced benzyl viologen (A) or

lapachol (B) as the electron donor and TMAO as the electron acceptor. The total

units of activity (µ moles of benzyl viologen or lapachol oxidized/min) and the

specific activity (expressed as units/mg of protein) in the membrane and soluble

fractions are indicated. The % distribution of activity in the membrane and

soluble fractions is shown in parenthesis. N.D: not detected.

Table 2: Comparison of DMSO reductase activity under control of Lac promoter.

E. coli DSS301 carrying the various recombinant plasmids was grown

anaerobically in TB medium for 8 hours at 370C. Assay of the reductase activity,

calculation of the total units, specific activity and distribution of the activity in

membrane and soluble fractions were as described in Table 1.


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Plasmid Sequence

pDMS160 M KTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHA VDS

pTDMS10 orpDMS189

VDSM ghmsrqlaspg---------------------------------

pDMS190 MvKTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHAvlg VDS

pDMSM1 Stop VDS* KTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRIAHA

pDMSA43N M KTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRInHA VDS

pDMSR17D M KTKIPDAVLAAEVSRdGLVKTTAIGGLAMASSALTLPFSRIAHA VDS

1 17 30 45 46

pDMS191 MvNNNDLFQA SRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQAATDAVISig VDS

Twin arginine consensus sequence T/SRRXFXK

pDMSA43N:A45N M KTKIPDAVLAAEVSRRGLVKTTAIGGLAMASSALTLPFSRInHn VDS

Sambasivarao et al, Fig. 1


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pTZ18R pDMS223 pT7R17-D

DmsA

DmsB

0.0

1.0

0.5

21.0

2.0

0.0

1.0

0.5

21.0

2.0

0.0

1.0

0.5

21.0

2.0

0.0

1.0

0.5

21.0

2.0

Time(h)

pT7M-1Stop

Sambasivarao etal, Figure 2


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0 50

100

150

010

2030

4050

pDM

S190

pDM

S189

pDM

S191

0 50

100

150

Klett Units

010

2030

40

pDM

SA

43N:A

45N

pDM

S160

DS

S301

pTD

MS

10pD

MS

R17D

pDM

SM

1 Stop

AB

Tim

e (hours)T

ime (hours)

Sam

basivarao

etal, Figure 3


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DMSO STD

pDMS189

pDMS190

pDMS191

M

D

DmsA

m s m ms s

B

m s

pDMSR17D

P

DMSO STD

pDMS160

pDMS190

pDMS43N

pDMS43N:45N

pDMSM1Stop

DmsA

M

Dm s m m m ms s s s

A

Sambasivarao etal, Figure 4


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Table 1

DMSO Reductase Specific Activity

A. Benzyl viologen assay B. Lapachol assay

Plasmid Total Units Membrane(%) Soluble(%) Total Units Membrane(%)

pDMS160 593 25.0 (93) 0.49 (7) 27.0 1.25 (91)

pDMS190 520 19.0 (94) 0.43 (6) 26.0 1.02 (80)

pDMSA43N:A45N 630 27.0 (92) 0.6 (8) 34.0 1.62 (94)

pDMSA43N 778 27.0 (94) 0.66 (6) 39.0 1.44 (96)

pDMSR17D 43 0.20 (13) 0.5 (87) N.D

pDMSM1 Stop 5 0.2 (100) N.D N.D

pTDMS10 N.D N.D N.D N.D


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Table 2

DMSO Reductase Specific Activity

Plasmid Total Units Membrane (%) Soluble(%)

pDMS189 35 0.20 (24) 0.20 (76)

pDMS190 519 11.0 (97) 0.10 (3)

pDMS191 111 2.10 (90) 0.13 (10)


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and Joel H WeinerDamaraju Sambasivarao, Raymond J Turner, Joanne L Simala-Grant, Gillian Shaw, Jing Hu

Reductase of Escherichia coliMultiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide

published online May 8, 2000J. Biol. Chem.

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multiple roles for the twin arginine leader sequence of dimethyl

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