Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction b
Trang 1and TatB from mutagenesis of the TatABC subunits
of the twin-arginine translocase
Claire M L Barrett and Colin Robinson
Department of Biological Sciences, University of Warwick, Coventry, UK
The twin-arginine translocation (Tat) system operates
in the plasma membranes of a wide range of bacteria
as well as the thylakoid membrane in plant
chloro-plasts (reviewed in [1,2]) Working in parallel with the
Sec system, it is responsible for the export of a subset
of proteins into the periplasm, outer membrane or
extracellular medium, and the primary defining
attrib-ute of the system is its ability to transport proteins in
a fully folded state [3,4] Particular attention has
centred on a series of periplasmic proteins that are
exported only after binding redox cofactors such as
FeS or molybdopterin centres [5–8] although it should
also be emphasized that the system also transports proteins that do not bind cofactors [1,2]
Substrates for the Tat pathway are exported post-translationally [8] after synthesis with cleavable, N-ter-minal signal peptides that almost invariably contain an essential twin-arginine motif in the N-terminal domain [9,10] They then interact with a translocon in the inner membrane that consists, minimally, of three sub-units (TatABC) in Escherichia coli and several other Gram-negative bacteria studied to date Genetic stud-ies indicate that the tatABC genes are all important for Tat activity although a fourth gene, tatE, encodes
Keywords
green fluorescent protein (GFP); Tat system;
twin-arginine; protein transport; signal
peptide
Correspondence
C Robinson, Department of Biological
Sciences, University of Warwick, Coventry,
CV4 7AL, UK
Fax: +44 2476523701
Tel: +44 2476523557
E-mail: Crobinson@bio.warwick.ac.uk
(Received 13 December 2004, revised 25
February 2005, accepted 8 March 2005)
doi:10.1111/j.1742-4658.2005.04654.x
The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane Three subunits, TatA, B and C, are known to be involved but their modes of action are poorly understood, as are the inter-subunit interactions occurring within Tat complexes We have generated mutations in the single transmembrane (TM) spans of TatA and TatB, with the aim of generating structural distortions We show that sub-stitution in TatB of three residues by glycine, or a single residue by proline, has no detectable effect on translocation, whereas the presence of three gly-cines in the TatA TM span completely blocks Tat translocation activity The results show that the integrity of the TatA TM span is vital for Tat activity, whereas that of TatB can accommodate large-scale distortions Near-complete restoration of activity in TatA mutants is achieved by the simultaneous presence of a V12P mutation in the TatB TM span, strongly implying a direct functional interaction between the TatA⁄ B TM spans
We also analyzed the predicted amphipathic regions in TatA and TatB and again find evidence of direct interaction; benign mutations in either subunit completely blocked translocation of two Tat substrates when present in combination Finally, we have re-examined the effects of previously ana-lyzed TatABC mutations under conditions of high translocation activity Among numerous TatA or TatB mutations tested, TatA F39A alone blocked translocation, and only substitutions of P48 and F94 in TatC blocked translocation activity
Abbreviations
GFP, green fluorescent protein; Tat, twin-arginine translocation; TM, transmembrane; TMAO, trimethylamine N-oxide; TorA, TMAO
reductase.
Trang 2a TatA paralog of minor importance in some species
[7,8,11–13] Only two tat genes (designated tatC and
tatA) are thought to be important in some
Gram-positive species, with a single gene product apparently
fulfilling both TatA and TatB functions [14–16]
Studies on the Tat mechanism are at an early stage
The Tat subunits are not related to any proteins in the
database and most studies point to a mechanism that
is unique among known protein transport systems
However, recent studies have begun to unravel some
salient features of this system Protein expression⁄
puri-fication approaches have resulted in the
characteriza-tion of two distinct complexes in E coli: a TatABC
complex and homo-oligomeric TatA complex The
TatABC complex has a mass of 600 kDa in
deter-gent and contains multiple copies of TatABC; within
this complex, TatB and TatC are in stoichiometric
amounts and the two subunits appear to function as a
unit [17] Approximately equal numbers of TatA
sub-units are present [17,18] but the vast majority of TatA
is found as separate, apparently homo-oligomeric
com-plexes [17,19,20] In vitro cross-linking studies on the
plant thylakoid [21] or E coli Tat system [22] have
shown that substrates initially bind to the TatB and
TatC subunits, and it thus appears that these subunits
cooperate to form the substrate binding site In plants,
the TatA homolog was only found to cross-link to
the Hcf106⁄ cpTatC complex (corresponding to bacterial
TatBC) in the presence of substrate and a proton
motive force [23] On the basis of these studies, it has
been proposed that binding of substrate to the TatBC
subunits triggers the recruitment of the separate TatA
complex to form an active translocation system
In an effort to pinpoint important regions of the Tat
subunits, the three proteins have been subjected to
site-specific mutagenesis and a number of key regions
or residues have been identified [24–27] TatA and
TatB are single-span proteins with C-terminal,
cyto-plasmic domains and each has also been truncated
from the C-terminus in order to delineate the regions
important for activity [28] Site-specific mutagenesis
has also been used to assess the importance of residues
in the predicted amphipathic domains and cytoplasmic
regions of TatA and TatB, and the highly conserved
residues of TatC have also been probed [24–27] In this
report we have analyzed the transmembrane regions of
TatA and TatB, in an effort to analyze their
import-ance for Tat function We show that mutations
designed to destabilize the TatB TM span through
sub-stitution by proline or multiple glycine residues have
no detectable effect, whereas some of the TatA
mutants are severely affected or blocked in
transloca-tion activity We also present evidence for interactransloca-tions
between the TM spans of TatA and TatB, and between the amphipathic regions Finally, we have re-examined the numerous mutations made previously
in TatA, B and C and we present new information on potentially important TatA and TatC mutants
Results
Analysis of TatA and TatB mutants The overall structures of the TatA and TatB subunits are similar: both contain a single TM span, with very short periplasmic N-terminal regions and cytoplasmic domains that are relatively small in the case of TatA ( 40 residues) and larger in TatB ( 90 residues) The TM spans and cytoplasmic domains are separated
by regions that are strongly predicted to form amphi-pathic a-helices [19,20] In the present study we have generated mutations in the TM and amphipathic regions of TatA and TatB (see below) in order to probe the importance of this region, especially with respect to a possible role for TatA as the translocation channel In order to present a comprehensive analysis
we have analyzed in parallel the translocation activity
of TatABC mutants described in several previous stud-ies [24–27] This was considered important because one
of the mutants exhibited unexpected properties when compared with previous findings
The effects of the mutations were analyzed using two types of export assay The first involves expression
of the mutated tatABC operon in the arabinose-indu-cible pBAD24 vector in a tat null background (Dtat-ABCDE strain) The cells were fractionated and the distribution of a known Tat substrate, trimethylamine N-oxide (TMAO) reductase (TorA) was analysed using
a native gel activity assay This assay is not quantita-tive but defects in translocation are usually apparent through an increased accumulation of TorA in the cytoplasm It should be noted that this vector expres-ses the tatABC operon by a factor of 10–20 fold higher compared with wild-type TatABC levels This means that minor, and even moderate defects in trans-location activity may not be revealed because the higher levels of Tat apparatus might be able to com-pensate for defects Moreover, this assay is qualitative rather than quantitative because the appearance of the TorA signal in the native gels is not linear with time
In summary, this assay is best suited for identification
of major defects in translocation activity
The second assay involves synthesis of a construct comprising the presequence of TorA linked to green fluorescent protein (GFP), which is efficiently exported
by the Tat pathway under these conditions [24,26,28]
Trang 3In these experiments, synthesis of TorA-GFP was
induced for 2 h using the pBAD vector, after which
the arabinose was removed and IPTG was added to
induce expression of tatABC from the compatible
pEXT22 plasmid (although this plasmid is relatively
leaky, thus TatABC are synthesized at appreciable
rates throughout the growth of the cells) Membranes
were isolated after a 3 h induction with IPTG This
assay is effectively semiquantitative (as the cells are
again analyzed over a relatively long period) but is
more reproducible than the TorA export assay and the
pEXT22 plasmid produces TatABC at lower levels (we
estimate approximately three- to fivefold more than
wild-type [28]) Most of the data presented below
involve the use of this assay but it should be
empha-sized that all mutants were tested several times using
the both types of export assay TorA export data are
shown only where there were minor discrepancies with
the TorA-GFP data
The experimental system varied from those of
previ-ous studies [24,26] in important respects We recently
found [28] that the Tat system is inhibited by the
pres-ence of arabinose (for unknown reasons) and TorA
export assays were conducted in a slightly different
manner compared to previous studies: only 50 lm
ara-binose was used for induction (instead of 200 lm)
With the TorA-GFP export assays, TorA-GFP was induced with arabinose for 2 h, after which the arabi-nose was removed and the cells incubated with IPTG for 3 h to induce expression of the mutated tatABC operons We have found that these conditions give more reproducible results and the Tat pathway of wild-type cells is shown below to be highly active at the time of analysis
First we analyzed TatABC levels in cells expressing the various mutated subunits, and the data for the TatA and TatB mutants (expressed using the pBAD vector) are shown in Fig 1 The expression of the wild-type tatABC from the pBAD-ABC is illustrated
in the indicated lane, with wild-type cells in the adja-cent MC4100 lane; it is evident that the TatA and TatB proteins are produced from pBAD-ABC at eleva-ted levels as described above No TatC signal is evi-dent in wild-type cells because this protein is detected using antibodies to the Strep-tag II on the TatC sub-unit in the pBAD-ABC vector In the other control lane, no Tat components are detected using mem-branes from DtatABCDE cells (denoted DABCDE in Fig 1 and other figures) as expected The remaining lanes contain membrane samples from DtatABCDE cells expressing pBAD-ABC in which mutations are present in the TatA or TatB subunit as indicated
Fig 1 TatABC expression levels in cells expressing wild-type or mutated TatA ⁄ B subunits Membranes were isolated from wild-type MC4100 cells, DtatABCDE cells (DABCDE) and DtatABCDE cells expressing pBAD-ABC containing mutations in the TatA or TatB subunit as indicated Samples were immunoblotted using antibodies to TatA, TatB or the Strep-tag II on TatC Asterisks denote strains in which a Strep-tag II is not detected by immunoblotting.
Trang 4Although the expression levels vary to some extent, all
of the mutant proteins are present at similar levels,
with the possible exception of TatA⁄ G2A which
exhib-its a relatively low TatB signal for unknown reasons
One anomaly is, however, evident with two newly
gen-erated TatB mutants, E8Q and L63A: TatA and TatB
are formed at typical levels but the TatC signal is
com-pletely absent (lanes denoted by asterisks) This is
reproducible and, because these mutants display high
levels of Tat activity (see below), we assume that the
C-terminal Strep-tag II has been removed from the
proteins after expression Some of the other mutants also have this property (see below)
Although the primary aim was to analyze new mutants affected in the amphipathic or TM regions, previously analysed TatA mutants containing single amino acid changes were also analyzed in terms of their ability to export TorA and TorA-GFP, and the data for the TatA mutants are shown in Fig 2 With the TorA export assays in Fig 2A, it is observed that the bulk of the activity is found in the periplasm in wild-type cells and cells expressing pBAD-ABC, as expected (lanes P) with very little cytoplasmic signal evident TorA is found exclusively in the cytoplasm in DtatABCDE cells, where it migrates more slowly in the gel system (denoted by an asterisk) The TatA mutants all export TorA with high efficiency with the exception
of F39A, where all of the TorA is present as the cyto-plasmic form Some cytocyto-plasmic TorA is also evident with L25A
The TorA-GFP export assays are in good agreement with the TorA data (Fig 2B) In pEXT-ABC-expres-sing cells, the bulk of GFP is found as mature-size protein in the periplasm (P), whereas GFP is found only in the cytoplasm and membrane fractions (C, F)
in cells expressing the pEXT22 vector Some of this protein is present as precursor form (TorA-GFP) and some mature-size GFP is also present, presumably due to proteolytic clipping No signal is observed in DtatABCDE cells that do not synthesize TorA-GFP (a control for the specificity of the GFP antibodies) All
of the TatA mutants export TorA-GFP with high effi-ciency except F39A, which is again completely defect-ive in translocation Whereas some cytoplasmic TorA
A
B
C
Fig 2 The TatA F39A mutant is inactive whereas other TatA ⁄ B mutants show no detectable loss of translocation activity (A) Dtat-ABCDE cells expressing pBAD-ABC or the same vector containing mutations in tatA were induced using 50 lM arabinose for 3.5 h, and cytoplasmic (C) and periplasmic fractions (P) were prepared as detailed in Experimental procedures These fractions were electro-phoresed on native polyacrylamide gels that were subsequently stained for TMAO reductase (TorA) activity Asterisk denotes slower-migrating cytoplasmic form of TorA (B) A TorA-GFP con-struct was expressed in DtatABCDE cells using an arabinose-inducible vector for 2 h The arabinose was then washed out and wild-type or tatABC operons containing the same tatA mutations as
in (A) were expressed for 3 h using the isopropyl thio-b-D-galacto-side-inducible pEXT22 plasmid as detailed in Experimental proce-dures (pEXT-ABC plasmid contains wild-type tatABC operon) Cytoplasmic, membrane and periplasmic fractions (C, M, P), were isolated and immunoblotted using antibodies to GFP The mobility
of mature-size GFP is indicated (C) TatB mutants described in Fig 1 were analyzed for export of TorA-GFP (B) exactly as des-cribed in (B) for TatA mutants.
Trang 5was evident with the L25A mutation, as described
above, no defect is apparent using TorA-GFP as a
substrate Because signal strengths are not linearly
related to protein activity in the native gel TorA assay,
we are more inclined to regard the TorA-GFP data as
evidence of high translocation activity, although other
possibilities can not be excluded
Similar tests on the TatB mutants are shown in
Fig 2C The results show that all of the strains
effi-ciently export TorA-GFP, including the E8Q and
L63A mutants that exhibited no signal with the
Strep-tag II immunoblots for TatC in Fig 1
Inhibitory effects of mutations in the predicted
amphipathic regions of TatA and TatB
Considerable attention has centred on possible roles of
conserved predicted amphipathic regions that are
highly conserved in both TatA and TatB These
regions effectively bridge the transmembrane helices
and soluble cytoplasmic domains, and truncation
ana-lysis [29] has shown that they are essential for
translo-cation activity Their sequences are shown in Fig 3A
In a previous report [24], we analyzed the effects of
changing three lysine residues in TatA (residues 37, 40
and 41) to glutamine, and in a second mutant we addi-tionally changed K24 to alanine These mutants, denoted TatA⁄)3K and TatA ⁄ )4K previously [24] were shown to be active albeit with reduced efficien-cies These TatA mutants have been made (and re-named TatA⁄ 3K > Q and TatA ⁄ K24A,3K > Q) after finding several revertants in recent studies of previ-ously analyzed mutants In the case of TatB, it was previously found that changing two arginines (residues
37 and 40) to asparagine (TatB⁄ 2R > N), or three lysines (residues 65, 67 and 68) to glutamine (TatB⁄ 3K > Q) had little effect on the efficiency of Tat-dependent export [24] In the present report we have re-assessed these mutants and another mutant combining the two sets of mutations in TatB (TatB⁄ 2R > N,3K > Q) This new mutant is indica-ted by ‘’ in Fig 3 The expression profiles are shown
in Fig 3B, which shows TatA and TatB to be synthes-ized in all cases, although again at slightly varying lev-els Strep-tagged TatC is formed in every case except TatA⁄ K24A,3K > Q, but because this mutant is act-ive (see below) we again believe that the TatC protein
is present but lacking the Strep-tag II
Export assays using these mutants are shown in Fig 4A The data show that all three TatB mutants
TatA
TatA
TatB
TatC
TatA
TatB
TatC
pB AD-AB
Cs
pB AD-AB
Cs
2R>N K24AK24A K24A
3K>Q 3K>Q 2R>N 3K>Q/ 2R>N
3K>Q 3K>Q 3K>Q
3K>Q
2R>N 3K>Q 2R>N 3K>Q ∆ABCDE
∆ABCDE
TatB
16
25
30
*
A
Fig 3 Mutations in the predicted
amphi-pathic regions of TatA and TatB (A) Primary
sequences of the amphipathic regions with
the targeted residues indicated by arrows
and numbered The changes introduced in
the various mutants are indicated and
under-lined (B) pBAD-ABC cells, DtatABCDE cells
and DtatABCDE cells expressing pBAD-ABC
containing the ‘amphipathic’ mutations
shown in (A) were analyzed by
immunoblot-ting with antibodies to TatA, TatB and the
Strep-tag II on TatC Asterisks denote
strains that exhibit no signal with Strep-tag
II antibodies (C) as in (B), except with
strains expressing combinations of
tions in TatA + TatB ‘^’ denotes new
muta-tions analyzed in this study Mutamuta-tions in
TatA are shown underlined and in grey font.
Mobilities of molecular mass markers are
given on the left.
Trang 6exhibit efficient export of TorA-GFP, in that the vast
majority of protein is found in the periplasmic fraction
This includes the new TatB mutant (TatB⁄ 2R > N,
3K > Q) in which five basic residues are substituted;
surprisingly, there is little evident effect Among the
TatA mutants, TatA⁄ 3K > Q was previously described
as active [24] but Fig 4A shows this not to be the case
with the newly generated mutant, which fails to export
TorA-GFP to any detectable extent No export of TorA
was observed either (data not shown) Surprisingly, the
presence of an additional mutation (K24A) enables
export to take place, with the TatA⁄ K24A,3K > Q
quadruple mutant exhibiting moderate export activity
Some precursor form of TorA-GFP is present in the
cytoplasm and⁄ or membrane fractions but considerable
amounts of periplasmic TorA and GFP are present It is
likely that the original TatA⁄ 3K > Q mutant [24]
simi-larly acquired an additional mutation prior to analysis
that enabled translocation to occur, although this has
yet to be confirmed Other TatABC mutations described
previously [24,26] have been remade and shown to have
unchanged properties
We also expressed tatABC operons in which these multiple mutations in the amphipathic regions were combined; in the relevant Figures the TatA mutations are shown underlined for simplicity We previously reported data [24] on two such combinations: (TatA⁄ K24A, 3K > Q + TatB ⁄ 3K > Q) and (TatA ⁄ 3K > Q + TatB⁄ 2R > N), and both mutants were described as active In this report we have reassessed the effects of these mutations (as the former TatA mutant TatA⁄ 3K > Q is now known to be inactive on its own, as shown above) and have constructed several new permutations as detailed in Fig 3 These new mutations are again denoted by ‘’ Figure 3C shows that strains synthesizing all of the multiple TatAB mutations contain TatABC at similar levels and activ-ity assays are shown in Fig 4(B,C)
These ‘mixed amphipathic’ mutations exhibit very interesting properties Figures 2 and 4A showed that the TatA⁄ K24A and TatB ⁄ 2R > N mutations support wild-type levels of export activity but Fig 4B shows that the combined (TatA⁄ K24A + TatB ⁄ 2R > N) mutations severely disrupt activity, with no periplasmic
A
B
C
Fig 4 Combinations of mutations in the TatA ⁄ TatB amphipathic regions have partic-ularly severe effects on translocation activ-ity (A) Mutants containing alterations in the predicted amphipathic regions of either TatA
or TatB (as detailed in Fig 3) were analyzed for export of TorA-GFP using the assay pro-tocols detailed in Fig 2 For clarity, muta-tions in TatA are shown underlined and in grey (B) Combinations of mutations in the amphipathic regions of TatA and TatB, whose structures and expression profiles are illustrated in Fig 3, were tested for the export of TorA-GFP Mutations in TatA are shown underlined and in grey font; addition-ally, labels on the right indicate whether mutations are in TatA or TatB (C) Dtat-ABCDE cells, or DtatABCDE expressing pBAD-ABC or the same vector containing mutations in both TatA and TatB as indica-ted, were assayed for export of TorA using the protocol described in Fig 2.
Trang 7mature-size GFP detected at all after synthesis of
TorA-GFP Note that mature-size GFP accumulates in
the cytoplasm, and not the full precursor protein; this
is due to proteolytic clipping of the signal peptide
when export is blocked [28,30] Figure 4C shows TorA
export assays in which the vast majority of TorA is
found in the cytoplasm A minor fraction of TorA
is found in the periplasm, indicating that the mutant
is not completed blocked in Tat-dependent
transloca-tion, but this mutant is clearly badly compromised An
almost identical result is obtained with (TatA⁄ K24A
+ TatB⁄ 3K > Q), and it should again be noted that
the individual TatA and TatB mutants show no
appar-ent defects These data show that the mutations have
synergistic effects and the particularly severe effects on
TorA-GFP export raise the intriguing possibility that
these mutations somehow affect the export of GFP
differently when compared to TorA
The next two mutants in Fig 4B (from left to right)
contain TatA⁄ 3K > Q in combination with either
TatB⁄ 2R > N or TatB ⁄ 3K > Q Both combinations
exhibit no detectable Tat activity and this is
unsurpris-ing as we showed above that the ‘new’ TatA⁄ 3K > Q
mutant is inactive on its own Of the remaining
mutants (TatA⁄ K24A, 3K > Q + TatB ⁄ 3K > Q) is
completely inactive, although the individual TatA and
TatB mutants did exhibit activity, and the final mutant
containing five changes in TatA plus four in TatB is
likewise totally inactive In all, these mutants
empha-size the importance of the amphipathic region but they
also show for the first time that combinations of
TatA⁄ TatB mutations can have far more dramatic
effects than the individual mutations
Mutations in the transmembrane spans
of TatA and TatB
Deletion of the TM spans of TatA or TatB leads to a
loss of activity [31] but the important characteristics of
these regions have not been probed We constructed
a series of new TatA⁄ B mutants containing changes
within the TM spans, for two reasons First, it has
been suggested that TatA may form the translocation
channel, in which case drastic structural alterations
may be expected to selectively block the translocation
event Secondly, mutations affecting the structure and
orientation of the TM span may be expected to disrupt
the interactions with other Tat components, and this
would provide information on the inter-subunit
associ-ations occurring within and between Tat complexes
Both of these areas are poorly understood at present
It has been shown with other proteins that the
intro-duction of proline residues has a marked effect on the
structures of TM spans [32,33], usually introducing dis-tortions of major proportions, and such substitutions were made in the TM spans of TatA and TatB in the present study We also substituted three residues in the TatA and TatB TM spans by glycine The presence
of glycine can also lead to increased flexibility in TM helices [34] However, glycine residues can also play important roles in modulating inter-helix interactions [35] and the effects of inserting or removing these resi-dues may therefore be less predictable than with pro-line mutations Nevertheless, the presence of three consecutive glycines should lead to a significant struc-tural effect in either case The proline and glycine sub-stitutions were made near the centre of the TM span
in order to maximize possible structural effects Figure 5A shows the sequences of the TatA and TatB TM spans, together those of the mutated forms With TatA, residues 11–13 were changed to glycine in one case (TatA⁄ 3Gly) and a single residue was chan-ged to proline in the centre of the TM span in another (TatA⁄ I12P) We also changed three additional residues to proline in the TatA⁄ 3Gly mutant (TatA ⁄ 3Pro3Gly) and then a further three residues to glycine
in the same subunit (TatA⁄ 3Pro6Gly) With TatB, resi-dues 11–13 were changed to glycine (TatB⁄ 3Gly) or a single residue to proline (TatB⁄ V12P)
The expression characteristics of these mutants are shown in Fig 5B With the simplest TatA mutants, TatA⁄ 3Gly and TatA ⁄ I12P, TatABC are all present
at expected levels With TatA⁄ 3Pro3Gly, TatA and TatB are present at the usual levels but TatC is not detected at all However, as this mutant is highly act-ive (see below) it appears that this is another example
of the C-terminal Strep-tag II being clipped or modi-fied With the most drastic of the TatA mutants, TatA⁄ 3Pro6Gly, TatB and TatC are synthesized but
no TatA is detected Fractionation of cells synthes-izing TatA⁄ 3Pro3Gly shows the presence of full-length protein in the cytosol, consistent with a slight defect in membrane-insertion; in contrast, smaller fragments of the TatA⁄ 3Gly6Pro protein are found in the cytosol (data not shown) This suggests that the TatA⁄ 3Gly6Pro protein is degraded either within the membrane or, perhaps more likely, after failure to insert into the membrane
Cells expressing the simpler of the tatB mutants, TatB⁄ V12P, appear to contain TatABC at expected levels but the TatB⁄ 3Gly mutations result in a much-reduced TatB signal, presumably reflecting problems in insertion and⁄ or stability No TatC signal is evident, but this again appears to reflect problems in detection
of the Strep-tag II as this mutant is active in Tat-dependent transport (see below)
Trang 8Figure 6 shows TorA and TorA-GFP export assays
for these TM span mutants Of the TatA mutants,
TatA⁄ I12P shows no detectable defect because GFP
and TorA are found predominantly in the periplasm
The TatA⁄ 3Gly mutant, on the other hand is blocked
in export and no translocation activity can be detected
The same applies to the TatA⁄ 3Pro6Gly mutant,
although this is expected because the immunoblots in
Fig 5 show no signal for TatA The real surprise is
the TatA⁄ 3Pro3Gly mutant, which is shown to export
both TorA and TorA-GFP with high efficiency Given
that the parent TatA⁄ 3Gly mutant is completely inac-tive, this result indicates that the three proline residues somehow compensate for the inhibitory effects of the glycine residues and enable translocation to occur Finally, Fig 6 shows that the two TatB mutants, TatB⁄ 3Gly and TatB ⁄ V12P, are highly active in export; perhaps surprisingly given the drastic effects of the 3Gly mutations in TatA
We also tested the effects of expressing tatABC operons carrying combinations of these mutations in the TM spans of both TatA and TatB, namely (TatA⁄ 3Gly + TatB⁄ 3Gly) (TatA ⁄ 3Gly + TatB ⁄ V12P) (TatA ⁄ I12P + TatB⁄ 3Gly) and (TatA ⁄ I12P + TatB ⁄ V12P) Immunoblots confirmed that the TatABC were syn-thesized at approximately the same levels as the wild-type subunits generated from pBAD-ABC (data not shown), and activity assays are shown in Fig 7 With the TorA assays, the control tests show efficient export with wild-type TatABC and a complete block in export
in the tat null mutant (denotedDABCDE), as expected The combination of (TatA⁄ 3Gly + TatB ⁄ 3Gly) is blocked in Tat function, and this is not unexpected given that the TatA⁄ 3Gly mutant itself shows no export activity However, a combination of (TatA⁄ 3Gly + TatB⁄ V12P) displays very efficient export of TorA and this shows that the TatB⁄ V12P mutation compensates for the drastic effects of the 3Gly muta-tions in TatA This is confirmed by the TorA-GFP export assays, which reveal a complete block in export with the (TatA⁄ 3Gly + TatB ⁄ 3Gly) mutant but near wild-type export efficiency with (TatA⁄ 3Gly + TatB ⁄ V12P) The remaining (TatA⁄ I12P + TatB ⁄ 3Gly) and (TatA⁄ I12P + TatB ⁄ V12P) mutants export both TorA and TorA-GFP efficiently, in keeping with the finding that none of the mutations affect export to a detect-able extent when present in TatA or TatB alone (see Fig 6) In conjunction with the data shown in Fig 6, these data show that the severe effects of the TatA⁄ 3Gly mutations can be rescued by the presence
of additional mutations either elsewhere in the TatA
TM span or in the TatB TM span (TatB⁄ V12P mutation)
Mutagenesis of TatC TatC has also been studied in previous reports and a number of mutations have been characterized [26,27], but some apparent differences were reported in studies
on the same mutants by different groups (see below) Few TatC residues are highly conserved but of these, a high proportion is clustered in the N-terminal cyto-plasmic domain and the first cytocyto-plasmic loop In our previous analysis [26] only two residues were found to
A
B
Fig 5 Expression of TatA and TatB mutants containing alterations
in the transmembrane spans (A) Sequences of the TM regions of
TatA and TatB, with residues numbered and the substitutions
indi-cated (B) Expression of the various mutations constructed within
the pBAD-ABC vector Samples were analyzed by immunoblotting
with antibodies to TatA, TatB and the Strep-tag II on TatC
Mobili-ties of molecular mass markers are given on the left.
Trang 9be absolutely essential for TatC function: R17A (N-terminal cytoplasmic region) and P48A (first peri-plasmic loop) The deletion of three residues (20–22) also disrupted function, prompting the suggestion that this cytoplasmic domain played an important role In
a separate study [27], R17 was again found to be important but two acidic residues, E103 and D211 were found to be particularly critical; E103A⁄ Q ⁄ R mutants and D211A were completely inactive, although D211E⁄ N mutants were partially active These residues are located on the first cytoplasmic loop and third periplasmic loop, respectively Substitution
of F94 (at the interface between cytoplasmic loop I and the membrane bilayer) was also reported to block transport activity [27] D211 was not analyzed in our previous study [26] but we did find that E103A showed
no translocation defect at all, and so we have made new mutants in all three residues in order to analyse the effects using our expression and assay systems
As with other mutants, we carried out expression studies using all of the TatC mutants; in each case the TatABC subunits were present at similar levels and in
A
B
Fig 6 Effects of mutations in the TM
regions of TatA and TatB The TatA and
TatB mutants containing alterations in TM
spans (detailed in Fig 5) were tested for
effects on translocation activity (A)
Muta-tions within the pBAD-ABC vector were
assayed for export of TorA (B) mutations
within pEXT-ABC were assayed for export
of TorA-GFP, as detailed in Fig 2 Cells
expressing pBAD-ABC or pEXT-ABC without
mutations were analyzed as controls, and
DtatABCDE cells were analyzed for export
of TorA, again as a control Other symbols
are as in Fig 2.
A
B
Fig 7 A mutation in the TatB TM span can compensate for the
severe effects of the TatA ⁄ 3Gly mutation This figure illustrates the
translocation activities of four DtatABCDE strains expressing
pBAD-ABC or pEXT-pBAD-ABC in which mutations are present in the TM spans
of both TatA and TatB (mutations in TatA are shown underlined and
labels on the right indicate whether the mutations are in TatA or
TatB) Cells were analyzed for the export of TorA (A) or TorA-GFP
(B) using protocols described in Fig 2.
Trang 10these instances the TatC Strep-tag II were all found to
be intact (data not shown)
Mutations in the first cytoplasmic loop are
charac-terized in Fig 8(A) None of these mutants display
any detectable defects in translocation efficiency,
inclu-ding the E103A and E103Q mutants which do not
even contain elevated levels of the precursor protein
TorA-GFP These data do not agree with reports that
the two residues are critical [27], and with this
appar-ent contradiction in mind we made each mutant again
and again found them to be highly active (data not
shown) Figure 8(B) shows the effects of mutations in
TM spans two to six Most of these mutations again
have no detectable effect with the notable exception of
F94A which is completely inactive These results agree
with those reported in [27]
The final study on TatC is shown in Fig 9, where
mutations in the periplasmic loops are analyzed We
have previously shown that the P48A mutation
des-troys activity, and the data confirm this result with no
export detected using either assay system The K73A
and Y154S mutants are active, as expected from
previ-ous studies [26] and so too are the D211A and D211N
mutants that were described as completely or partially
inactive, respectively, in a previous study [27] In Fig 9, no translocation defects are apparent with either D211A or D211N and these mutants were again
A
B
Fig 8 Mutations in several residues of the cytoplasmic loops in TatC cause no detect-able loss of translocation activity (A) TatC mutants carrying substitutions in the 1st cytoplasmic loops were tested for export of TorA-GFP as described in Fig 2; mutations were made within the pEXT-ABC vector and the Figure shows assays using pEXT-ABC
as a control (B) Similar tests carried out using TatC mutants carrying substitutions in
TM spans (numbered above the lanes).
A
B
Fig 9 Effects of mutations in the periplasmic loops of TatC Muta-tions in the three periplasmic loops of TatC (numbers indicated above the lanes) were assessed using export assays for TorA (upper panel) or TorA-GFP (lower panel) as described earlier for other mutants (Fig 2).