From the INSERM U447, Institut Pasteur de Lille - Institut de Biologie de Lille, 59019 Lille, France, ¶ School of
Biosciences, The University of Birmingham, Edgbaston, B15 2TT
Birmingham, United Kingdom, and
Laboratoire de Chimie
Bactérienne, UPR9043 CNRS, Institut de Biologie Structurale et
Microbiologie, 13402 Marseille, France
Received for publication, August 4, 2002, and in revised form, October 16, 2002
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ABSTRACT |
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Dolichol
phosphate-mannose (Dol-P-Man) is a mannose
donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the
way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment
(TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the
small hydrophobic proteins Dpm3 to be properly stabilized in the
endoplasmic reticulum membrane. In Mycobacterium
tuberculosis, the Polyprenol-P-Man synthase
MtPpm1 is involved in the biosynthesis of the cell
wall-associated glycolipid lipoarabinomannan. MtPpm1 is
composed of two domains. The C-terminal catalytic domain is homologous
to eukaryotic Dol-P-Man synthases. The N-terminal domain of
MtPpm1 contains six TMS that anchor the enzyme in the
cytoplasmic membrane. In contrast, in Mycobacterium
smegmatis, orthologs of the two domains of MtPpm1 are
encoded by two distinct open reading frames, Msppm1 and
Msppm2, organized as an operon. No TMS are predicted in
MsPpm1, and subcellular fractionation experiments indicate
that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline
phosphatase insertions showed that MsPpm2 is an integral
membrane protein. Using a recently developed bacterial two-hybrid
system, it was found that MsPpm2 interacts with
MsPpm1 to stabilize the synthase MsPpm1 in the
bacterial membrane. This interaction is reminiscent of that of
mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1
as demonstrated by the capacity of the two domains of
MtPpm1 to spontaneously interact when co-expressed
in E. coli.
Dolichol phosphate mannose
(Dol-P-Man)1
synthase transfers mannose (Man) from GDP-Man to polyisoprenoid
dolichol phosphate (Dol-P). Dol-P-Man is a
mannosyl donor in pathways leading to N-glycosylation
(reviewed in Refs. 1 and 2), the synthesis of
glycosylphosphatidylinositol anchors (3, 4), O-mannosylation of fungal proteins (4), and the construction of bacterial cell walls
and protozoan glycocalyx (5, 6).
The well characterized Dol-P-Man synthases fall into two
distinct groups. One group contains the Schizosaccharomyces
pombe, Caenorhabditis briggsiae, and mammalian
Dol-P-Man synthases. The other group includes the
Saccharomyces cerevisiae, Ustilago maydis, and
Trypanosoma brucei Dol-P-Man synthases. Enzymes
of the mammalian class lack the C-terminal hydrophobic domain that is
characteristic of the S. cerevisiae class of
Dol-P-Man synthases (7). The C-terminal hydrophobic domain
has been proposed to stabilize the yeast enzyme by anchoring it in the
cytoplasmic membrane. More recently, the mammalian Dol-P-Man
synthase Dpm1 has been shown to interact with the transmembrane protein
Dpm3 that retains and stabilizes the synthase in the endoplasmic
reticulum membrane (8, 9), thereby functionally replacing the
missing C-terminal hydrophobic domain.
In mycobacteria, polyprenol phosphate mannose
(Polyprenol-P-Man), closely related to Dol-P-Man,
is involved in lipoarabinomannan biosynthesis (10, 11), a key
immunomodulator implicated in tuberculosis pathogenesis (12). Based on
its homology with the human Dol-P-Man synthase Dpm1, we
recently identified and characterized a Polyprenol-P-Man
synthase (MtPpm1) from Mycobacterium tuberculosis (13). Unexpectedly, MtPpm1 is composed of two domains. The
Dol-P-Man homologous domain is located in the C-terminal
part of the protein, whereas the N-terminal domain possesses a low
degree of similarity with a variety of bacterial acyltransferases.
Interestingly, when genetically disconnected from the N-terminal
domain, the catalytic C-terminal domain of MtPpm1 was still
able to catalyze Polyprenol-P-Man synthesis in mycobacteria.
However, further experiments also demonstrated that the addition of the
disconnected N-terminal domain somehow increased the transferase
activity of the C-terminal domain without displaying catalytic
activity by itself (13).
Surprisingly, in related mycobacterial species, such as
Mycobacterium leprae, Mycobacterium avium, and
Mycobacterium smegmatis, orthologs of the two domains are
encoded by two distinct open reading frames organized as an operon.
This observation suggests that MtPpm1 has resulted from the
fusion of two ancestral neighboring open reading frames, still
separated in some related mycobacterial species. According to the
"Rosetta stone theory" (14, 15), the presence in the M. smegmatis genome of adjacent genes encoding MsPpm1 and
MsPpm2 that are both homologs of MtPpm1 encoded
by a single gene in M. tuberculosis suggests that
MsPpm1 and MsPpm2 interact with each other to
exert a function similar to that of MtPpm1.
In this report, we show that MsPpm2 is an integral membrane
protein and, using a bacterial two-hybrid system (16), we demonstrate that the synthase MsPpm1 binds to MsPpm2 in
vivo. As observed with mammalian Dol-P-Man synthases,
MsPpm1 also lacks the characteristic hydrophobic C terminus.
Thus, the MsPpm1-MsPpm2 interaction is reminiscent of that of the mammalian Dpm1 with Dpm3 (8, 9). In
contrast, MsPpm1 is functionally active when expressed in
Escherichia coli, similarly to the S. cerevisiae
group of Dol-P-Man synthases (7, 17). As a consequence,
MsPpm1 may constitute a new intermediate group of
Polyprenol-P-Man synthases.
Bioinformatics--
The transmembrane segments (TMS) of
MtPpm1 and MsPpm2 were predicted using the
following seven methods available on the World Wide Web: TMHMM
(www.cbs.dtu.dk/services/TMHMM-2.0/), HMMTOP
(www.enzim.hu/hmmtop/index.html), DAS (www.sbc.su.se/~miklos/DAS/),
TMPRED (www.ch.embnet.org/software/TMPRED_form.html), MEMSAT2
(bioinf.cs.ucl.ac.uk/psiform.html), TOPPRED
(www.sbc.su.se/~erikw/toppred2/), and PHDHTM
(www.embl-heidelberg.de/predictprotein/submit_adv.html#top).
Bacterial Strains and Growth Conditions--
All cloning steps
were performed in E. coli XL1-Blue (Stratagene, La Jolla,
CA). M. smegmatis mc2155 was a generous gift
from W. R. Jacobs, Albert Einstein College of Medicine, Bronx, NY
(18) and was transformed as described previously (19). Recombinant
clones were selected on Middlebrook 7H10 agar supplemented with oleic
acid-albumin-dextrose-catalase enrichment (Difco, Detroit, MI)
containing 25 µg/ml kanamycin (Sigma). Liquid cultures of recombinant
M. smegmatis were grown at 37 °C in Luria-Bertani (LB)
broth (Difco) supplemented with 25 µg/ml kanamycin and 0.05% Tween
80. Liquid cultures of E. coli (pUC8) and E. coli
(pUC8-MtPpm1/D2) were grown in LB broth at 37 °C with 100 µg/ml ampicillin to an optical density (OD) at 600 nm of 0.4 and
induced for 4 h with 1 mM
isopropyl- Insertion of phoA Cassettes--
To fuse phoA to
codons 465 or 600 of MtPpm1, phoA was first
amplified by PCR from E. coli XL1-Blue using primers N°226
(5'-GCCATTAAGTCTGGATCCTAACAG-3') and N°228
(5'-CGGACACCAGAGGCGCCTGTTCT-3') and the TaqDNA polymerase (Qiagen, Courtaboeuf, France). The 1421-bp PCR fragment was inserted into the commercially linearized pCR2.1Topo (Invitrogen) to
create pCR2.1-PhoA (alkaline phosphatase). pMV261-MtPpm1
(13) was cut by Asp718 and blunted using the Klenow
polymerase. The resulting 1802-bp fragment was inserted into
pCR2.1-PhoA cleaved with EcoRV. The resulting construct was
then digested with Asp718 and BglII, and the
fragment was gel-purified and inserted into pMV261 (20) cleaved with
the same enzymes to create
pMV261-MtPpm1/PhoA465.
To produce pMV261-MtPpm1/PhoA600, both
pMV261-MtPpm1 and pCR2.1-PhoA were cut with BamHI
and KasI. The 6716-bp fragment from pMV261-MtPpm1
and the 1392-bp fragment carrying phoA were gel-purified and
ligated to produce pMV261-MtPpm1/PhoA600.
Plasmids for the Two-hybrid System--
The region coding
for MtPpm1/D1 (corresponding to
Met1 to Trp593) was amplified by PCR using
primers N°148 (5'-GGACTGCAGCGCAGCTACCCACCA-3') and N°151
(5'-TTGGATCCGGTGGTCATGTAACTCCTC-3'). The amplified 1780-bp fragment was cleaved by PstI and BamHI and
inserted into PstI-BamHI-restricted pT25 (16) to
yield pT25-MtPpm1/D1.
The DNA region corresponding to MtPpm1/D2
(Met594 to Glu874) was amplified by PCR using
primers N°153 (5'-AGCGGTACCGAGGAGTTACATGACCAC-3') and N°154
(5'-GAGCGCCTCGAGCATTCGGTCAC-3'). The 876-bp PCR product was cleaved by
XhoI and KpnI and inserted into pT18 (16)
digested previously with the same enzymes. The resulting plasmid was
termed pT18-MtPpm1/D2.
A similar strategy was used for Msppm1 and
Msppm2. Primers N°200
(5'-AAGGTACCGAGCGTCCCAGGTGAACGTGA-3') and N°201
(5'-CAAAGCTTACCACGCCCCTGGCCCGGTCGA-3') were used to amplify
Msppm1 from M. smegmatis chromosomal DNA. The
805-bp PCR product was cleaved by KpnI and
HinDIII and inserted into pT18 digested previously with the
same enzymes. The resulting plasmid was termed pT18-MsPpm1.
Primers N°198 (5'-CCCTGCAGTCACCGATGACGACCCCCTC-3') and N° 199 (5'-GGGTCGGGCGGACGGTGGCAGGA-3') were used to amplify a 1853-bp fragment
of Msppm1. Subsequently, the DNA fragment was cut with
PstI and BamHI and then inserted into pT25 cut
with the same enzymes to yield pT25-MsPpm2. All inserts were
verified by DNA sequencing.
Random phoA Insertion into pT25-MsPpm2--
TnTap is a
Tn5-based mini-transposon carrying the signal sequenceless
phoA reporter gene, lacking a promoter and translation initiation signals (21). To construct a transposable element suitable
for in vitro transposition, the phoA-neo cassette
was amplified by PCR using tap-xba
(5'-GCTCTAGATTTGCGGCCGCGTCGACCTGCA-3') and tap-sac
(5'-TCCGAGCTCTTTGAAAACCTGTACTTCCAG-3') as primers and TnTap as
the template. The PCR product was digested with
XbaI-SacI and inserted into pMOD<MCS>
(Epicentre, Madison, WI) to yield pMODTap. The transposable element
carrying the phoA-neo cassette flanked by hyperactive 19-bp
mosaic end (ME) was amplified by PCR using pMODTap as template and
pmod-fp (5'-CGGAATTCATTCAGGCTGCGCAACTGT-3') and pmod-rp
(5'-CGGGATCCGTCAGTGAGCGAGGAAGCGGAAG-3') as primers. The random in
vitro transposition of the ME-phoA-neo-ME transposable element into pT25-MsPpm2 was performed by using the
EZ::TN transposase according to the manufacturer's
instructions (Epicentre, Madison, WI). The transposition reaction
product was introduced into E. coli CC118
(araD139 Alkaline Phosphatase Activity Assay--
Alkaline phosphatase
activity was assayed in M. smegmatis and in E. coli by measuring the rate of p-nitrophenyl-phosphate (Sigma) hydrolysis in intact cells as described previously (22, 23).
Enzymatic reactions were performed in triplicate in the dark at
37 °C. Reactions were stopped with 100 µl of 1 M
KH2PO4, 0.1 M EDTA (pH 8.0), and
the OD at the appropriate wavelength was measured. The activity is
expressed in arbitrary units. Units of activity for M. smegmatis = A420 nm × mg of
protein Preparation of Enzyme Fractions and Polyprenol Phosphate Mannose
Synthase Assay--
E. coli(pT18-MtPpm1/D2),
E. coli(pT25-MtPpm1/D2), E. coli(pT18-MtPpm1/D2 + pT25-MtPpm1/D2), and
E. coli(pT18-MsPpm1) were grown, washed, and
sonicated as described previously (13). For further fractionation, the
lysate of E. coli(pT18-MsPpm1) was centrifuged at
27,000 × g for 20 min at 4 °C. The membrane
fraction was obtained by further centrifugation of the 27,000 × g supernatant at 100,000 × g for 1 h
at 4 °C. The supernatant was carefully removed, and the membranes
were gently resuspended in buffer 50 mM MOPS (adjusted to
pH 8.0 with KOH), 5 mM Computer-predicted Topology of Mycobacterial Polyprenol-P-Man
Synthases--
Consistent with the previously shown membrane
association of the Polyprenol-P-Man synthase activity in
M. tuberculosis (13), MtPpm1 contains TMS located
in its N-terminal domain (D1). Since MtPpm1 uses GDP-Man as
a substrate (11), its catalytic domain (D2) would be expected to be
cytoplasmic where GDP-Man is readily available. To predict the topology
of MtPpm1, we used seven different programs available
on the internet (TMHMM, HMMTOP, DAS, TMPRED, MEMSAT2,
TOPPRED, and PHDHTM) since the reliability of topology predictions
increases substantially when different prediction methods are compared
(25). When applied to MtPpm1, all seven programs converged
to the presence of several TMS, most of which was located in the
N-terminal half of the protein (D1), strongly suggesting that
MtPpm1 is an integral membrane protein (Fig.
1A). However, the algorithms
did not converge on the precise number of TMS and on the orientation of
the catalytic domain (D2) of MtPpm1.
In other mycobacterial species, such as M. leprae, M. avium, and M. smegmatis, the orthologs of the two
MtPpm1 domains, referred to as D1 and D2, are encoded by
separate genes. The orthologs of the catalytic domain (D2) of
MtPpm1 are named Ppm1, and the orthologs of the N-terminal
non-catalytic domain of MtPpm1 (D1) are named Ppm2.
Application of the topology prediction algorithms on Ppm2 from M. smegmatis (MsPpm2) (Fig. 1B), M. leprae (MlPpm2), and M. avium
(MaPpm2) (data not shown) strongly suggests that these
proteins, similar to the MtPpm1/D1 domain, are anchored in
the bacterial membrane. In contrast, none of the seven methods predicted the presence of TMS in MsPpm1, MlPpm1,
or MaPpm1, nor were signal sequences predicted in the
N-terminal part of these proteins. These results suggest a cytoplasmic
localization for these proteins and, by analogy, a cytoplasmic
localization for the catalytic domain of MtPpm1.
Experimental Determination of the Cellular Localization of the
Catalytic Domain of MtPpm1--
In Gram-negative bacteria, the
topology of integral membrane proteins has largely been studied using
PhoA fusions since the sites at which alkaline phosphatase has high
enzymatic activity normally correspond to periplasmic domains of the
membrane protein. In a previous report, we have shown that PhoA fusions
can be used in mycobacteria to determine the cytoplasmic or
extracytoplasmic location of proteins or protein domains (22). To
determine the cellular location of the catalytic domain of
MtPpm1, we made use of PhoA fusions to various parts of the
protein. The phoA gene was thus inserted into
pMV261-MtPpm1, a plasmid previously shown to allow for
overexpression of Mtppm1 in mycobacteria (13). The
phoA gene was inserted in-frame with
Mtppm1 either before (codon 465) or after (codon
600) the last predicted TMS (amino acids 509-527) of MtPpm1
(Fig. 1A). The resulting constructs, named
pMV261-MtPpm1/PhoA465 and
pMV261-MtPpm1/PhoA600, respectively, were
introduced into M. smegmatis mc2155, and the
recombinant clones were tested for their ability to hydrolyze
X-phosphate. When compared with untransformed bacteria, M. smegmatis
mc2155(pMV261-MtPpm1/PhoA465)
exhibited an increased capacity to hydrolyze
X-phosphate (25.7·10 Experimental Determination of the Localization of
MsPpm1--
Computer predictions suggested a cytoplasmic location
for MsPpm1. These predictions are consistent with the
cytoplasmic location of GDP-Man and, by analogy, is strengthened by the
cytoplasmic orientation of MtPpm1/D2. To experimentally
localize MsPpm1, E. coli(pT18-MsPpm1)
cells were fractionated, and both the soluble and membrane fractions
were tested for their respective Polyprenol-P-Man synthase
activities. Experiments performed in triplicate on two independent
clones indicated that 94.6% (average [14C]Man
incorporation, 276,249 cpm) of the Polyprenol-P-Man
transferase activity is associated with the cytosolic fraction as
compared with 5.4% (average [14C]Man incorporation,
15,719 cpm) associated with the membrane fraction. These data confirm
that MsPpm1, when expressed independently of
MsPpm2 in E. coli, is located in the bacterial cytosol.
In Vivo Interaction between MsPpm1 and MsPpm2--
The existence
of a strictly conserved operon architecture for ppm2-ppm1 in
M. leprae, M. avium, and M. smegmatis
suggests that the two corresponding proteins participate in a common
structural complex or metabolic pathway, consistent with the presence
in M. tuberculosis of a single gene (Mtppm1)
resulting from the fusion of the two corresponding open reading frames.
It is thus likely that MsPpm1 and MsPpm2 are able
to directly interact with each other. Among the general methodologies
to identify interactions between proteins, the yeast two-hybrid system
represents the most powerful in vivo approach. However, the
detection of the interaction between MsPpm1 and
MsPpm2 using a yeast two-hybrid system may be impaired by
the association of MsPpm2 with the membrane. Therefore, to
test whether MsPpm2 can interact with MsPpm1, a
recently described bacterial two-hybrid system (16) was used, which is
suitable to study protein interactions even if one of the partners is
membrane-associated. In this system, the interaction of two proteins
results in functional complementation between two domains of the
adenylate cyclase (CyaA) from Bordetella pertussis, leading
to cAMP synthesis. As a soluble regulatory molecule, cAMP is then able
to activate cAMP-dependent transcriptional events, which
can be easily monitored (16).
The entire MsPpm1 was thus genetically fused to the 18-kDa
domain of CyaA to produce pT18-MsPpm1, and MsPpm2
was fused to the 25-kDa domain of CyaA to produce
pT25-MsPpm2. The cyclase-deficient E. coli strain
DHP1 was co-transformed with the two constructs, and the resulting
recombinant colonies were tested for their ability to metabolize
maltose on McConkey agar plates supplemented with ampicillin and
chloramphenicol. As shown in Fig.
2A, E. coli DHP1 containing both fusions were able to metabolize maltose and appeared red, whereas colonies containing pT18-MsPpm1 with pT25 or
pT25-MsPpm2 with pT18 were not able to metabolize maltose
and remained white on McConkey-maltose plates. These results
demonstrate specific interactions between MsPpm1 and
MsPpm2.
Interaction between the Two Domains of MtPpm1 Is
Conserved--
Since MsPpm1 and MsPpm2
correspond to domains D2 and D1 of MtPpm1, respectively,
encoded by a single gene, we wanted to test whether D1 and D2 can also
interact with each other, even in the absence of a covalent link
between them. We therefore genetically disconnected the two domains and
co-expressed them within the bacterial two-hybrid system.
MtPpm1/D2 was fused to T18, and MtPpm1/D1 was
fused to T25. After introduction of both constructs into E. coli DHP1, the colonies were found to be able to metabolize
maltose on McConkey agar plates, in contrast to the cells containing
pT18-MtPpm1/D2 and pT25 or pT25-MtPpm1/D1 and
pT18 (Fig. 2B). This result demonstrates that the two
domains D1 and D2 of MtPpm1 can specifically interact with
each other, even if they are not covalently linked.
Trans-species in Vivo Interaction--
We have previously shown
that MtPpm1/D1 has no Dol-P-Man synthase activity
but increases Dol-P-Man production when its gene is
overexpressed in M. smegmatis, suggesting that the M. tuberculosis MtPpm1/D1 may interact with the M. smegmatis
MsPpm1 enzyme and may thereby increase its enzymatic activity. To
test this hypothesis, pT18-MsPpm1 and
pT25-MtPpm1/D1 were both introduced into E. coli DHP1. The recombinant bacteria showed a red color on McConkey-maltose plates (Fig. 2C), demonstrating that MsPpm1 can
interact with MtPpm1/D1. Vice versa, when
pT18-MtPpm1/D2 and pT25-MsPpm2 were introduced
into E. coli DHP1, the recombinant bacteria also displayed a
red color on McConkey-maltose plates (Fig. 2C).
Partial Determination of the Topology of T25-MsPpm2 in E. coli--
MsPpm2 and MtPpm1/D1 were both predicted to
contain many TMS, suggesting that they are integral membrane proteins.
Moreover, PhoA fusions demonstrated that MtPpm1 is
associated with the membrane. Here, PhoA fusions were used to determine
the global topology of MsPpm2. TnTap (21) was randomly
transposed in vitro into pT25-MsPpm2 using the
EZ::TN transposase (Epicentre, Madison, WI), and the
recombinant plasmids were introduced into E. coli CC118.
Blue colonies obtained as a result of PhoA activity were selected on
agar plates containing X-phosphate. The insertion positions of the
mini-transposon were determined by DNA sequencing for 25 blue colonies
that contained the phoA cassette in the Msppm2 gene. Activities were measured according to Manoil (24). The results
confirm that MsPpm2 is an integral membrane protein and provide some insight into the topology of the protein (Fig.
3). The blue color obtained by insertion
of phoA after codon 495 is similar to the results obtained
after insertion of phoA in the corresponding region of
MtPpm1, indicating the extracytoplasmic location of this
region for both proteins. Four of the seven algorithms predicted the
existence of a TMS at approximately residue 450 (Fig. 1B).
However, the PhoA fusion data indicate that this TMS is unlikely since
PhoA fusions upstream and downstream of residue 450 resulted in strong
phosphatase activity. No TnPhoA insertions leading to a PhoA-positive
phenotype were obtained in the region between Ala160 and
Val222. However, based on the phosphatase activity of the
PhoA insertions in the Gly88 to Gly116 region
and the unanimous prediction of TMS Tyr117 to
Ser134 and Ala160 to Ala139, this
region of MsPpm1 is likely to be periplasmic. It is possible that insertions of TnTap in this region lead to unstable hybrid proteins. One colony with a very low phoA activity (phoA activity = 1.15 units) obtained after transposition revealed an insertion of
TnTap in the region corresponding to Trp239 to
Ala252, confirming that this portion of the protein is
cytoplasmic (Fig. 3). Interestingly, the topology determined by the
PhoA fusions together with the TMS predicted by all the algorithms used
indicate that only very few amino acids of MsPpm2
are located at the cytoplasmic side of the membrane and may
thus be available for the interaction with MsPpm1 (Figs. 3
and 5).
Influence of T25-MtPpm1/D1 on the Catalytic Activity of
T18-MtPpm1/D2--
To test whether the physical interaction
between MtPpm1/D2 and MtPpm1/D1 has an impact on
the Polyprenol-P-Man synthase activity, we measured
Dol-P-Man synthesis in E. coli transformed with
pT18-MtPpm1/D2 and compared it with that measured in
E. coli co-transformed with both pT18-MtPpm1/D2
and pT25-MtPpm1/D1. As shown in Fig.
4, E. coli co-producing
T18-MtPpm1/D2 and T25-MtPpm1/D1 synthesized 4.8-fold more Dol-P-Man than E. coli cells
producing T18-MtPpm1/D2 alone, demonstrating that the
interaction between MtPpm1/D1 and MtPpm1/D2 leads
to an increase in the catalytic activity of MtPpm1/D2.
Due to the lack of a transporter, GDP-Man, a widely used mannosyl
donor, is generally unable to cross biological membranes. Consequently,
the enzymes that use GDP-Man are predicted to be located in the
cytosol. In eukaryotes, Dol-P-Man synthase (EC 2.4.1.83)
catalyzes the transfer of Man from GDP-Man to dolichol monophosphate,
forming Dol-P-Man, which is subsequently translocated through the endoplasmic reticulum membrane to be used as a mannosyl donor in the lumen of the endoplasmic reticulum. The
Dol-P-Man synthases are usually associated with the
cytoplasmic side of the membrane, and two different mechanisms have
been proposed for this membrane association (7, 17). In S. cerevisiae, U. maydis, and T. brucei Dpm1
contains a C-terminal hydrophobic domain proposed to anchor the enzyme
into the lipid bilayer of the membrane. In contrast, mammalian
Dol-P-Man synthases are associated with the endoplasmic
reticulum membrane through specific interactions with the hydrophobic
protein Dpm3, in turn, stabilized by Dpm2 (8, 9). In mycobacteria, the
biosynthesis of cell wall glycoconjugates, such as lipoarabinomannan,
also requires the transfer of Man residues through the bacterial
membrane. The Man carriers in mycobacteria are
Polyprenol-P-Man, isoprenoid derivatives that are shorter than Dol-P-Man and contain an unsaturated
In this study, we found that the synthesis of
Polyprenol-P-Man in M. smegmatis is also mediated
by two proteins, one being the catalytic MsPpm1 and the
other being the "helper" protein MsPpm2.
MsPpm1 and MsPpm2 interact in vivo
when co-produced in E. coli, and MsPpm2 enhances
the catalytic activity of MsPpm1. Thus, as for mammalian
Dpm1, MsPpm1 lacks the C-terminal hydrophobic domain but
interacts instead with the integral membrane protein MsPpm2.
This finding suggests a role for the two-domain structure of Ppm1 from
M. tuberculosis, in which the first domain would anchor
the protein into the bacterial membrane. As shown by the bacterial
two-hybrid assay, the two domains of MtPpm1 are able to
interact with each other even in the absence of covalent linkage between them. Moreover, the interaction between the two domains of
MtPpm1 independently produced in E. coli results
in enhanced Polyprenol-P-Man production. In addition, the
capacity of interaction has been conserved between two mycobacteria
that differ with respect to the genetic structure of their
ppm1 genes, as illustrated by the capacity of
MsPpm1 and MsPpm2 to interact with
MtPpm1/D1 and MtPpm1/D2, respectively.
By analogy with the S. cerevisiae Dpm1 (ScDpm1),
MtPpm1 contains a hydrophobic region responsible for its
attachment to the bacterial membrane. However, in M. tuberculosis, the hydrophobic region is located in the N-terminal
portion of the protein and is larger than the C-terminal hydrophobic
domain of ScDpm1. On the other hand, MsPpm1 may
be, to some extent, compared with the human Dpm1 as they both lack the
C-terminal transmembrane domain and interact with a polypeptide
(MsPpm2 versus human Dpm3) that is localized in
the membrane. In contrast, MtPpm1/D2 and MsPpm1 are active when produced in E. coli, whereas human Dpm1 is
not. Thus, as illustrated in Fig. 5, we
propose to extend the family of Polyprenol-P-Man synthases by including
two new members that use two original strategies of membrane
association.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranose. Large scale cultures
of bacteria were grown as described above, harvested by centrifugation,
washed with phosphate-buffered saline, and stored at
20 °C until
further use. E. coli DHP1 is an adenylate cyclase deficient
(cya) derivative of DH1 (F
, glnV44(AS),
recA1, endA1, gyrA96 (Nalr), thi1, hsdR17, spoT1, rfbD1) (16).
Protein-protein interactions leading to the cytoplasmic production and
assembly of a functional adenylate cyclase in E. coli DHP1
were detected by the ability to ferment maltose. Thus, clones were
checked for their ability to form red colonies at 30 °C on freshly
prepared MacConkey agar plates containing 1% maltose and supplemented
with 25 µg/ml kanamycin, 25 µg/ml chloramphenicol, and 100 µg/ml
ampicillin when appropriate.
(ara, leu)7697
lacX74
phoA
20 galE galK thi rpsE rpoB
argEam recA1) by electroporation. Blue
AmpR and KanR colonies were selected on agar
plates containing corresponding antibiotics and 40 µg/ml of
X-Phosphate (Sigma).
1 × min
1 (23), units of
activity for E. coli = (A420
nm
(1.75 × A550 nm)) × 103 × min
1 × A600
nm
1 × ml of culture
1 (24).
-mercaptoethanol, 10 mM MgCl2 at a protein concentration of 20 mg/ml. Protein concentrations were determined using the BCA protein
assay reagent kit (Pierce). Reaction mixtures for assessing
[14C]Man transfer consisted of 2.4 µM
GDP-[14C]Man (PerkinElmer Life Sciences, 321 mCi/mmol, 0.125 µCi), 62.5 µM ATP, 10 µM
MgCl2, and crude or fractionated subcellular preparations corresponding to 80 µg of protein in a final volume of 80 µl. Exogenous lipid monophosphate substrates (C95, dolichol
monophosphate) were added to the reaction mixtures at a final
concentration of 0.125 mM in 0.25% CHAPS. The reaction
mixtures were then incubated at 37 °C for 30 min. The reaction was
terminated, and the lipids were extracted as described previously (13).
Thin-layer chromatography using 10% of the reaction mixture were
conducted on aluminum-backed plates of Silica Gel 60 F254
(Merck) using
CHCl3/CH3OH/NH4OH/H2O (65/25/0.4/3.6). Autoradiograms were obtained by exposing chromatograms to Kodak X-Omat AR films at
70o for 4-5 days. In
parallel, autoradiograms were exposed to a PhosphorImager screen, and
radioactivity was quantified using a PhosphorImager detector (Storm,
Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, topology prediction of
MtPpm1. The long gray rectangle corresponds to
the first domain (Met1-Tyr593) of
MtPpm1 (MtPpm1/D1). The dashed
rectangle depicts the catalytic domain
(Met594-Glu874)
(MtPpm1/D2). The black boxes correspond
to predicted transmembrane -helices. The proposed location of the N
terminus by the various programs is indicated as intracytoplasmic
(i) or extracytoplasmic (o). Precise positions of
TMS are provided as Supplemental Data. Vertical arrows show
locations of PhoA insertions (amino acid 465 and amino acid 600). B,
topology prediction for MsPpm2. Precise positions of TMS are
provided as Supplemental Data.
2 ± 0.011 OD
unit(s)·min
1·mg
1). In contrast,
M. smegmatis mc2155
(pMV261-MtPpm1/PhoA600) did not hydrolyze
X-phosphate (
0.178·10
2 ± 0.014 OD
unit(s)·min
1·mg
1). These results
indicate that amino acid 465 of MtPpm1 is localized in a
portion of the protein facing the outside of the cell membrane, whereas
the region surrounding amino acid 600 is cytoplasmic. This topology is
in agreement with four out of the seven computer-assisted predictions
(HMMTOP, DAS, MEMSAT, and PHDHTM). Moreover, these results are
consistent with the predicted cytoplasmic location of the
MtPpm1/D2 orthologs, Ppm1 from M. smegmatis,
M. avium, and M. leprae.
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Fig. 2.
Bacterial two-hybrid system assays.
A, in vivo interaction between MsPpm1
and MsPpm2. pT18-MsPpm1 carries a fusion between
Msppm1 and a fragment corresponding to the T18 fragment of
B. pertussis CyaA. pT25-MsPpm2 carries a fusion
between Msppm2 and a fragment corresponding to the T25
fragment of CyaA. No red color on McConkey/Maltose agar plates was
detected when pT18-MsPpm1 + pT25 or pT25-MsPpm2 + pT18 were introduced into E. coli DHP1. B,
in vivo interaction assay between the two domains of
MtPpm1. When co-introduced into E. coli DHP1,
pT25-MtPpm1/D1 and pT18-MtPpm1/D2 yielded red
transformants on MacConkey/maltose. C, trans-species
in vivo interaction assay. The two domains of
MtPpm1 (MtPpm1/D1 and MtPpm1/D2) were
tested for their capacity to interact with MsPpm1 and
MsPpm2, respectively.
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Fig. 3.
Proposed model of MsPpm2
membrane topology based on TnPhoA insertions and computer-assisted TMS
predictions. The six putative trans-membrane -helices are
boxed, and their positions are labeled in white.
Arrows indicate the positions of the alkaline phosphatase
insertions into MsPpm2. Numbers in
parentheses represent the PhoA activity units for the
corresponding MsPpm2-PhoA fusions. The background PhoA
activity for E. coli CC118 is 0.4 ± 0.1 units.
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Fig. 4.
Incorporation of [14C]Man from
GDP-[14C]Man into exogenous C95
Dol-P using extracts from E. coli(pT18-MtPpm1/D2), E. coli(pT25-MtPpm1/D1), and E. coli(pT18-MtPpm1/D2 + pT25-MtPpm1/D1). Arrows indicate the
origin of the migration, the solvent front, and the C95
Dol-P-Man. Radioactivity was quantified using a
PhosphorImager detector (Storm, Amersham Biosciences). The average
incorporation of [14C]Man was as follows: E. coli(pT18-MtPpm1/D2), 18,080 cpm; E. coli(pT18-MtPpm1/D2 + pT25-MtPpm1/D1),
87,330 cpm; and E. coli(pT25-MtPpm1/D1), 205 cpm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-isoprene residue.
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Fig. 5.
Proposed comparative model for the
stabilization of Dol-P-Man synthases and
Polyprenol-P-Man synthases in membranes. As shown
in A, the M. smegmatis
Polyprenol-P-Man synthase MsPpm1 interacts with
membrane-associated MsPpm2. LAM,
lipoarabinomannan. As shown in B, the M. tuberculosis Polyprenol-P-Man synthase is a
single-component enzyme harboring multiple TMS at the N terminus. As
shown in C, the S. cerevisiae
Dol-P-Man synthase is a single-component enzyme with a TMS
near the C terminus (7). As shown in D, human
Dol-P-Man synthase consists of three subunits. Dpm2
associates with Dpm3, which in turn stabilizes the catalytic subunit
Dpm1 (8, 9).
MsPpm1 is a cytoplasmic soluble protein, whereas MsPpm2 is a hydrophobic protein containing of 6-8 predicted TMS. Due to the hydrophobic nature of MsPpm2, we have not been able to purify MsPpm2 to test physical interactions with MsPpm1 in vitro. However, the interactions could be studied in vivo by using a bacterial two-hybrid system based on the functional complementation between two domains of the cyaA gene from B. pertussis, which catalyzes the production of cAMP from ATP. Both the substrate and the product of the reaction are soluble in the cytosol, even if one of the partners of the two-hybrid system is anchored in the inner face of the membrane. This approach allowed us to demonstrate a specific interaction between the transmembrane protein MsPpm2 and its soluble catalytic partner MsPpm1. The bacterial two-hybrid system did not abolish the enzymatic activity of MtPpm1/D2 or MsPpm1, nor did it affect the enhancing effects of MtPpm1/D1 and MsPpm2 on the enzymatic activities of MtPpm1/D2 and MsPpm1, respectively.
Based on both the TMS prediction methods and the PhoA insertions, we deduced a topological model of MsPpm2 (Fig. 3). Surprisingly, the proposed model suggests that only few amino acids of MsPpm2 are located at the cytoplasmic side of the membrane and may thus be available for the interaction with MsPpm1. Mutagenesis of residues of the N-terminal tail and of the first and second intracellular predicted loops of MsPpm2 may help to identify amino acids implicated in the interaction with MsPpm1.
The physical interaction demonstrated between MsPpm1 and MsPpm2 and the existence of MtPpm1 in M. tuberculosis containing the two domains fused into a single protein are an illustration of the Rosetta stone theory (15). This theory proposes that interactions between a protein of unknown function and a well characterized protein suggest that the function of the former is somewhat related to that of the latter. In accordance with this theory, the implication of MsPpm2 in the Polyprenol-P-Man synthesis pathway may have been inferred from the architecture of MtPpm1 (14).
In conclusion, the interaction of MsPpm1 with
MsPpm2 allows us now to understand how the
Polyprenol-P-Man synthase activity of M. smegmatis is associated with the membrane in the absence of a TMS
in MsPpm1. Therefore, M. smegmatis uses a
membrane-targeting strategy similar to that of the mammalian
Dol-P-Man synthases.
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ACKNOWLEDGEMENTS |
---|
We thank D. Ladant and G. Karimova for the gift of plasmids pT25 and pT18 and of E. coli DHP1.
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FOOTNOTES |
---|
* This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Medical Research Council Co-operative Group (Grant 49343)(UK), and The Wellcome Trust (Grant 058972).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary data showing the precise
positions of TMS depicted in Fig. 1.
§ To whom correspondence should be addressed. Tel.: 33-320871155; Fax: 33-320871158; E-mail: alain.baulard@pasteur-lille.fr.
** Supported as a Lister Institute-Jenner Research Fellow.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M207922200
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ABBREVIATIONS |
---|
The abbreviations used are: Dol-P-Man, dolichol phosphate-mannose; Polyprenol-P-Man, polyprenol phosphate mannose; PhoA, alkaline phosphatase; X-phosphate, 5-bromo-4-chloro-3-indolyl phosphate; CyaA, adenylate cyclase; TMS, transmembrane segment(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; Sc, Saccharomyces cerevisiae; Mt, Mycobacterium tuberculosis; Ms, Mycobacterium smegmatis, Ma, Mycobacterium avium; Ml, Mycobacterium leprae..
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REFERENCES |
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