In Vivo Interaction between the Polyprenol Phosphate Mannose Synthase Ppm1 and the Integral Membrane Protein Ppm2 from Mycobacterium smegmatis Revealed by a Bacterial Two-hybrid System*,

Alain R. BaulardDagger §, Sudagar S. Gurcha, Jean Engohang-NdongDagger , Kamila Gouffi||, Camille LochtDagger , and Gurdyal S. Besra**

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-beta -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.

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 Delta (ara, leu)7697 Delta lacX74 Delta phoADelta 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).

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-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).

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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 alpha -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.

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 (25.7·10-2 ± 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.

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.


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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.

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).


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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 alpha -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.

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.


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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

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 alpha -isoprene residue.

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.


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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.

    ACKNOWLEDGEMENTS

We thank D. Ladant and G. Karimova for the gift of plasmids pT25 and pT18 and of E. coli DHP1.

    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

    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..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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