Mannitol Permease Research Articles

Phospholipid dependency of membrane protein insertion by the Sec translocon

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli.

The effect of antibiotics on protein diffusion in the Escherichia coli cytoplasmic membrane.

Accumulating evidence suggests that molecular motors contribute to the apparent diffusion of molecules in cells. However, current literature lacks evidence for an active process that drives diffusive-like motion in the bacterial membrane. One possible mechanism is cell wall synthesis, which involves the movement of protein complexes in the cell membrane circumferentially around the cell envelope and may generate currents in the lipid bilayer that advectively transport other transmembrane proteins. We test this hypothesis in Escherichia coli using drug treatments that slow cell wall synthesis and measure their effect on the diffusion of the transmembrane protein mannitol permease using fluorescence recovery after photobleaching. We found no clear decrease in diffusion in response to vancomycin and no decrease in response to mecillinam treatment. These results suggest that cell wall synthesis is not an active contributor to mobility in the cytoplasmic membrane.

Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER.

The mannitol utilization system of the marine bacterium Zobellia galactanivorans.

Mannitol is a polyol that occurs in a wide range of living organisms, where it fulfills different physiological roles. In particular, mannitol can account for as much as 20 to 30% of the dry weight of brown algae and is likely to be an important source of carbon for marine heterotrophic bacteria. Zobellia galactanivorans (Flavobacteriia) is a model for the study of pathways involved in the degradation of seaweed carbohydrates. Annotation of its genome revealed the presence of genes potentially involved in mannitol catabolism, and we describe here the biochemical characterization of a recombinant mannitol-2-dehydrogenase (M2DH) and a fructokinase (FK). Among the observations, the M2DH of Z. galactanivorans was active as a monomer, did not require metal ions for catalysis, and featured a narrow substrate specificity. The FK characterized was active on fructose and mannose in the presence of a monocation, preferentially K(+). Furthermore, the genes coding for these two proteins were adjacent in the genome and were located directly downstream of three loci likely to encode an ATP binding cassette (ABC) transporter complex, suggesting organization into an operon. Gene expression analysis supported this hypothesis and showed the induction of these five genes after culture of Z. galactanivorans in the presence of mannitol as the sole source of carbon. This operon for mannitol catabolism was identified in only 6 genomes of Flavobacteriaceae among the 76 publicly available at the time of the analysis. It is not conserved in all Bacteroidetes; some species contain a predicted mannitol permease instead of a putative ABC transporter complex upstream of M2DH and FK ortholog genes.

Isolation of Escherichia coli mannitol permease, EIImtl, trapped in amphipol A8-35 and fluorescein-labeled A8-35.

Amphipols (APols) are short amphipathic polymers that keep integral membrane proteins water-soluble while stabilizing them as compared to detergent solutions. In the present work, we have carried out functional and structural studies of a membrane transporter that had not been characterized in APol-trapped form yet, namely EII(mtl), a dimeric mannitol permease from the inner membrane of Escherichia coli. A tryptophan-less and dozens of single-tryptophan (Trp) mutants of this transporter are available, making it possible to study the environment of specific locations in the protein. With few exceptions, the single-Trp mutants show a high mannitol-phosphorylation activity when in membranes, but, as variance with wild-type EII(mtl), some of them lose most of their activity upon solubilization by neutral (PEG- or maltoside-based) detergents. Here, we present a protocol to isolate these detergent-sensitive mutants in active form using APol A8-35. Trapping with A8-35 keeps EII(mtl) soluble and functional in the absence of detergent. The specific phosphorylation activity of an APol-trapped Trp-less EII(mtl) mutant was found to be ~3× higher than the activity of the same protein in dodecylmaltoside. The preparations are suitable both for functional and for fluorescence spectroscopy studies. A fluorescein-labeled version of A8-35 has been synthesized and characterized. Exploratory studies were conducted to examine the environment of specific Trp locations in the transmembrane domain of EII(mtl) using Trp fluorescence quenching by water-soluble quenchers and by the fluorescein-labeled APol. This approach has the potential to provide information on the transmembrane topology of MPs.

Membrane sequestration by the EIIB domain of the mannitol permease MtlA activates the Bacillus subtilismtl operon regulator MtlR

In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIB(Mtl) domain of the mannitol permease MtlA (EIICB(Mtl) ). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3' part (EIIB(Mtl) ) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIB(Mtl) domain with the two C-terminal domains of MtlR. Complementation of the Δ3'-mtlA ΔmtlF or ΔmtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3'-mtlA had no effect. Moreover, synthesis of EIIB(Mtl) in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIB(Mtl) fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIB(Mtl) lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIB(Mtl) . Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICB(Mtl) in order to be functional.

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by theEscherichia colisignal recognition particle

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.

Spatial and temporal organization of the E. coli PTS components

The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to β-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.

Structure of the Cytoplasmic Loop between Putative Helices II and III of the Mannitol Permease ofEscherichia coli: A Tryptophan and 5-Fluorotryptophan Spectroscopy Study

In this work, four single tryptophan (Trp) mutants of the dimeric mannitol transporter of Escherichia coli, EII(mtl), are characterized using Trp and 5-fluoroTrp (5-FTrp) fluorescence spectroscopy. The four positions, 97, 114, 126, and 133, are located in a region shown by recent studies to be involved in the mannitol translocation process. To spectroscopically distinguish between the Trp positions in each subunit of dimeric EII(mtl), 5-FTrp was biosynthetically incorporated because of its much simpler photophysics compared to those of Trp. The steady-state and time-resolved fluorescence methodologies used point out that all four positions are in structured environments, both in the absence and in the presence of a saturating concentration of mannitol. The fluorescence decay of all 5-FTrp-containing mutants was highly homogeneous, suggesting similar microenvironments for both probes per dimer. However, Stern-Volmer quenching experiments using potassium iodide indicate different solvent accessibilities for the two probes at positions 97 and 133. A 5 A two-dimensional (2D) projection map of the membrane-embedded IIC(mtl) dimer showing 2-fold symmetry is available. The results of this work are in better agreement with a 7 A projection map from a single 2D crystal on which no symmetry was imposed.

Improving d-mannitol productivity of Escherichia coli: Impact of NAD, CO 2 and expression of a putative sugar permease from Leuconostoc pseudomesenteroides

The highly productive whole-cell biotransformation of d-fructose to d-mannitol with recombinant, resting cells of Escherichia coli BL21(DE3) requires the combined expression of mdh, fdh and glf which encode mannitol and formate dehydrogenases and a sugar facilitator, respectively. However, long-term stability of the system was restricted, possibly due to loss of the cofactor NAD, high concentrations of formate, formation of CO 2 affecting the internal pH of the cells, accumulation of high intracellular concentrations of d-mannitol, and export of d-mannitol. Downstream of the mdh gene of Leuconostoc pseudomesenteroides, we identified an open reading frame encoding for a putative mannitol permease. The gene was cloned and expressed in E. coli. Biochemical analyses revealed an activity as secondary carrier for d-fructose. Therefore, the carrier was named FupL and participation in d-mannitol transport was excluded. In biotransformation experiments, the productivity of d-mannitol formation obtained with the strain expressing the additional fupL gene was enhanced by 20%.

Structural and Mechanistic Characterization of the Mannitol Transporter from E. coli using 5-fluorotryptophan as a Spectroscopic Probe

The mannitol permease (EIImtl) of E. coli is an integral membrane protein re- sponsible for the active transport of mannitol over the cytoplasmic membrane. It is composed of three domains: two cytosolic domains A and B, and trans- membrane C domain. The structures of A an B domains were solved by X- ray crystallography and NMR spectroscopy. For the transmembrane C domain a 5A 2D projection map is available and several topology models. EIImtl is functional as a dimer. A dozen single Trp mutants of EIImtl were made and 5-fluoroTrp was in- corporated in the C domain with R 95% efficiency. Compared to Trp, 5- fluoroTrp shows the advantage that the fluorescence decay kinetics is much more homogeneous. 5-fluoroTrp is also a good energy donor, which makes it suitable for resonance energy transfer (RET) experiments. An an- alogue of mtl, azi-mtl, was used as an acceptor. Steady state fluorescence spectroscopy was used to characterize the solvent exposure of specific po- sitions within the transmembrane C domain. Time resolved fluorescence spectroscopy was used to probe the local microenvironment of the residues as well as the distance between 5-fluoroTrp residues and the mannitol binding site. Our results show that mannitol binding induces large conformational changes in EIImtl, that the C domain shows a rigid structure and that the binding site is asymmetrically positioned in the EIImtl dimer.

Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of approximately 140-kDa was observed, which probably corresponds to a monomeric SecYEG-substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.

Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli

This paper presents domain complementation studies in the mannitol transporter, EII mtl, from Escherichia coli. EII mtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EII mtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EII mtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7–28 fold by forming heterodimers with either the C domain of W97 (IIC mtlW97) or the inactive EII mtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EII mtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.

High yield cell-free production of integral membrane proteins without refolding or detergents

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570 ± 50 μg/mL and 130 ± 30 μg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

A Derivative of Lipid A Is Involved in Signal Recognition Particle/SecYEG-dependent and -independent Membrane Integrations

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Substrate-induced Conformational Changes in the Membrane-embedded IICmtl-domain of the Mannitol Permease from Escherichia coli, EnzymeIImtl, Probed by Tryptophan Phosphorescence Spectroscopy

Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII(mtl), of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC(mtl)-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of beta-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII(mtl).

Double-spanning Plant Viral Movement Protein Integration into theEndoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent,Translocon-mediated, andConcerted

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeII mtl, from Escherichia coli

Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeII mtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with K D-values in the nano- and micromolar range, respectively. However, reported K D-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in K D-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeII mtl. Furthermore, the affinity of EnzymeII mtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeII mtl in the same range as mannitol.

Analysis of Transport Activity of Arabidopsis Sugar Alcohol Permease Homolog AtPLT5

The transporters responsible for sugar uptake into non-photosynthetic sink tissues in plants, such as roots and flowers, have not been fully identified and analyzed. Plants encode around 100 putative sugar transporters within the major facilitator superfamily, yet only a few have been studied. Here we report the analysis of a sugar alcohol permease homolog (AtPLT5, At3g18830) from Arabidopsis. A wide range of sugars including hexoses, pentoses, tetroses, a sugar acid, and sugar alcohols but not disaccharides induced inward currents in oocytes expressing AtPLT5. AtPLT5 expression also resulted in 14C-labeled substrate uptake in oocytes, indicating that AtPLT5 encodes an ion-coupled uptake transporter. K(0.5) values for glucose and sorbitol were highly dependent on external pH. Expression of AtPLT5 was found primarily in sink tissues: in the elongation zone of roots, in the inflorescence stem, and several floral structures, especially in the floral abscission zone. Expression was induced by mechanical wounding and insect feeding. Analysis of transport properties and expression in Arabidopsis indicate that AtPLT5 functions to transport a wide range of sugars into specific sink tissues in the plant.

The First Cytoplasmic Loop of the Mannitol Permease from Escherichia coli is Accessible for Sulfhydryl Reagents from the Periplasmic Side of the Membrane

The mannitol permease (EII(Mtl)) from Escherichia coli couples mannitol transport to phosphorylation of the substrate. Renewed topology prediction of the membrane-embedded C domain suggested that EII(Mtl) contains more membrane-embedded segments than the six proposed previously on the basis of a PhoA fusion study. Cysteine accessibility was used to confirm this notion. Since cysteine 384 in the cytoplasmic B domain is crucial for the phosphorylation activity of EII(Mtl), all cysteine mutants contained this activity-linked cysteine residue in addition to those introduced for probing the membrane topology of the protein. To distinguish between the activity-linked cysteine and the probed cysteine, either trypsin was used to specifically digest the two cytoplasmic domains (A and B), thereby removing Cys384, or Cys384 was protected by phosphorylation from alkylation by N-ethylmaleimide (NEM). Our data show that upon phosphorylation EII(Mtl) undergoes major conformational changes, whereby residues in the putative first cytoplasmic loop become accessible to NEM. Other residues in this loop were accessible to NEM in intact cells and inside-out membrane vesicles, but cysteine residues at these positions only reacted with the membrane-impermeable sulfhydryl reagent from the periplasmic side of the protein. These and other results suggest that the predicted loop between TM2 and TM3 may fold back into the membrane and form part of the translocation path.