Insertion and folding of the amino-terminal amphiphilic signal sequences of the mannitol and glucitol permeases of Escherichia coli.

Peptides which correspond to the NH2-terminal 23 or 22 residues of the mannitol and glucitol permeases (enzymes IImtl and IIgut of the bacterial phosphotransferase system; mtl-23 and gut-22) and which are believed to function in envelope targeting were synthesized chemically, and their interactions with lipid model membranes were studied. Both wild-type peptides penetrated phospholipid monolayers up to high surface pressures, and partition constants of 8.0 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively, were derived from the incorporation isotherms of mtl-23 and gut-22 with monolayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine at 32 mN/m or bilayers of the same lipid. The mtl-23 peptide was highly alpha-helical in trifluoroethanol, sodium dodecyl sulfate, lysolecithin, or vesicles of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol, with estimated percentages of alpha-helix ranging between 60 and 85%. The interactions with model membranes of several single site mutants (S3P, D4P, and D4K) of mtl-23 which were defective in properly assembling the mannitol permease in the cytoplasmic membrane of Escherichia coli were also studied. The contents of alpha-helix of these peptides in detergent micelles or phospholipid bilayers were not significantly changed compared with those of the wild type, suggesting that the amphiphilic NH2-terminal membrane-targeting domain could still be formed in these mutants. However, the mutants which contained a proline in positions 3 or 4, i.e. NH2-terminal to the proposed amphiphilic alpha-helix, partitioned into phospholipid monolayers with partition constants that were 2 or 4 times smaller than those of the wild type. Based on these data, a model of the amphiphilic structure of the NH2-terminal domain of the mannitol permease is discussed. This domain may interact physiologically with amphiphilic interfaces of lipids and/or proteins during membrane insertion.     

Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions.

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Hydrophilic C-terminal domain of the Escherichia coli mannitol permease: phosphorylation, functional independence, and evidence for intersubunit phosphotransfer

The mannitol-specific enzyme II (mannitol permease) of the Escherichia coli phosphotransferase system (PTS) catalyzes the concomitant transport and phosphorylation of D-mannitol. Previous studies have shown that the mannitol permease (637 amino acid residues) consists of 2 structural domains of roughly equal size: an N-terminal, hydrophobic, membrane-bound domain and a C-terminal, hydrophilic, cytoplasmic domain. The C-terminal domain can be released from the membrane by mild proteolysis of everted membrane vesicles [Stephan, M.M., & Jacobson, G.R. (1986) Biochemistry 25, 8230-8234]. In this report, we show that phosphorylation of the intact permease by [32P]HPr (a general phosphocarrier protein of the PTS) followed by tryptic separation of the two domains resulted in labeling of only the C-terminal domain. Phosphorylation of the C-terminal domain occurred even in the complete absence of the N-terminal domain, showing that the former contains most, if not all, of the critical residues comprising the interaction site for phospho-HPr. The phosphorylated C-terminal domain, however, could not transfer its phospho group to mannitol, suggesting that the N-terminal domain is necessary for mannitol binding and/or phosphotransfer from the enzyme to the sugar. The elution profile of the C-terminal domain after molecular sieve chromatography showed that the isolated domain is monomeric, unlike the native permease which is likely a dimer in the membrane. Experiments employing a deletion mutation of the mtlA gene, which encodes a protein lacking the first phosphorylation site in the C-terminal domain (His-554) but retaining the second phosphorylation site (Cys-384), demonstrated that a phospho group could be transferred from phospho-HPr to Cys-384 of the deletion protein, and then to mannitol, only in the presence of the full-length permease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.     

The membrane-bound domain of the phosphotransferase enzyme IImtl of Escherichia coli constitutes a mannitol translocating unit

The orientation of the mannitol binding site on the Escherichia coli phosphotransferase enzyme IImtl in the unphosphorylated state has been investigated by measuring mannitol binding to cytoplasmic membrane vesicles with a right-side-out and inside-out orientation. Enzyme IImtl is shown to catalyze facilitated diffusion of mannitol at a low rate. At equilibrium, bound mannitol is situated at the periplasmic side of the membrane. The apparent binding constant is 40 nM for the intact membranes. Solubilization of the membranes in detergent decreases the affinity by about a factor of 2. Inside-out membrane vesicles, treated with trypsin to remove the C-terminal cytoplasmic domain of enzyme IImtl, showed identical activities. These experiments indicate that the translocation of mannitol is catalyzed by the membrane-bound N-terminal half of enzyme IImtl which is a structurally stable domain.     

Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.

Uracil permease is a multispanning protein of the Saccharomyces cerevisiae plasma membrane which is encoded by the FUR4 gene and produced in limited amounts. It has a long N-terminal hydrophilic segment, which is followed by 10 to 12 putative transmembrane segments, and a hydrophilic C terminus. The protein carries seven potential N-linked glycosylation sites, three of which are in its N-terminal segment. Overexpression of this permease and specific antibodies were used to show that uracil permease undergoes neither N-linked glycosylation nor proteolytic processing. Uracil permease N-terminal segments of increasing lengths were fused to a reporter glycoprotein, acid phosphatase. The in vitro and in vivo fates of the resulting hybrid proteins were analyzed to identify the first signal anchor sequence of the permease and demonstrate the cytosolic orientation of its N-terminal hydrophilic sequence. In vivo insertion of the hybrid protein bearing the first signal anchor sequence of uracil permease into the endoplasmic reticulum membrane was severely blocked in sec61 and sec62 translocation mutants.     

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.     

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane

Summary The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).Deceased June 5, 1988     

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.     

Translocation of N-terminal tails across the plasma membrane.

Previously we have shown that the first hydrophobic domain of leader peptidase (lep) can function to translocate a short N-terminal 18 residue antigenic peptide from the phage Pf3 coat protein across the plasma membrane of Escherichia coli. We have now examined the mechanism of insertion of N-terminal periplasmic tails and have defined the features needed to translocate these regions. We find that short tails of up to 38 residues are efficiently translocated in a SecA- and SecY-independent manner while longer tails are very poorly inserted. Efficient translocation of a 138 residue tail is restored and is Sec-dependent by the addition of a leader sequence to the N-terminus of the protein. We also find that while there is no amphiphilic helix requirement for N-terminal translocation, there is a charge requirement that is needed within the tail; an arginine and lysine residue can inhibit or completely block translocation when introduced into the tail region. Intriguingly, the membrane potential is required for insertion of a 38 residue tail but not for a 23 residue tail.     

A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli

Basic amino acid residues were introduced into an extracellular (periplasmic) domain, preceding a membrane-spanning hydrophobic domain, of SecY, an integral cytoplasmic membrane protein. The localization of the domain was monitored as to the alkaline phosphatase activity of TnPhoA fused adjacent to the domain. The alkaline phosphatase activity of such Escherichia coli cells drastically decreased when positive charges were introduced, indicating that on the introduction the SecY domain showed a change in localization from the periplasm to the cytoplasm. In another experiment, positive charges were introduced to the same periplasmic domain of another SecY-PhoA fusion protein, in which PhoA is fused to the cytoplasmic domain of SecY following the particular hydrophobic domain. The alkaline phosphatase activity increased drastically when positive charges were introduced, indicating that the SecY domain fused to PhoA showed a change in localization from the cytoplasm to the periplasm. In both experiments, the removal of a large amino-terminal portion of the SecY domain did not alter the effect of the positive charge introduction. Changes in localization of SecY domains thus demonstrated were also supported by a protease accessibility test on spheroplasts. It is proposed that a positively charged region adjacent to a membrane-embedded hydrophobic region tends to be stabilized on the cytoplasmic surface of the membrane, which in turn endows the hydrophobic region with the ability to act as a stop-transfer sequence or a signal sequence and consequently determines the orientation of the hydrophobic region in the membrane.     

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.     

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

Summary A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the thylakoid-transfer domain of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.     

The Tsr chemosensory transducer of Escherichia coli assembles into the cytoplasmic membrane via a SecA-dependent process

The Tsr protein of Escherichia coli is a chemosensory transducer that mediates taxis toward serine and away from certain repellents. Like other bacterial transducers, Tsr spans the cytoplasmic membrane twice, forming a periplasmic domain of about 150 amino acids and a cytoplasmic domain of about 300 amino acids. The 32 N-terminal amino acids of Tsr resemble the consensus signal sequence of secreted proteins, but they are not removed from the mature protein. To investigate the function of this N-terminal sequence in the assembly process, we isolated translational fusions between tsr and the phoA and lacZ genes, which code for the periplasmic enzyme alkaline phosphatase and the cytoplasmic enzyme beta-galactosidase, respectively. All tsr-phoA fusions isolated code for proteins whose fusion joints are within the periplasmic loop of Tsr, and all of these hybrid proteins have high alkaline phosphatase activity. The most N-terminal fusion joint is at amino acid 19 of Tsr. Tsr-lacZ fusions were found throughout the tsr gene. The beta-galactosidase activity of the LacZ-fusion proteins varies greatly, depending on the location of the fusion joint. Fusions with low activity have fusion joints within the periplasmic loop of Tsr. The expression of these fusions is most likely reduced at the level of translation. In addition, one of these fusions markedly reduces the export and processing of the periplasmic maltose-binding protein and the outer membrane protein OmpA, but not of intact PhoA or of the outer membrane protein LamB. A temperature-sensitive secA mutation, causing defective protein secretion, stops expression of new alkaline phosphatase activity coded by a tsr-phoA fusion upon shifting to the nonpermissive temperature. The same secA mutation, even at the permissive temperature, increases the activity and the level of expression of LacZ fused to the periplasmic loop of Tsr relative to a secA+ strain. We conclude that the assembly of Tsr into the cytoplasmic membrane is mediated by the machinery responsible for the secretion of a subset of periplasmic and outer membrane proteins. Moreover, assembly of the Tsr protein seems to be closely coupled to its synthesis.     

Membrane incorporation and induction of secondary structure of synthetic peptides corresponding to the N-terminal signal sequences of the glucitol and mannitol permeases of Escherichia coli

The 22-residue synthetic signal peptide of the glucitol permease (Enzyme IIgut of the bacterial phosphotransferase system; gut22), which in the intact protein is believed to function in envelope targeting, was found to insert into phospholipid monolayers of various phospholipid compositions up to high limiting pressures (36-41 milliNewton/m). The partition coefficient, derived from monolayer area expansion experiments, was greatest for the negatively charged gut22 when partitioning into monolayers of the zwitterionic lipid 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (about 1.1 X 10(5] as compared with that obtained with a mixture of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine and the negatively charged lipids 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol and cardiolipin. Gut22 contains a titratable histidyl residue (pKa = 6.8), and its protonation decreased the relative monolayer area increase 3-fold. Circular dichroism spectra showed that gut22 formed an amphiphilic alpha-helix when incorporated into lipid membranes (estimated percent helix = 65%). Fluorescence measurements indicated that tryptophan 11 is in a more hydrophobic environment in the presence of lipid than in its absence, with the environment being more hydrophobic at pH 5 than at pH 8. The more hydrophilic 15-residue signal peptide of the mannitol permease (mtl15) also incorporated into monolayers and detergent micelles (although to a lesser extent) with induction of secondary structure. Based on these results and a parallel with mitochondrial targeting in eucaryotes, we suggest that the induction of N-terminal amphiphilic structures and their association with a hydrophobic-hydrophilic interface are important for envelope targeting and the initiation of the membrane insertion of bacterial phosphoenol-pyruvate-dependent phosphotransferase system permeases.     

Mechanistic coupling of transport and phosphorylation activity by enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Mannitol bound to enzyme IImtl could be trapped specifically by rapid phosphorylation with P-HPr. The assay was used to demonstrate transport of mannitol across the cytoplasmic membrane with and without phosphorylation of mannitol. The latter was 2-3 orders of magnitude slower. The fraction of bound mannitol molecules that was actually phosphorylated, the efficiency of the trap, was less than 50%. The efficiency was not very different for enzyme IImtl embedded in the membrane of vesicles with an inside-out orientation or solubilized in detergent. Subsequently, it is argued that the fraction of the bound mannitol molecules that was not phosphorylated dissociated into the cytoplasmic space. A model for the catalytic mechanism of enzyme IImtl is proposed on the basis of interpretations of the present experiments. The main features of the model are the following: (i) mechanistically, the coupling between transport and phosphorylation is less than 50%; (ii) in the physiological steady state of mannitol transport and metabolism, the coupling is 100%; (iii) phosphorylated enzyme IImtl catalyzes facilitated diffusion at a high rate; (iv) the state of phosphorylation of the cytoplasmic domain modulates the activity of the translocator domain; (v) the enzyme catalyzes phosphorylation of free cytoplasmic mannitol at least as fast as it catalyzes transport plus phosphorylation of free periplasmic mannitol.     

Molecular cloning and sequence analysis of human placental alkaline phosphatase

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phosphatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage lambda gt11 placental cDNA library with antibodies against CNBr fragments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental alkaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn122 and Asn249 and a highly hydrophobic membrane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between placental alkaline phosphatase and Escherichia coli alkaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.     

Use of phoA and lacZ fusions to study the membrane topology of ProW, a component of the osmoregulated ProU transport system of Escherichia coli.

The Escherichia coli ProU system is a member of the ATP-binding cassette (ABC) superfamily of transporters. ProU consists of three components (ProV, ProW, and ProX) and functions as a high-affinity, binding protein-dependent transport system for the osmoprotectants glycine betaine and proline betaine. The ProW protein is the integral inner membrane component of the ProU system. Its hydropathy profile predicts seven transmembrane spans and a hydrophilic amino terminus of approximately 100 residues, and it suggests the presence of an amphiphilic alpha-helix (L-61 to F-97) in close proximity to the first strongly hydrophobic segment of ProW. We have studied the membrane topology of the ProW protein by the phoA and lacZ gene fusion approach. A collection of 10 different proW-phoA fusions with alkaline phosphatase activity and 8 different proW-lacZ fusions with beta-galactosidase activity were isolated in vivo after TnphoAB and TnlacZ mutagenesis of a plasmid-encoded proW gene. The recovery of both enzymatically active ProW-PhoA and ProW-LacZ hybrid proteins indicates that segments of ProW are exposed on both sides of the cytoplasmic membrane. To compare the enzymatic activities of each of the indicator proteins joined at a particular site in ProW, we switched the phoA and lacZ reporter genes in vitro in each of the originally in vivo-isolated gene fusions. A mirror-like pattern in the enzyme activity of the resulting new ProW-PhoA and ProW-LacZ hybrid proteins emerged, thus providing positive signals for the location of both periplasmic and cytoplasmic domains in ProW. The protease kallikrein digests the amino-terminal tail of a ProW-LacZ hybrid protein in spheroplasts, suggesting that the amino terminus of ProW is located on the periplasmic side of the cytoplasmic membrane. From these data, a two-dimensional model for ProW was constructed; this model consists of seven transmembrane alpha-helices and an unusual amino-terminal tail of approximately 100 amino acid residues that protrudes into the periplasmic space.