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.     

Mechanics of solute translocation catalyzed by enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The kinetics of binding of mannitol to enzyme IImtl embedded in the membrane of vesicles with an inside-out or a right-side-out orientation were analyzed at 4 degrees C in the absence of the phosphoryl group donor, P-HPr. The binding to the right-side-out oriented vesicles equilibrated too fast to be monitored by the flow dialysis technique. On the other hand, with the inside-out oriented membrane vesicles two conformational changes of the enzyme could be detected kinetically. One change involved a recruitment of binding sites from a state of the enzyme where the binding sites were inaccessible from the cytoplasmic volume. The second change involved a conformational change of the enzyme that followed upon the initial binding to the cytoplasmic-facing binding site leading to a state with a higher affinity for mannitol. Equilibrium binding to the inside-out and right-side-out oriented membrane vesicles at 4 degrees C indicated that the two transitions did not represent the translocation of the binding site, free and with mannitol bound to it, to the other side of the membrane. Instead, a model is proposed in which the conformational changes represent transitions from states with the binding pocket opened to the cytoplasmic side of the membrane to occluded states of the enzyme in which the binding sites, with or without mannitol bound, are not accessible to either side of the membrane.     

Interaction between the cytoplasmic and membrane-bound domains of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system

Sulfhydryl reagents affected the binding properties of the translocator domain, NIII, of enzyme IImtl in two ways: (i) the affinity for mannitol was reduced, and (ii) the exchange rate of bound and free mannitol was increased. The effect on the affinity was very much reduced after solubilization of enzyme IImtl in the detergent decylPEG. The effects were caused exclusively by reaction of the sulfhydryl reagents with the cysteine residue at position 384 in the primary sequence. Interaction between two domains is involved, since Cys384 is located in the cytoplasmic domain, CII. When Cys384 was mutated to serine, the enzyme exhibited the same binding properties as the chemically modified enzyme. The data support our proposal that phosphorylation of enzyme IImtl drastically reduces the activation energy for the translocation step through interaction between domains CII and NIII [Lolkema J. S., ten Hoeve-Duurkens, R. H., Swaving Dijkstra, D., & Robillard, G. T. (1991) Biochemistry (preceding paper in this issue)]. Functional interaction between the translocator domain, NIII, and domain CI was investigated by phosphorylation of His554, located in domain CI, in the C384S mutant. No effect on the binding properties was observed. In addition, the binding properties were insensitive to the presence of the soluble phosphotransferase components enzyme I and HPr.     

Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence

The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Immunological approaches to the transmembrane topology and conformational changes of the carboxyl-terminal regulatory domain of yeast plasma membrane H(+)-ATPase.

Molecular genetic experiments have suggested that the carboxyl terminus of the Saccharomyces cerevisiae plasma membrane H(+)-ATPase is an inhibitory domain involved in the "in vivo" regulation of the enzyme by glucose metabolism. An antibody prepared against a fusion protein including the last 59 amino acids of the ATPase sequence has been affinity purified to yield a preparation which requires the 18 carboxyl-terminal amino acids for recognition. Antibody binding experiments show that the carboxyl-terminal domain of the ATPase can be selectively exposed by concentrations of the detergent Tween-20 which do not break down the permeability barrier of the plasma membrane to the antibody. Both enzyme-linked immunosorbent assay and immunofluorescence analysis demonstrate that the accessibility of the carboxyl-terminal domain in isolated plasma membranes depends on the physiological state of the cell being increased by glucose metabolism. Immunofluorescence analysis of isolated plasma membrane vesicles, using a dual labeling protocol with concanavalin A and antibody to reveal the orientation of individual vesicles, and colloidal gold immunoelectron microscopy of ultrathin cryosections of whole yeast cells separately demonstrate that the ATPase carboxyl terminus is located in the cytoplasmic compartment. The application of a mutant deleted of the epitope(s) recognized by the affinity purified carboxyl-terminal antibody eliminates the possibility of artifacts arising from nonspecific antibody binding. The accessibility properties and cytoplasmic location of the carboxyl-terminal domain appear to be consistent with its role as a negative regulator of the ATPase.     

Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent

The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted [Saier, M.H. (1980) J. Supramol. Struct. 14, 281-294]. The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed. Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions. The dimer is favored over the monomer at high ionic strength and basic pH. Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization. A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer. Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions. The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3. The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed. The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction.     

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)     

Selective modulation of band 4.1 binding to erythrocyte membranes by protein kinase C

We have studied the effects of band 4.1 phosphorylation on its association with red cell inside-out vesicles stripped of all peripheral proteins. Band 4.1 bound to these vesicles in a saturable manner, and binding was characterized by a linear Scatchard plot with an apparent Kd of 1-2 x 10(-7) M. Phosphorylation of band 4.1 by purified protein kinase C reduced its ability to bind to membranes, resulting in a reduction in the apparent binding capacity of the membrane by 60-70% but little or no change in the apparent Kd of binding. By contrast, phosphorylation of band 4.1 by cAMP-dependent kinase had no effect on membrane binding. Digestion of the stripped inside-out vesicles with trypsin cleaved 100% of the cytoplasmic domain of band 3 but had little or no effect on glycophorin. Binding of band 4.1 to these digested vesicles was reduced by 70%. Phosphorylation of band 4.1 by protein kinase C had no effect on its binding to the digested vesicles, suggesting that the cytoplasmic domain of band 3 contained the phosphorylation-sensitive binding sites. This was confirmed by direct measurement of band 4.1 binding to the purified cytoplasmic domain of band 3. Phosphorylation of band 4.1 by protein kinase C reduced its binding to the purified 43-kDa domain by as much as 90%, while phosphorylation by cAMP-dependent kinase was without effect. These results show a selective effect of protein kinase C phosphorylation on the binding of band 4.1 to one of its membrane receptors, band 3, and suggest a mechanism whereby one of the key red cell-skeletal membrane associations may be modulated.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Fusion between disk membranes and plasma membrane of bovine photoreceptor cells is calcium dependent.

Disk membranes and plasma membrane vesicles were prepared from bovine retinal rod outer segments (ROS). The plasma membrane vesicles were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C and in the presence of micromolar calcium, an increase in R18 fluorescence with time was observed when R18-labeled plasma membrane vesicles were introduced to a suspension of disks. This result was interpreted as fusion between the disk membranes and the plasma membranes, the fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes as a result of lipid mixing during membrane fusion. While the disk membranes exposed exclusively their cytoplasmic surface, plasma membrane vesicles were found with both possible orientations. These vesicles were fractionated into subpopulations with homogeneous orientation. Plasma membrane vesicles that were oriented with the cytoplasmic surface exposed were able to fuse with the disk membranes in a Ca(2+)-dependent manner. Fusion was not detected between disk membranes and plasma membrane vesicles oriented such that the cytoplasmic surface was on the interior of the vesicles. ROS plasma membrane-disk membrane fusion was stimulated by calcium, inhibited by EGTA, and unaffected by magnesium. Rod photoreceptor cells of vertebrate retinas undergo diurnal shedding of disk membranes containing the photopigment rhodopsin. Membrane fusion is required for the shedding process.     

Purification and properties of human erythrocyte band 4.2. Association with the cytoplasmic domain of band 3

We have purified the human erythrocyte membrane protein band 4.2 to greater than 85% homogeneity. The protein was extracted from spectrin-actin-depleted inside-out vesicles in a pH 11 medium and purified by gel filtration in the presence of 1 M KI. The purified protein was heterogeneous and had an average S20,w of 5.5 and an average Stokes radius of 82 A. By electron microscopy, the protein appeared heterogeneous in size and shape, having a diameter ranging from 80 to 150 A. The protein bound saturably to band 4.2-depleted red cell inside-out vesicles, and the binding exhibited a concave Scatchard plot. Binding was reduced greater than 90% by proteolytic digestion of membranes. Digestion studies suggested that there are two classes of binding sites for band 4.2 on the cytoplasmic aspect of red cell membranes, one of which is likely to be band 3. The purified 43-kDa cytoplasmic domain of band 3 competed for band 4.2 binding to red cell membranes and could completely abolish binding when added at a concentration of greater than 200 micrograms/ml. The purification of band 4.2 and the characterization of its association with red cell membranes should facilitate the discovery of the function of this major red cell membrane protein.     

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.     

Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.     

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.     

Cytochemistry of cytochrome oxidase in the cytoplasmic and intracytoplasmic membranes of Azotobacter vinelandii

Vegetative cells of Azotobacter vinelandii contain a system of intracytoplasmic membranes in the form of numerous internal vesicles. The three-dimensional morphology of these internal vesicles was established by an examination of stereopair electron micrographs of negatively stained cells. The vesicles assumed a variety of forms ranging from nearly spherical units to short, curved tubules. These structures were found at the periphery of the cytoplasm, subjacent to the cytoplasmic membrane. Large flattened cisternae were also present in some cells. The amount of intracytoplasmic membrane varied widely even among individual cells from the same culture. The total surface area of the intracytoplasmic membranes was greater than that of the cytoplasmic membrane in many cells. To assess the possible association of cytochrome oxidase activity with the intracytoplasmic membranes, enzyme localization experiments were conducted with the cytochemical substrate 3,3'-diaminobenzidine. The results showed that a cyanide-sensitive cytochrome oxidase activity is located at the intracytoplasmic membrane. The quantity of cytochrome oxidase activity present in the internal membranes is probably less than that present in the cytoplasmic membrane.     

Calcium-Dependent Self-Association of Synaptotagmin I

Abstract: Synaptotagmin I, an integral membrane protein of secretory vesicles, appears to have an essential role in calcium-triggered hormone and neurotransmitter release. The large cytoplasmic domain of synaptotagmin I has two C2 domains that are thought to mediate calcium and phospholipid binding. A recombinant protein (p65 1–5) comprised of the cytoplasmic domain was previously shown to aggregate purified chromaffin granules and artificial phospholipid vesicles in a calcium-dependent manner. p65 1–5 may be able to aggregate membrane vesicles by a self-association reaction. This hypothesis led us to investigate the ability of synaptotagmin I protein fragments to multimerize in vitro. We found that p65 1–5, in the absence of membranes, was able to self-associate to form large aggregates in a calcium-dependent manner as shown by light-scattering assays and electron microscopy. In addition, a recombinant protein comprised of only the second half of the cytoplasmic domain, including the second C2 domain, was also able to self-associate and aggregate phospholipid vesicles in a calcium-dependent manner. A recombinant protein comprised of only the first C2 domain was not able to self-associate or aggregate vesicles. These results suggest that synaptotagmin I is able to bind calcium in the absence of membranes and that the second half of the cytoplasmic domain is able to bind calcium and mediate its multimerization in a calcium-dependent manner. The ability of synaptotagmin I protein fragments to multimerize in a calcium-dependent manner in vitro suggests that multimerization may have an important function in vivo.     

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.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB_1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB_1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.