MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: topography of francisella novicida LpxE expressed in Escherichia coli

The lipid A anchor of Francisella tularensis lipopolysaccharide (LPS) lacks both phosphate groups present in Escherichia coli lipid A. Membranes of Francisella novicida (an environmental strain related to F. tularensis) contain enzymes that dephosphorylate lipid A and its precursors at the 1- and 4'-positions. We now report the cloning and characterization of a membrane-bound phosphatase of F. novicida that selectively dephosphorylates the 1-position. By transferring an F. novicida genomic DNA library into E. coli and selecting for low level polymyxin resistance, we isolated FnlpxE as the structural gene for the 1-phosphatase, an inner membrane enzyme of 239 amino acid residues. Expression of FnlpxE in a heptose-deficient mutant of E. coli caused massive accumulation of a previously uncharacterized LPS molecule, identified by mass spectrometry as 1-dephospho-Kdo2-lipid A. The predicted periplasmic orientation of the FnLpxE active site suggested that LPS export might be required for 1-dephosphorylation of lipid A. LPS and phospholipid export depend on the activity of MsbA, an essential inner membrane ABC transporter. Expression of FnlpxE in the msbA temperature-sensitive E. coli mutant WD2 resulted in 90% 1-dephosphorylation of lipid A at the permissive temperature (30 degrees C). However, the 1-phosphate group of newly synthesized lipid A was not cleaved at the nonpermissive temperature (44 degrees C). Our findings provide the first direct evidence that lipid A 1-dephosphorylation catalyzed by LpxE occurs on the periplasmic surface of the inner membrane.     

ATPase activity of the MsbA lipid flippase of Escherichia coli

Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to approximately 95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent K(m) of 878 microm and a V(max) of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mm Mg(2+). Preincubation of MsbA-containing liposomes with 3-deoxy-d-mannooctulosonic acid (Kdo)(2)-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 microm Kdo(2)-lipid A. Addition of Kdo(2)-lipid A increased the V(max) to 154 nmol/min/mg and decreased the K(m) to 379 microm. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IV(A). MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30 degrees C but was significantly reduced at 42 degrees C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator.     

MsbA is not required for phospholipid transport in Neisseria meningitidis

The outer membrane of Gram-negative bacteria contains phospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively. Little is known about the transport of the phospholipids from their site of synthesis to the outer membrane. The inner membrane protein MsbA of Escherichia coli, which is involved in the transport of LPS across the inner membrane, has been reported to be involved in phospholipid transport as well. Here, we have reported the construction and the characterization of a Neisseria meningitidis msbA mutant. The mutant was viable, and it showed a retarded growth phenotype and contained very low amounts of LPS. However, it produced an outer membrane, demonstrating that phospholipid transport was not affected by the mutation. Notably, higher amounts of phospholipids were produced in the msbA mutant than in its isogenic parental strain, provided that capsular biosynthesis was also disrupted. Although these results confirmed that MsbA functions in LPS transport, they also demonstrated that it is not required for phospholipid transport, at least not in N. meningitidis.     

Biochemical characterization of MsbA from Pseudomonas aeruginosa

Lipopolysaccharide of Pseudomonas aeruginosa is a major constituent of the outer membrane, and it is composed of three distinct regions: lipid A, core oligosaccharide, and O antigen. Lipid A and core oligosaccharides (OS) are synthesized and assembled at the cytoplasmic side of the inner membrane and then translocated to the periplasmic side of the membrane where lipid A-core becomes the acceptor of the O antigens. Here we show that MsbA encoded by pA4997 of the P. aeruginosa genome is a member of the ABC transporter family, but this protein has distinctive features when compared with other MsbA proteins. msbA is an essential gene in this organism since mutation in this gene is lethal to the bacterium. Disruption of the chromosomal msbA was achieved only when a functional copy of the gene was provided in trans. msbA from Escherichiacoli (msbA(Ec)) could not cross complement the msbA merodiploid cells of P. aeruginosa. MsbA was expressed and purified, and the kinetic of its ATPase activity is vastly different than that of MsbA(Ec). The activity of MsbA could be selectively stimulated by different truncated versions of core OS of P. aeruginosa LPS. Specifically, phosphate substituents in the lipid A-core are important for stimulating ATPase activity of MsbA. Expression of MsbA(Ec) but not MsbA(Pa) conferred resistance to erythromycin in P. aeruginosa.     

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.     

Complement attack of altered outer membrane areas synthesized after inhibition of the 3-deoxy-D-manno-octulosonate pathway leads to cell death

Salmonella typhimurium containing specific genes coding for either temperature-sensitive (TS) 3-deoxy-D-manno-octulosonate (KDO) 8-phosphate synthetase or TS cytidine monophosphate-KDO synthetase grow normally when incubated at 30 degrees C and are resistant to C-mediated killing. However, bacteria become avirulent and sensitive to C-mediated killing upon thermal inhibition of TS KDO-8-phosphate synthetase (incubation at 38 degrees C) or TS cytidine monophosphate-KDO synthetase (incubation at 42 degrees C). Such thermal inhibition concurrently causes synthesis of an altered outer membrane which we now show is the site that renders cells susceptible to C-mediated killing. After incubation of cells in serum, the altered outer membrane area contains C9 in a trypsin-resistant state and membrane attack complex (MAC) lesions observable by electron microscopy. Trypsin-resistant C9 and MAC lesions were also observed in the inner membrane fraction from such serum-treated cells. In contrast, little C9 and few MAC lesions were associated with unaltered outer membrane areas present on these same serum treated cells. Control cells, grown at 30 degrees C and treated with serum (1) bound one-fifth as much C9 as was bound to cells incubated at 42 degrees C, (2) contained only a rare MAC lesion in the outer membrane, and (3) no observable MAC lesions in the inner membrane. We conclude that the altered outer membrane area is the site that renders cells susceptible to insertion of the MAC into both the outer and inner membrane resulting in cell death.     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).     

Single amino acid substitutions in either YhjD or MsbA confer viability to 3-deoxy-d-manno-oct-2-ulosonic acid-depleted Escherichia coli

The Escherichia coli K-12 strain KPM22, defective in synthesis of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), is viable with an outer membrane (OM) composed predominantly of lipid IV(A), a precursor of lipopolysaccharide (LPS) biosynthesis that lacks any glycosylation. To sustain viability, the presence of a second-site suppressor was proposed for transport of lipid IV(A) from the inner membrane (IM), thus relieving toxic side-effects of lipid IV(A) accumulation and providing sufficient amounts of LPS precursors to support OM biogenesis. We now report the identification of an arginine to cysteine substitution at position 134 of the conserved IM protein YhjD in KPM22 that acts as a compensatory suppressor mutation of the lethal DeltaKdo phenotype. Further, the yhjD400 suppressor allele renders the LPS transporter MsbA dispensable for lipid IV(A) transmembrane trafficking. The independent derivation of a series of non-conditional KPM22-like mutants from the Kdo-dependent parent strain TCM15 revealed a second class of suppressor mutations localized to MsbA. Proline to serine substitutions at either residue 18 or 50 of MsbA relieved the Kdo growth dependence observed in the isogenic wild-type strain. The possible impact of these suppressor mutations on structure and function are discussed by means of a computationally derived threading model of MsbA.     

Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.     

Membrane-bound enterotoxin of Vibrio cholerae

The mode of transport of the complex toxin molecule of Vibrio cholerae (which has a mol. wt of 84000 and consists of several subunits) across the inner and outer membranes of V. cholerae is not known. In this study we found two peptides in the outer and inner membranes of V. cholerae which may be the form in which the toxin subunits are transported across the membrane. We examined two growth conditions: aerobic growth at 37 degrees C, when most of the synthesized toxin is membrane-bound; and anaerobic growth at 37 degrees C, when little toxin remains membrane-bound, the toxin being released into the growth medium. When V. cholerae was grown aerobically at 37 degrees C, the outer and the inner membranes contained two peptides with mol. wts of approximately 22000 and 6000 which were not found in the outer or the inner membrane of anaerobically grown cells. Sodium deoxycholate, which releases membrane-bound toxin, released several peptides including the 22000 and the 6000 mol. wt peptides. Trypsin also released the 22000 and 6000 mol. wt peptides. Purified cholera toxin had three kinds of peptides, of mol. wt 21000 (A1 peptide), 11000 (B subunit) and 5000 (A2 peptide). We postulate that the membrane peptides may be precursors of the A subunit of the toxin molecule.     

Translocation of phospholipids is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic membrane

The mechanism by which phospholipids are transported across biogenic membranes, such as the bacterial cytoplasmic membrane, is unknown. We hypothesized that this process is mediated by the presence of the membrane-spanning segments of inner membrane proteins, rather than by dedicated flippases. In support of the hypothesis, it was demonstrated that transmembrane alpha-helical peptides, mimicking the membrane-spanning segments, mediate flop of 2-6-(7-nitro-2,1,3-benzoxadiazol-4-yl) aminocaproyl (C6-NBD)-phospholipids (Kol, M. A., de Kroon, A. I., Rijkers, D. T., Killian, J. A., and de Kruijff, B. (2001) Biochemistry 40, 10500-10506). Here the dithionite reduction assay was used to measure transbilayer equilibration of C6-NBD-phospholipids in proteoliposomes, composed of Escherichia coli phospholipids and a subset of bacterial membrane proteins. It is shown that two well characterized integral proteins of the bacterial cytoplasmic membrane, leader peptidase and the potassium channel KcsA, induce phospholipid translocation, most likely by their transmembrane domains. In contrast, the ATP-binding cassette transporter from the E. coli inner membrane MsbA, a putative lipid flippase, did not mediate phospholipid translocation, irrespective of the presence of ATP. OmpT, an outer membrane protein from E. coli, did not facilitate flop either, demonstrating specificity of protein-mediated phospholipid translocation. The results are discussed in the light of phospholipid transport across the E. coli inner membrane.     

The Physical and Genetic Organization of A Viral Genom

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Proteins that bind and move lipids: MsbA and NPC1

Membrane proteins that bind and transport lipids face special challenges. Since lipids typically have low water solubility, both accessibility of the substrate to the protein and delivery to the desired destination are problematical. The amphipathic nature of membrane lipids, and their relatively large molecular size, also means that these proteins must possess substrate-binding sites of a different nature than those designed to handle small polar molecules. This review considers two integral proteins whose function is to bind and transfer membrane lipids within or across a membrane. The first protein, MsbA, is a putative lipid flippase that is a member of the ATP-binding cassette (ABC) superfamily. The protein is found in the inner (cytoplasmic) membrane (IM) of Gram-negative bacteria such as E. coli, where it is proposed to move lipid A from the inner to the outer membrane (OM) leaflet, an important step in the lipopolysaccharide biosynthetic pathway. Cholesterol is a major component of the plasma membrane in eukaryotic cells, where it regulates bilayer fluidity. The other lipid-binding protein discussed here, mammalian NPC1 (Niemann-Pick disease, Type C1), binds cholesterol inside late endosomes/lysosomes (LE/LY) and is involved in its transfer to the cytosol as part of a key intracellular sterol-trafficking pathway. Mutations in NPC1 lead to a devastating neurodegenerative condition, Niemann-Pick Type C disease, which is characterized by massive cholesterol accumulation in LE/LY. The accelerating pace of membrane protein structure determination over the past decade has allowed us a glimpse of how lipid binding and transfer by membrane proteins such as MsbA and NPC1 might be achieved.     

Asymmetric distribution and temperature-dependent clustering of filipin-sterol complexes in the nuclear membrane of Ehrlich ascites tumor cells

When Ehrlich ascites tumor (EAT) cells were fixed at 4 degrees C and freeze-fractured, patchy areas having no intramembrane particles were visible in the nuclear envelope. The particle free areas (PFAs) were not seen on fixation at 28 degrees C, indicating that appearance of PFAs was caused by a kind of thermotropic phase separation. The PFAs were detected only in the nuclear membrane, and not in the plasma membrane. Most of them were present in the outer membrane of the nuclear envelope. In cells fixed at 4 degrees C, and treated with filipin all the filipin-sterol complexes appeared in clusters located in the PFAs. In contrast, the filipin-sterol complexes were evenly distributed in cells fixed at 28 degrees C. This suggests that at low temperature, molecules of cholesterol gather in the PFAs. Temperature-dependent cluster formation was seen only in the complexes of the nuclear membrane, suggesting that the cholesterol in the nuclear membrane is more mobile than that in the plasma membrane. In addition, the distribution of filipin-sterol complexes in the nuclear envelope was asymmetric. The complexes were seen only in the outer (cytoplasmic), but not in the inner (nucleoplasmic) membrane of the nuclear envelope, reflecting differences in the structural, and presumably functional, characteristics of the outer and inner nuclear membranes.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

Functional characterization of Escherichia coli MsbA: interaction with nucleotides and substrates

The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of approximately 2 mm and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 microm. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 microm. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.     

Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling

MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang, and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein. The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques. SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface. The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined. Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.     

Membrane proteins correlated with expression of the polysialic acid capsule in Escherichia coli K1.

Growth of Escherichia coli K1 strains at 15 degrees C results in a defect in the synthesis or assembly of the K1 polysialic acid capsule. Synthesis is reactivated in cells grown at 15 degrees C after upshift to 37 degrees C, and activation requires protein synthesis (Whitfield et al., J. Bacteriol. 159:321-328, 1984). Using this temperature-induced defect, we determined the molecular weights and locations of membrane proteins correlated with the expression of K1 (polysialosyl) capsular antigen. Pulse-labeling experiments demonstrated the presence of 11 proteins whose synthesis was correlated with capsule appearance at the cell surface. Using the differential solubility of inner and outer membranes in the detergent Sarkosyl, we localized five of the proteins in the outer membrane and four in the inner membrane. The subcellular location of two of the proteins was not determined. Five proteins appeared in the membrane simultaneously with the initial expression of the K1 capsule at the cell surface. One of these proteins, a 40,000-dalton protein localized in the outer membrane, was identified as porin protein K, which previously has been shown to be present in the outer membrane of encapsulated E. coli. The possible role of these proteins in the synthesis of the polysialosyl capsule is discussed.     

Gramicidin-induced enhancement of transbilayer reorientation of lipids in the erythrocyte membrane

Incorporation of the channel-forming antibiotic gramicidin into the membrane of human erythrocytes highly (up to 30-fold) enhances rates of reorientation (flip) of lysophosphatidylcholine and palmitoylcarnitine to the inner membrane layer after their primary incorporation into the outer layer. Despite the high increase of flip rates by gramicidin, the asymmetric orientation of the inner membrane layer phospholipids phosphatidylethanolamine and phosphatidylserine is stable as demonstrated by the lack of accessibility of these lipids toward cleavage by exogenous phospholipase A2. On the other hand, gramicidin enhances the rate of cleavage of outer membrane layer phosphatidylcholine by phospholipase A2, which indicates changes in the packing of phosphatidylcholine following gramicidin binding. The increase of flip becomes detectable when about 10(5) copies of gramicidin per cell have been bound (gramicidin to membrane phospholipid ratio of 1:2000). This is a 1000-fold higher concentration than that required for an increase of K+ permeability mediated by the gramicidin channel. Acceleration of flip is thus not simply correlated with channel formation. The enhancement of flip is markedly dependent on structural details of gramicidin. Formylation of its four tryptophan residues abolishes the effect. Even at high concentrations of formylated gramicidin at which the extents of binding of native and of formylated gramicidin to the membrane are comparable, no flip acceleration is produced. Enhancement of flip by gramicidin occurs after a temperature-dependent lag phase. At 37 degrees C, flip rates begin to increase within a few minutes and at 25 degrees C, only after 3 h. This lag phase is most likely not due to limitations by the rate of binding of gramicidin to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.