Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein

Due to its extreme insolubility, Fe3+ is not transported as a monoatomic ion. In microbes, iron is bound to low molecular weight carriers, designated siderophores. For uptake into cells of Escherichia coli Fe3+ siderophores have to be translocated across two membranes. Transport across the outer membrane is receptor-dependent and energy-coupled; transport across the cytoplasmic membrane seems to follow a periplasmic binding protein-dependent transport mechanism. In support of this notion we demonstrate specific binding of the Fe3+ hydroxamate compounds ferrichrome, aerobactin, and coprogen, which are transported via the Fhu system, to the periplasmic FhuD protein, and no binding of the transport inactive ferrichrome A, ferric citrate, and iron sulfate. About 10(4) ferrichrome molecules were bound to the FhuD protein of cells which overproduced plasmid-encoded FhuD. Binding depended on transport across the outer membrane mediated by the FhuA receptor and the TonB protein. Binding to FhuD was supported by the exclusive resistance of FhuD to proteinase K in the presence of the transport active hydroxamates. The overproduced precursor form of the FhuD protein was not protected by the Fe3+ hydroxamates indicating a conformation different to the mature form. The FhuD protein apparently serves as a periplasmic carrier for Fe3+ hydroxamates with widely different structures.     

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.     

Evidence for post-translational membrane insertion of the integral membrane protein bacterioopsin expressed in the heterologous halophilic archaeon Haloferax volcanii

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.     

Membrane targeting of a folded and cofactor-containing protein.

Targeting of proteins to and translocation across the membranes is a fundamental biological process in all organisms. In bacteria, the twin arginine translocation (Tat) system can transport folded proteins. Here, we demonstrate in vivo that the high potential iron-sulfur protein (HiPIP) from Allochromatium vinosum is translocated into the periplasmic space by the Tat system of Escherichia coli. In vitro, reconstituted HiPIP precursor (preHoloHiPIP) was targeted to inverted membrane vesicles from E. coli by a process requiring ATP when the Tat substrate was properly folded. During membrane targeting, the protein retained its cofactor, indicating that it was targeted in a folded state. Membrane targeting did not require a twin arginine motif and known Tat system components. On the basis of these findings, we propose that a pathway exists for the insertion of folded cofactor-containing proteins such as HiPIP into the bacterial cytoplasmic membrane.     

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.     

Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.     

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.     

The interaction of mammalian medium-chain hydrolase with yeast fatty acid synthetase

The prolipoprotein, a secretory precursor of the outer membrane lipoprotein of Escherichia coli, is known to be accumulated in the cell envelope when cells are grown in the presence of a cyclic antibiotic, globomycin. The prolipoprotein was localized in the cytoplasmic membrane when it was separated from the outer membrane by sucrose-density gradient centrifugation. However, when the envelope fraction was treated with sodium sarcosinate, the prolipoprotein was found almost exclusively in the sarcosinate-insoluble outer membrane fraction. The prolipoprotein separated in the cytoplasmic membrane by sucrose-density gradient centrifugation was soluble in sarcosinate and could not form a complex with the outer membrane once solubilized in sarcosinate. Labeling of the two lysine residues at positions 2 and 5 of the prolipoprotein with [3H]dinitrophenylfluorobenzene was enhanced 26-fold when the cells were disrupted by sonication. On the other hand, a tryptic fragment of the ompA protein, which is known to exist in the periplasmic space, increased its susceptibility to [3H]dinitrophenylfluorobenzene only 5.3-times upon disruption of the cell structure. These results indicate that the prolipoprotein accumulated in the presence of globomycin is translocated across the cytoplasmic membrane and interacts with the outer membrane. At the same time, it is attached to the cytoplasmic membrane with its amino-terminal signal peptide in such a way that the amino-terminal portion of the signal peptide containing two lysine residues is left inside the cytoplasm.     

Energy-dependent in vitro translocation of a model protein into Escherichia coli inverted membrane vesicles can take place efficiently in the complete absence of the cytosol fraction

The translocation of the precursor of a secretory protein into Escherichia coli inverted membrane vesicles was demonstrated in the absence of the cytosol fraction. A small, hydrophilic chimeric protein, OmpF-Lpp, possessing an uncleavable signal peptide was used as the model protein. As much as 80% translocation of the precursor protein into membrane vesicles was observed within 6 min in the absence of the cytosol fraction, when the precursor protein purified by means of immunoaffinity chromatography was used. The translocation was dependent on both ATP and respiratory substrates such as succinate. ATP could be replaced by a higher concentration of CTP or UTP, whereas GTP was inactive. Trichloroacetic acid treatment of the precursor protein, which is reported to result in removal of the trigger factor that is attached to a precursor protein (Crooke, E., and Wickner, W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5216-5220), did not lower the translocation efficiency significantly. Finally, the precursor protein, which was highly purified by means of successive immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, could still be efficiently translocated into the membrane vesicles. The precursor proteins purified in the presence and absence of bovine serum albumin were both active. Neither washing of the membrane vesicles for prevention of possible contamination by cytosolic factors nor the addition of the cytosol fraction to the reaction mixture affected the translocation efficiency. These results indicate that the in vitro translocation of the OmpF-Lpp precursor protein can take place in the complete absence of cytosolic soluble proteins.     

Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase

The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its "ancestral" assembly pathway.     

Identification of FtsW as a transporter of lipid-linked cell wall precursors across the membrane

Bacterial cell growth necessitates synthesis of peptidoglycan. Assembly of this major constituent of the bacterial cell wall is a multistep process starting in the cytoplasm and ending in the exterior cell surface. The intracellular part of the pathway results in the production of the membrane-anchored cell wall precursor, Lipid II. After synthesis this lipid intermediate is translocated across the cell membrane. The translocation (flipping) step of Lipid II was demonstrated to require a specific protein (flippase). Here, we show that the integral membrane protein FtsW, an essential protein of the bacterial division machinery, is a transporter of the lipid-linked peptidoglycan precursors across the cytoplasmic membrane. Using Escherichia coli membrane vesicles we found that transport of Lipid II requires the presence of FtsW, and purified FtsW induced the transbilayer movement of Lipid II in model membranes. This study provides the first biochemical evidence for the involvement of an essential protein in the transport of lipid-linked cell wall precursors across biogenic membranes.     

Protein import into yeast mitochondria: the inner membrane import site protein ISP45 is the MPI1 gene product.

Protein import across both mitochondrial membranes is mediated by the cooperation of two distinct protein transport systems, one in the outer and the other in the inner membrane. Previously we described a 45 kDa yeast mitochondrial inner membrane protein (ISP45) that can be cross-linked to a partially translocated precursor protein (Scherer et al., 1992). We have now purified ISP45 to homogeneity and identified it as the product of the nuclear MPI1 gene. Identity of ISP45 with the MPI1 gene product was shown by microsequencing of three tryptic ISP45 peptides and by demonstrating that an antibody against an Mpi1p-beta-galactosidase fusion protein specifically recognizes ISP45. Antibodies monospecific for ISP45 inhibited protein import into right-side-out mitochondrial inner membrane vesicles, but not into intact mitochondria. On solubilizing mitochondria, ISP45 was rapidly converted to a 40 kDa proteolytic fragment unless mitochondria were first denatured with trichloroacetic acid. The combined genetic and biochemical evidence identifies ISP45/Mpi1p as a component of the protein import system of the yeast mitochondrial inner membrane.     

Inhibition of protein translocation in permeabilized cells of Schizosaccharomyces pombe by puromycin.

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-alpha-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Skp is a periplasmic Escherichia coli protein requiring SecA and SecY for export

Skp of Escherichia coli (OmpH of Salmonella typhimurium) is a protein whose precise function has been obscured by its ubiquity in a wide range of subcellular fractions such as those containing DNA, ribosomes, and outer membranes. Combining in vitro and in vivo techniques we show that Skp is synthesized as a larger precursor that is processed upon translocation across the plasma membrane. Translocation is dependent on the H(+)-gradient, ATP, SecA, and SecY. Upon cellular subfractionation (avoiding non-specific electrostatic interactions) Skp partitions with beta-lactamase into the fraction of soluble, periplasmic proteins. In the context of the export factor properties of Skp previously demonstrated in vitro it is conceivable that this protein is involved in the later steps of protein translocation across the plasma membrane and/or sorting to the outer membrane.     

Organic anion transport in macrophage membrane vesicles

Cells of the J774 mouse macrophage-like cell line possess organic anion transporter that transport fluorescent dyes such as Lucifer Yellow out of the cytoplasmic matrix of the cells; the dye is both sequestered in endosomes and secreted into the extracellular medium. Lucifer Yellow that is sequestered within endosomes is subsequently delivered to the lysosomal compartment. In the present studies we demonstrated that probenecid inhibited removal of Lucifer Yellow from the soluble cytoplasm and sequestration into membrane bound organelles by quantitating Lucifer Yellow fluorescence in both soluble and membrane-associated fractions of J774 cells. In addition, we examined the uptake of Lucifer Yellow into isolated subcellular organelles derived from J774 cells. Lucifer Yellow transport in the organellar fraction of J774 cell homogenates was temperature- and pH-dependent and did not require ATP. Subcellular organelles from J774 cells were fractionated into endosome- and lysosome-enriched fractions by Percoll density gradient centrifugation. Lucifer Yellow was preferentially taken up by vesicles of the endosome-enriched fraction, and this transport was inhibited by probenecid. These studies provide direct evidence that probenecid inhibits Lucifer Yellow transport out of the cytoplasmic matrix and into cytoplasmic vacuoles in J774 cells and that organic anion transport in isolated organelles derived from J774 cells occurs preferentially in endosome, rather than in lysosome-enriched fractions; they suggest that Lucifer Yellow is carried across membranes via a secondary active transport process that requires proton symptom or hydroxyl anion antiport.