Inhibition of secretion by 1,3-Cyclohexanebis(methylamine), a dibasic compound that interferes with coatomer function.

We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the epsilon-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1, 3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane.

Interaction of coatomer with aminoglycoside antibiotics: evidence that coatomer has at least two dilysine binding sites.

Coatomer is the soluble precursor of the COPI coat (coat protein I) involved in traffic among membranes of the endoplasmic reticulum and the Golgi apparatus. We report herein that neomycin precipitates coatomer from cell extracts and from purified coatomer preparations. Precipitation first increased and then decreased as the neomycin concentration increased, analogous to the precipitation of a polyvalent antigen by divalent antibodies. This suggested that neomycin cross-linked coatomer into large aggregates and implies that coatomer has two or more binding sites for neomycin. A variety of other aminoglycoside antibiotics precipitated coatomer, or if they did not precipitate, they interfered with the ability of neomycin to precipitate. Coatomer is know to interact with a motif (KKXX) containing adjacent lysine residues at the carboxyl terminus of the cytoplasmic domains of some membrane proteins resident in the endoplasmic reticulum. All of the antibiotics that interacted with coatomer contain at least two close amino groups, suggesting that the antibiotics might be interacting with the di-lysine binding site of coatomer. Consistent with this idea, di-lysine itself blocked the interaction of antibiotics with coatomer. Moreover, di-lysine and antibiotics each blocked the coating of Golgi membranes by coatomer. These data suggest that certain aminoglycoside antibiotics interact with di-lysine binding sites on coatomer and that coatomer contains at least two of these di-lysine binding sites.

Elevation of vacuolar pH inhibits the cytotoxic activity of furin-cleaved exotoxin A.

Exotoxin A (ETA) inhibits protein synthesis in cells by a process that involves receptor-mediated endocytosis and the transport of a 37-kDa proteolytic fragment across a membrane into the cytoplasm. The fragment is apparently generated by the endoprotease furin after the toxin has been endocytosed. Cleavage of ETA by furin requires a low pH in vitro, and presumably also in vivo. Drugs that raise the pH of intracellular compartments are known to protect cells from ETA. The simplest hypothesis to explain this protection has been that the drugs interfere with furin cleavage. To test this idea, we measured the effect of pH-elevating drugs on the action of ETA that had been precleaved with recombinant furin before addition to cells. Surprisingly, we found that pH-elevating drugs protected cells from precleaved ETA as well as intact ETA. These results suggest that the process by which ETA intoxicates cells requires a low vacuolar pH for another event in addition to proteolysis by furin.

The nature of major histocompatibility complex recognition by gamma delta T cells.

Despite intensive efforts, the general rules for gamma delta T cell recognition remain undefined. Here, we take advantage of the detailed knowledge of the molecular structure and biosynthetic pathways of major histocompatibility complex (MHC) molecules to analyze the recognition properties of the gamma delta T cell clones LBK5 (specific for the class II MHC, IEk) and G8 (specific for the nonclassical class I MHC, TL10b). We find that the activation of these clones requires neither class I nor class II antigen-processing and that peptides do not confer specificity. Epitope mapping also shows that the topology of gamma delta T cell receptor interaction with the MHC is distinct from that of alpha beta T cells. These results suggest that the molecular nature of gamma delta T cell recognition is fundamentally different than that of alpha beta T cells.

Ricin intoxicates End4 mutants that have an aberrant Golgi complex.

Ricin is a protein toxin that arrests protein synthesis in mammalian cells by catalytically inactivating ribosomes. To interact with ribosomes, the A chain of the toxin must enter the cytosol. Entry involves receptor-mediated endocytosis and penetration of the toxin through an intracellular membrane, but the identity of the compartment where penetration occurs is unknown. It has been suggested that the A chain penetrates from the endoplasmic reticulum, implying the existence of a pathway from endosomes to the endoplasmic reticulum, perhaps via, retrograde transport through the Golgi. (For a recent review, see Pelham, H. R. B., Roberts, L. M., and Lord, J. M. (1992) Trends Cell Biol. 2, 183-185.) To investigate the role of the Golgi in the intoxication process of ricin, we studied the effect of ricin on mutants of the End4 complementation group of Chinese hamster ovary cells. End4 mutants express a temperature-sensitive block in secretion that is correlated with the disappearance of the Golgi stacks at the level of fluorescence microscopy. We found that End4 cells and wild-type cells were equally sensitive to ricin at the restrictive temperature, although the minimum lag before inhibition of protein synthesis was longer in the mutant cells. The simplest interpretation of these data is that ricin does not pass through the cis, medial, or trans stacks of the Golgi en route to the cytosol.

A variant of exotoxin A that forms potent and specific chemically conjugated immunotoxins.

To introduce a free sulfhydryl into Pseudomonas aeruginosa exotoxin A (ETA), methionine 161 in domain I of the toxin was changed to cysteine by site-directed mutagenesis. The free sulfhydryl provides a convenient site for covalent attachment of ETA to other proteins in the production of chimeric toxins. The mutation was then introduced into a variant of ETA that is impaired in receptor binding, termed ETA-60EF61, that has the dipeptide Glu-Phe inserted between residues 60 and 61. The resulting double mutant, ETA-60EF61 Cys161, was conjugated to three different monoclonal antibodies via a thioether linkage, and the immunotoxins were tested for cytotoxicity with cells in culture. Each immunotoxin was extremely potent against cells that expressed surface determinants for the monoclonal antibodies but had little cytotoxicity for cells that did not bind the antibodies. For comparison, we also conjugated ricin A chain to each of the three monoclonal antibodies and found that the resulting immunotoxins were at least two-orders of magnitude less potent than the corresponding immunotoxins made with ETA-60EF61Cys161. This study demonstrates that ETA-60EF61Cys161 makes potent and specific immunotoxins and may potentially be useful in selectively eliminating subpopulations of cells in vitro and in vivo.

Novel method for isolating mammalian cells defective in fluid-phase endocytosis.

A new method for isolating mutants defective in fluid-phase endocytosis has been developed based on the observation that endocytosed horseradish peroxidase can be made lethal to cells. The method was used to isolate a mutant from Chinese hamster ovary cells, termed HRP-1, that was temperature-sensitive for viability and had a 70% reduction in the rate of horseradish peroxidase endocytosis at the restrictive temperature. At high temperature, HRP-1 cells were also defective in the secretory path and their Golgi complex disappeared at the resolution of fluorescence microscopy. These properties are similar to two previously described mutants of CHO cells, DS28-6 and V.24.1. In complementation tests, mutants HRP-1, DS28-6, and V.24.1 all appeared to be in the same complementation group.

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells.

Mutant V.24.1, a member of the End4 complementation group of temperature-sensitive CHO cells, is defective in secretion at the restrictive temperature (Wang, R.-H., P. A. Colbaugh, C.-Y. Kao, E. A. Rutledge, and R. K. Draper. 1990. J. Biol. Chem. 265:20179-20187; Presley, J. F., R. K. Draper, and D. T. Brown. 1991. J. Virol. 65:1332-1339). We have further investigated the secretory lesion and report three main findings. First, the block in secretion is not due to aberrant folding or oligomerization of secretory proteins in the endoplasmic reticulum because the hemagglutinin of influenza virus folded and oligomerized at the same rate in mutant and parental cells at the restrictive temperature. Second, secretory proteins accumulated in a compartment intermediate between the ER and the Golgi. Several lines of evidence support this conclusion, the most direct being the colocalization by immunofluorescence microscopy of influenza virus hemagglutinin with a 58-kD protein that is known to reside in an intermediate compartment. Third, at the resolution of fluorescence microscopy, the Golgi complex in the mutant cells vanished at the restrictive temperature.

Turnover of the transferrin receptor is not influenced by removing most of the extracellular domain.

We treated intact cells with trypsin to remove most of the external domain of the transferrin receptor and investigated what effect the absence of the external domain had on the turnover of the fragment that remained associated with the cells. To detect the cell-associated tryptic fragment, which contains a small amount of the external domain, the transmembrane domain, and the cytoplasmic domain, we prepared an anti-peptide antibody against a segment of the cytoplasmic domain. This antibody specifically immunoprecipitated the intact transferrin receptor as well as a 21-kDa peptide from trypsin-treated HeLa cells. Several lines of evidence indicated that the 21-kDa peptide was the cell-associated tryptic fragment of the transferrin receptor. The fragment was only present in trypsin-treated cells; the fragment migrated as a dimer in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as it should if it were derived from the transferrin receptor; a goat antibody prepared to the purified human transferrin receptor also precipitated the 21-kDa peptide from trypsinized cells. In addition, treating the tryptic fragment with neuraminidase increased the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels, suggesting the fragment contained O-linked carbohydrate. When cells were trypsinized and then incubated at 37 degrees C, the half-life of the tryptic fragment (15 +/- 4 h) was not significantly different than the half-life of the intact receptor (19 +/- 6 h). This indicates that removing 95% of the external domain of the transferrin receptor has little effect on processes operating in the turnover of the receptor.

Biosynthesis of lysosomal enzymes in cells of the End3 complementation group conditionally defective in endosomal acidification.

Various aspects of lysosome biogenesis have been studied in Chinese hamster ovary (CHO) cells of the End3 complementation group (designated G.7.1 cells), which display a temperature-sensitive defect in the acidification of endosomes, but not lysosomes. In G.7.1 and normal wild-type cells grown at the permissive temperature (34 degrees C), the lysosomal enzymes alpha-glucosidase and cathepsin D were synthesized as high-molecular-weight precursors that subsequently underwent intracellular proteolytic processing to yield lower molecular weight mature forms. The mature forms of the enzymes were retained in cells, and small amounts of each precursor were secreted. However, in G.7.1 cells grown at the restrictive temperature (41 degrees C), there was a massive and inappropriate oversecretion of lysosomal enzyme precursors, which resulted in very little of the mature forms being processed and retained by the cells. This mistargeting of lysosomal enzymes was not due to an absence of phosphorylated oligosaccharides on the enzymes, nor to a defect in mannose 6-phosphate (Man6P) receptors. However, it was found that whereas G.7.1 cells had the same number of cell surface Man6P receptors at 34 degrees C and 41 degrees C, the rate of accumulation and degradation of Man6P-containing ligands was about two to three times more rapid in cells maintained at the permissive temperature. There did not appear to be any gross changes in Golgi function as the oligosaccharides of alpha-glucosidase and the Man6P receptor were processed in a similar fashion at both 34 degrees C and 41 degrees C. In addition to these studies, electron microscopic observations revealed that at 41 degrees C, G.7.1 cells accumulated inclusion-type bodies reminiscent of those found in I-cell disease fibroblasts. Thus, the biochemical and electron microscopic results on G.7.1 cells provide further evidence that acidified endosomes are important for the biogenesis of lysosomes.

Defective transport of Sindbis virus glycoproteins in End4 mutant Chinese hamster ovary cells.

Mutant V.24.1, a temperature-sensitive derivative of Chinese hamster ovary cells, defines the End4 complementation group of mutants selected for resistance to protein toxins and has defective lysosomes at the restrictive temperature (P. A. Colbaugh, M. Stookey, and R. K. Draper, J. Cell Biol. 108:2211-2219, 1989). We have investigated the biosynthesis of Sindbis virus envelope glycoproteins in V.24.1 cells. When the cells were infected at the restrictive temperature, the envelope glycoproteins E1 and E2 were undetectable on the cell surface and proteolytic processing of the precursor protein pE2 to envelope protein E2 did not occur. Protein retained intracellularly was sensitive to endoglycosidase H and, by immunofluorescence localization, appeared to accumulate in the endoplasmic reticulum. We conclude that the genetic defect in V.24.1 cells impairs the transport of Sindbis virus glycoproteins, apparently at the level of export from the endoplasmic reticulum.

Impaired secretion and fluid-phase endocytosis in the End4 mutant of Chinese hamster ovary cells.

Mutant V.24.1 defines the End4 complementation group of temperature-sensitive Chinese hamster ovary cell mutants selected for resistance to protein toxins. We investigated the secretory pathway in the mutant cells and found: 1) The hemagglutinin of influenza virus failed to reach the plasma membrane and was retained in a form sensitive to endoglycosidase H at the restrictive temperature. 2) Transferrin receptors synthesized at the restrictive temperature remained sensitive to endoglycosidase H. 3) Secretion of total soluble protein into the medium was strongly reduced at high temperature. These data indicate that V.24.1 cells are defective in secretion at the restrictive temperature. To see what effect the lesion had on the endocytic pathway, we measured the accumulation and recycling of the fluid-phase marker horseradish peroxidase. Accumulation was inhibited by 50% while recycling was barely affected, suggesting that the rate of fluid-phase endocytosis was reduced. We previously showed that the clathrin-coated pit pathway of endocytosis was not affected in the mutant, indicated by a normal transferrin cycle (Colbaugh, P. A., Stookey, M., and Draper, R. K. (1989) J. Cell Biol. 108, 2211-2219). Thus, the secretory lesion correlates with reduced fluid-phase endocytosis without impairing the clathrin-dependent pathway of receptor-mediated endocytosis. We also investigated the delivery of endocytosed material to lysosomes and found that delivery was partially, but not completely, impaired in the mutant. This suggests that endocytosed material can enter lysosomes, although slowly, in the absence of a functional secretory pathway.

Antibodies to clathrin inhibit endocytosis but not recycling to the trans Golgi network in vitro.

Mannose 6-phosphate receptors carry newly synthesized lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes and then return to the TGN to carry out another round of lysosomal enzyme delivery. Although clathrin-coated vesicles mediate the export of mannose 6-phosphate receptors from the TGN, nothing is known about the transport vesicles used to carry these receptors back to the TGN. Two different in vitro assays used in this study show that an antibody that interferes with clathrin assembly blocks receptor-mediated endocytosis of transferrin, but has no effect on the recycling of the 300-kilodalton mannose 6-phosphate receptor from prelysosomes to the TGN. These results suggest that the transport of mannose 6-phosphate receptors from prelysosomes to the TGN does not involve clathrin.

A dipeptide insertion in domain I of exotoxin A that impairs receptor binding.

Deletions within the structural exotoxin A gene of 27 or 119 amino acids in domain I of the mature polypeptide, or of 88 or 105 amino acids in domains I and II, resulted in the synthesis of exotoxin A (ETA) polypeptides that were not secreted from Pseudomonas aeruginosa hosts but were localized in the cell membrane. Insertions of a hexanucleotide sequence, either pCGAGCT or pCGAATT, at TaqI sites within the gene resulted in variant exotoxin A polypeptides which were secreted normally. pCGAGCT causes insertion of either Glu-Leu or Ser-Ser in the amino acid sequence of the toxin, while pCGAATT causes insertion of either Glu-Phe or Asn-Ser dipeptides. Although the cytotoxicity of eight variants was unimpaired, that of four others was reduced, and one variant which had a Glu-Phe insert between residues 60 and 61 (ETA-60EF61) was 500-fold less cytotoxic than wild-type exotoxin A. Purified ETA-60EF61 dissociated much faster from mouse LMTK- cells than wild-type ETA, suggesting that the insertion impaired the ability of ETA-60EF61 to interact with exotoxin A receptors. The location of the insert is within a major concavity on the surface of domain I of the exotoxin A molecule, suggesting that this concavity is important for toxin-receptor interaction.

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.

Three new complementation groups of temperature-sensitive Chinese hamster ovary cell mutants defective in the endocytic pathway.

We describe here the results of complementation studies with six mutant Chinese hamster ovary cells expressing temperature-sensitive lesions affecting the endocytic pathway. The mutants were crossed with representatives of the End1 and End2 complementation groups identified previously by Robbins et al. (J. Cell Biol. 99:1296-1308, 1984). Two mutants, G.8.1 and 31.1, were members of the End1 complementation group. One mutant, 25.2, was a member of the End2 complementation group. The other three mutants each defined new complementation groups, which we have designated End3 (mutant G.7.1), End4 (mutant V.24.1), and End5 (mutant 42.2). Previous work on mutants of the End1, End2, and End3 classes had shown that these mutants were defective in endosomal acidification. We prepared postnuclear supernatants from mutants harvested at the nonpermissive temperature and compared their acidification activities, assessed by ATP-stimulated quenching of acridine orange. Members of the End1, End2, and End2 groups had reduced acidification activity, correlating with the acidification defects known to be expressed by these mutants. Strain V.24.1 (End4) also expressed a 40% reduction in acidification activity, while strain 42.2 (End5) had no reduction of acidification activity.

Two pathways of transferrin recycling evident in a variant of mouse LMTK- cells.

We describe here the properties of a variant cell line, termed AF192, selected by exposing mouse LMTK- cells to a cytotoxic form of transferrin prepared by conjugating transferrin to diphtheria toxin. AF192 cells were mildly resistant to the transferrin-diphtheria toxin conjugate and were cross-resistant to the protein toxins modeccin, abrin, ricin, and Pseudomonas aeruginosa exotoxin A. AF192 cells had an aberrant transferrin cycle characterized by an approximately 50% reduction in the rate of iron uptake from diferric transferrin, an approximately 25% reduction in the number of surface transferrin receptors, and a time course for transferrin recycling that resolved into two apparent first-order rate processes. The aberrant transferrin cycle was not the result of a failure of endocytosed transferrin to discharge iron; rather, part, but not all, of the transferrin taken up by AF192 cells was diverted to an intracellular site from which it was recycled very slowly.

Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa.

The exotoxin A genes from Pseudomonas aeruginosa strains PA103 and PAO1 have been independently cloned in a pUC9-derived plasmid. In a non-toxigenic mutant of PAO1 as host, the cloned genes directed the synthesis of intact exotoxin A that expressed ADP-ribosyltransferase activity upon treatment with urea and dithiothreitol. Western-blot analysis of culture supernatants identified a polypeptide of 67 kDa, the molecular mass of intact exotoxin A. There was an approximately 15-fold increase in the toxin yield from P. aeruginosa cells carrying a cloned PA103 gene compared to PA103, and a 40-fold increase in the yield of toxin gene yielded about four times more toxin than those carrying the cloned PAO1 gene. Toxin expression was correlated with the presence of a transcript that was initiated 88 bp upstream from the translational start site. Little or no messenger RNA from either cloned gene could be detected in an Escherichia coli host, or in a P. aeruginosa host grown in the presence of 0.1 mM-Fe2+, a condition that inhibits toxin expression. The nucleotide sequences of two regions, each of approximately 500 bp, near the 5' and 3' termini of the structural gene were established. In these regions, three exotoxin A gene from PAO1 has ten base-pair differences compared to the PA103 gene, three in the non-coding region, and seven in the structural gene, four of which should lead to amino-acid differences. No apparent sequence similarities were found between the inferred promoter region of the exotoxin A gene and that of other Pseudomonas genes, nor with the consensus sequence of E. coli promoters.