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  • 1
    Electronic Resource
    Electronic Resource
    Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides (1992)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 100 (1992), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of α-amylase of β-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the β-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1992.tb14046.x
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  • 2
    Electronic Resource
    Electronic Resource
    Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell-bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb02164.x
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  • 3
    Electronic Resource
    Electronic Resource
    Dual regulation by heat and nutrient stress of the yeast HSP150 gene encoding a secretory glycoprotein (1993)
    Springer
    Molecular genetics and genomics 239 (1993), S. 273-280 
    Details
    ISSN: 1617-4623
    Keywords: Heat shock gene ; Heat shock protein ; Secretion ; Yeast ; Transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have cloned and characterized the HSP150 gene of Saccharomyces cerevisiae, which encodes a glycoprotein (hsp150) that is secreted into the growth medium. Unexpectedly, the HSP150 gene was found to be regulated by heat shock and nitrogen starvation. Shifting the cells from 24° C to 37° C resulted in an abrupt increase in the steady-state level of the HSP150 mRNA, and de novo synthesized hsp150 protein. Returning the cells to 24° C caused a rapid decrease in mRNA and protein synthesis to basal levels. The HSP150 5′-flanking region contains several heat shock element-like sequences (HSE). To study the function of these sequences, a strain bearing a disrupted copy of the HSP150 gene was transformed with plasmids in which the coding region of HSP150, or a HSP150-lacZ fusion gene, was preceded by 5′ deletion derivatives of the HSP150 promoter. Site-directed mutagenesis of one HSE-like element, located between the TATA box and transcription initiation sites, abolished heat activation of transcription. In addition to heat shock, the HSP150 gene is regulated by the availability of nutrients in the growth medium. The HSP150 mRNA level was increased by nitrogen limitation at 24° C, even when under the control of a HSP150 promoter region of 137 by carrying the mutagenized HSE.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00281628
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  • 4
    Electronic Resource
    Electronic Resource
    Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 10 (1994), S. 355-370 
    Details
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; protein secretion ; protein folding ; disulphide bond formation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress-protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine-free pro-α-factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320100308
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  • 5
    Electronic Resource
    Electronic Resource
    Structural features of a polypeptide carrier promoting secretion of a β-lactamase fusion protein in yeast (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1381-1391 
    Details
    ISSN: 0749-503X
    Keywords: secretion ; yeast ; glycosylation ; β-lactamase ; fusion protein ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Escherichia coli β-lactamase was secreted into the culture medium of Saccharomyces cerevisiae in biologically active form, when fused to the C-terminus of the hsp150δ-carrier. The hsp150δ-carrier is an N-terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 times in tandem. Here we expressed the hsp150δ-carrier fragment alone in S. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150δ-carrier were synthesized. About half of the de novo synthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre-Golgi compartment. The extensively O-glycosylated carrier fragment was purified from the culture medium under non-denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non-glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologous proteins attached to it.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320111406
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    Paper (German National Licenses)
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  • 6
    Electronic Resource
    Electronic Resource
    The hsp150Δ-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 12 (1996), S. 457-466 
    Details
    ISSN: 0749-503X
    Keywords: yeast ; NGFR ; secretion ; protein production ; hsp150 protein ; Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150Δ-carrier, the hsp150Δ-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150Δ-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150Δ-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150Δ-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150Δ-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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