Neil Bulleid - Academia.edu (original) (raw)

Papers by Neil Bulleid

Biochemical Journal, 1996

Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating ... more Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (Cterminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ' mini-gene ' which lacks part of the triplehelical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the Cpropeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed

Molecular Biology of the Cell, 2015

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in e... more The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

Biochemical Society Transactions, 2014

The efficient folding, assembly and secretion of proteins from mammalian cells is a critically im... more The efficient folding, assembly and secretion of proteins from mammalian cells is a critically important process for normal cell physiology. Breakdown of the ability of cells to secrete functional proteins leads to disease pathologies caused by a lack of protein function or by cell death resulting from an aggravated stress response. Central to the folding of secreted proteins is the formation of disulfides which both aid folding and provide stability to the protein structure. For disulfides to form correctly necessitates the appropriate redox environment within the endoplasmic reticulum: too reducing and disulfides will not form, too oxidizing and non-native disulfides will not be resolved. How the endoplasmic reticulum maintains the correct redox balance is unknown. Although we have a good appreciation of the processes leading to a more oxidizing environment, our understanding of how any counterbalancing reductive pathway operates is limited. The present review looks at potential mechanisms for introducing reducing equivalents into the endoplasmic reticulum and discusses an approach to test these hypotheses.

Protein Folding Handbook, 2005

Journal of Biological Chemistry

ABSTRACT

Biochemical Society Symposium

Formation of native disulphide bonds is a post-translational modification associated with the fol... more Formation of native disulphide bonds is a post-translational modification associated with the folding and assembly of secretory proteins. The process is catalysed within the lumen of the endoplasmic reticulum by the enzyme protein disulphide-isomerase (PDI), which is abundant in secretory cells and catalyses thiol: protein-disulphide interchange in vitro with very broad protein substrate specificity. The presence of PDI within microsomal vesicles is essential for efficient and rapid cotranslational disulphide bond formation during protein synthesis in vitro. The sequence of PDI is now known from several species, and shows the presence of two domains closely homologous to thioredoxins. Chemical modification data confirm the role of the thioredoxin domains in thiol:disulphide interchange activity, and structural models of these domains can be built based on homology with thioredoxin. Thus PDI is both strongly implicated in the process of native protein folding in vivo and well characterized at the molecular level. In addition to its disulphide-isomerase activity, PDI participates as a component of the enzyme prolyl-4-hydroxylase, and further functions have also been proposed.

Anticancer research

We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface ... more We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface protease associated with tumour cells. This protease has been referred to as guanidinobenzoatase (GB) and is now known to be functionally identical to tissue plasminogen activator (t-PA). The use of a fluorescent competitive inhibitor of GB enabled the enzymic status of GB to be determined, i.e. was the enzyme active, latent or removed from our test system. The cell surface GB was then demonstrated to interact with extractable cytoplasmic inhibitors obtained from these sections. We then used a protected form of the GB in the absence of these internal inhibitors; such sections were used to transfer the GB to fibrin fibrils, thus exposing the presumptive receptor on the tumour cell surfaces. Texas red labelled t-PA was then shown to bind to the tumour cells in these pretreated sections from which the GB had previously been removed. We believe that the surface of tumour cells can be used to study the interaction of the naturally occurring inhibitors with GB and also that the cell surface receptors for GB can be used to study the binding of t-PA to cell surfaces.

The assembly and peptide loading of MHC-Class I molecules within the endoplasmic reticulum is ess... more The assembly and peptide loading of MHC-Class I molecules within the endoplasmic reticulum is essential for antigen presentation at the cell-surface and is facilitated by the peptide loading complex. The formation of a mixed disulfide between the heavy chain of class I and components of the loading complex (ERp57, PDI and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is co-incident with the stabilisation of the α2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57 and tapasin was observed and shown to be stabilised by a disulfide between both tapasi...

Journal of Cell Science

Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fu... more Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that β1-integrins adopt the bent, inactive conformation after assembly with α-integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca(2+) ions. In addition, Ca(2+) bin...

The Biochemical journal, 2014

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide e... more Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as PDI (protein disulfide-isomerase) and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α (endoplasmic reticulum oxidase 1α) which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide (H2O2). Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactivate the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen nor H2O2 was able to oxidize Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family, such as PDI or ERp46 (endoplasmic reticulum-r...

Progress in nucleic acid research and molecular biology, 2001

Protein folding in living cells is a complex process involving many interdependent factors. The p... more Protein folding in living cells is a complex process involving many interdependent factors. The primary site for folding of nascent proteins destined for secretion is the endoplasmic reticulum (ER). Several disease states, including cystic fibrosis, are brought about because of irregularities in protein folding. Under normal cellular conditions, "quality control" mechanisms ensure that only correctly folded proteins are exported from the ER, with incorrectly folded or incompletely assembled proteins being degraded. Quality control mechanisms can be divided into two broad processes: (1) Primary quality control involves general mechanisms that are not specific for individual proteins; these monitor the fidelity of nascent protein folding in the ER and mediate the destruction of incompletely folded proteins. (2) Partially folded or assembled proteins may be subject to secondary quality control mechanisms that are protein- or protein-family-specific. Here we use the folding an...

The Biochemical journal, 1998

Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of cata... more Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of catalysing disulphide-bond formation and isomerization, and of participating as a non-catalytic subunit of prolyl 4-hydroxylase (P4-H) and microsomal triacylglycerol transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate its potential role as a molecular chaperone within a cellular context, we studied the folding, modification and assembly of type X collagen in semi-permeabilized cells. Using this approach, we demonstrate that depletion of ATP has no effect on the rate or extent of helix formation, indicating that the individual triple helical regions do not interact with the molecular chaperone immunoglobulin heavy-chain binding protein (BiP). However, PDI was shown to interact transiently with type X during helix formation in a role related to its function as the beta subunit of P4-H. Once the col...

The EMBO journal, Jan 17, 1997

The folding and assembly of procollagen occurs within the cell through a series of discrete steps... more The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; howeve...

The Biochemical journal, 1993

We describe here a cell-free system which will carry out the initial stages in the synthesis, pos... more We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of h...

The Biochemical journal, 1995

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, tr... more We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results sho...

The Biochemical journal, Jan 15, 1986

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified... more Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum M...

The Biochemical journal, 1989

The Biochemical journal, Jan 15, 1992

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two fo... more Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the fo...

Extracellular Matrix Protocols, 2000

ABSTRACT

Biochemical Journal, 1996

Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating ... more Procollagen assembly is initiated within the endoplasmic reticulum by three α-chains associating via their C-propeptides (Cterminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen ' mini-gene ' which lacks part of the triplehelical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the Cpropeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed

Molecular Biology of the Cell, 2015

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in e... more The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

Biochemical Society Transactions, 2014

The efficient folding, assembly and secretion of proteins from mammalian cells is a critically im... more The efficient folding, assembly and secretion of proteins from mammalian cells is a critically important process for normal cell physiology. Breakdown of the ability of cells to secrete functional proteins leads to disease pathologies caused by a lack of protein function or by cell death resulting from an aggravated stress response. Central to the folding of secreted proteins is the formation of disulfides which both aid folding and provide stability to the protein structure. For disulfides to form correctly necessitates the appropriate redox environment within the endoplasmic reticulum: too reducing and disulfides will not form, too oxidizing and non-native disulfides will not be resolved. How the endoplasmic reticulum maintains the correct redox balance is unknown. Although we have a good appreciation of the processes leading to a more oxidizing environment, our understanding of how any counterbalancing reductive pathway operates is limited. The present review looks at potential mechanisms for introducing reducing equivalents into the endoplasmic reticulum and discusses an approach to test these hypotheses.

Protein Folding Handbook, 2005

Journal of Biological Chemistry

ABSTRACT

Biochemical Society Symposium

Formation of native disulphide bonds is a post-translational modification associated with the fol... more Formation of native disulphide bonds is a post-translational modification associated with the folding and assembly of secretory proteins. The process is catalysed within the lumen of the endoplasmic reticulum by the enzyme protein disulphide-isomerase (PDI), which is abundant in secretory cells and catalyses thiol: protein-disulphide interchange in vitro with very broad protein substrate specificity. The presence of PDI within microsomal vesicles is essential for efficient and rapid cotranslational disulphide bond formation during protein synthesis in vitro. The sequence of PDI is now known from several species, and shows the presence of two domains closely homologous to thioredoxins. Chemical modification data confirm the role of the thioredoxin domains in thiol:disulphide interchange activity, and structural models of these domains can be built based on homology with thioredoxin. Thus PDI is both strongly implicated in the process of native protein folding in vivo and well characterized at the molecular level. In addition to its disulphide-isomerase activity, PDI participates as a component of the enzyme prolyl-4-hydroxylase, and further functions have also been proposed.

Anticancer research

We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface ... more We have used frozen sections of squamous cell carcinoma as a convenient source of a cell surface protease associated with tumour cells. This protease has been referred to as guanidinobenzoatase (GB) and is now known to be functionally identical to tissue plasminogen activator (t-PA). The use of a fluorescent competitive inhibitor of GB enabled the enzymic status of GB to be determined, i.e. was the enzyme active, latent or removed from our test system. The cell surface GB was then demonstrated to interact with extractable cytoplasmic inhibitors obtained from these sections. We then used a protected form of the GB in the absence of these internal inhibitors; such sections were used to transfer the GB to fibrin fibrils, thus exposing the presumptive receptor on the tumour cell surfaces. Texas red labelled t-PA was then shown to bind to the tumour cells in these pretreated sections from which the GB had previously been removed. We believe that the surface of tumour cells can be used to study the interaction of the naturally occurring inhibitors with GB and also that the cell surface receptors for GB can be used to study the binding of t-PA to cell surfaces.

The assembly and peptide loading of MHC-Class I molecules within the endoplasmic reticulum is ess... more The assembly and peptide loading of MHC-Class I molecules within the endoplasmic reticulum is essential for antigen presentation at the cell-surface and is facilitated by the peptide loading complex. The formation of a mixed disulfide between the heavy chain of class I and components of the loading complex (ERp57, PDI and tapasin) suggests that these molecules are involved in the redox regulation of components during assembly and peptide loading. We demonstrate here that a disulfide formed between heavy chain and tapasin can occur between cysteine residues located in the cytosolic regions of these proteins following translation of heavy chain in an in vitro translation system. The formation of this disulfide occurs after assembly into the loading complex and is co-incident with the stabilisation of the α2 disulfide bond within the peptide binding grove. A ternary complex between heavy chain, ERp57 and tapasin was observed and shown to be stabilised by a disulfide between both tapasi...

Journal of Cell Science

Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fu... more Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that β1-integrins adopt the bent, inactive conformation after assembly with α-integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca(2+) ions. In addition, Ca(2+) bin...

The Biochemical journal, 2014

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide e... more Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as PDI (protein disulfide-isomerase) and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α (endoplasmic reticulum oxidase 1α) which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide (H2O2). Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactivate the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen nor H2O2 was able to oxidize Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family, such as PDI or ERp46 (endoplasmic reticulum-r...

Progress in nucleic acid research and molecular biology, 2001

Protein folding in living cells is a complex process involving many interdependent factors. The p... more Protein folding in living cells is a complex process involving many interdependent factors. The primary site for folding of nascent proteins destined for secretion is the endoplasmic reticulum (ER). Several disease states, including cystic fibrosis, are brought about because of irregularities in protein folding. Under normal cellular conditions, "quality control" mechanisms ensure that only correctly folded proteins are exported from the ER, with incorrectly folded or incompletely assembled proteins being degraded. Quality control mechanisms can be divided into two broad processes: (1) Primary quality control involves general mechanisms that are not specific for individual proteins; these monitor the fidelity of nascent protein folding in the ER and mediate the destruction of incompletely folded proteins. (2) Partially folded or assembled proteins may be subject to secondary quality control mechanisms that are protein- or protein-family-specific. Here we use the folding an...

The Biochemical journal, 1998

Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of cata... more Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of catalysing disulphide-bond formation and isomerization, and of participating as a non-catalytic subunit of prolyl 4-hydroxylase (P4-H) and microsomal triacylglycerol transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate its potential role as a molecular chaperone within a cellular context, we studied the folding, modification and assembly of type X collagen in semi-permeabilized cells. Using this approach, we demonstrate that depletion of ATP has no effect on the rate or extent of helix formation, indicating that the individual triple helical regions do not interact with the molecular chaperone immunoglobulin heavy-chain binding protein (BiP). However, PDI was shown to interact transiently with type X during helix formation in a role related to its function as the beta subunit of P4-H. Once the col...

The EMBO journal, Jan 17, 1997

The folding and assembly of procollagen occurs within the cell through a series of discrete steps... more The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; howeve...

The Biochemical journal, 1993

We describe here a cell-free system which will carry out the initial stages in the synthesis, pos... more We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of h...

The Biochemical journal, 1995

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, tr... more We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results sho...

The Biochemical journal, Jan 15, 1986

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified... more Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum M...

The Biochemical journal, 1989

The Biochemical journal, Jan 15, 1992

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two fo... more Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the fo...

Extracellular Matrix Protocols, 2000

ABSTRACT