scholarly journals Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane.

1988 ◽  
Vol 107 (2) ◽  
pp. 457-470 ◽  
Author(s):  
S Monier ◽  
P Van Luc ◽  
G Kreibich ◽  
D D Sabatini ◽  
M Adesnik
Keyword(s):  
Endoplasmic Reticulum ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Signal Peptidase ◽  
Endoplasmic Reticulum Membrane ◽  
Peptide Cleavage ◽  
Amino Terminal ◽  
Transfer Signal ◽  
Membrane Anchoring

Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein.

scholarly journals The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.

1987 ◽  
Vol 104 (6) ◽  
pp. 1705-1714 ◽  
Author(s):  
J Finidori ◽  
L Rizzolo ◽  
A Gonzalez ◽  
G Kreibich ◽  
M Adesnik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Endoplasmic Reticulum Membrane ◽  
Hydrophobic Region ◽  
Amino Terminal ◽  
Influenza Hemagglutinin

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane

1987 ◽  
Vol 242 (3) ◽  
pp. 767-777 ◽  
Author(s):  
A Robinson ◽  
M A Kaderbhai ◽  
B M Austen
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Phospholipid Bilayer ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the ‘cold’ synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

scholarly journals Biosynthesis and processing of ribophorins in the endoplasmic reticulum.

1984 ◽  
Vol 99 (3) ◽  
pp. 1076-1082 ◽  
Author(s):  
M G Rosenfeld ◽  
E E Marcantonio ◽  
J Hakimi ◽  
V M Ort ◽  
P H Atkinson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Posttranslational Modifications ◽  
Rat Hepatocytes ◽  
Direct Analysis ◽  
In Vitro Translation ◽  
Endoplasmic Reticulum Membrane ◽  
Pituitary Cells ◽  
Amino Terminal

Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.

The Role of Intracellular Membranes in Protein Processing

1981 ◽  
Vol 39 ◽  
pp. 514-515
Author(s):  
Dennis Shields ◽  
Thomas G. Warren ◽  
Sara E. Roth ◽  
Reza F. Green
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Messenger Rna ◽  
Polypeptide Chain ◽  
Secretory Protein ◽  
Free Protein ◽  
Amino Terminal ◽  
Free Translation ◽  
Microsomal Membranes

Most polypeptides destined for secretion are synthesized on polyribosomes bound to the membrane of the endoplasmic reticulum (E.R.), in contrast, cytosolic proteins are made on free ribosomes. When the messenger RNA (mRNA) for a secretory protein is translated in a cell-free protein synthesizing system, the product is usually larger than the mature protein by about 3,000 daltons. Numerous studies have demonstrated that the higher molecular weight of the cell-free translation product can be attributed to an amino terminal extension of about 20-30 amino acids termed the “signal peptide”. This signal peptide is thought to mediate binding of ribosomes bearing the nascent polypeptide chain to the membrane of the endoplasmic reticulum. Upon interaction with the E.R., the polypeptide chain is translocated across the membrane usually resulting in proteolytic removal of the signal peptide and segregation of the “processed” polypeptide into the ER. cisternae. This series of reactions can be followed in vitro by supplementing the cell-free protein synthesizing system with heterologous microsomal membranes which have been stripped of their endogenous ribosomes.

scholarly journals Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein.

1981 ◽  
Vol 91 (2) ◽  
pp. 551-556 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Preceding Paper ◽  
Secretory Protein ◽  
Microsomal Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Lipid Signal ◽  
Recognition Protein

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.

scholarly journals Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation.

1992 ◽  
Vol 12 (7) ◽  
pp. 3288-3296 ◽  
Author(s):  
D Feldheim ◽  
J Rothblatt ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Carboxyl Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

scholarly journals Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals

2001 ◽  
Vol 12 (2) ◽  
pp. 487-501 ◽  
Author(s):  
Monika Anand ◽  
Jeffrey S. Rush ◽  
Sutapa Ray ◽  
Marie-Agnes Doucey ◽  
Jennifer Weik ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Product ◽  
Water Soluble ◽  
Predicted Amino Acid Sequence ◽  
Endoplasmic Reticulum Membrane ◽  
In Vitro System ◽  
Streptolysin O ◽  
Membrane Anchoring ◽  
In Vitro Data

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-β-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation

1992 ◽  
Vol 12 (7) ◽  
pp. 3288-3296
Author(s):  
D Feldheim ◽  
J Rothblatt ◽  
R Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Protein A ◽  
Integral Membrane Protein ◽  
Secretory Protein ◽  
Carboxyl Terminus ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Network Cell

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

scholarly journals Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum.

1988 ◽  
Vol 107 (5) ◽  
pp. 1697-1706 ◽  
Author(s):  
M S Poruchynsky ◽  
P H Atkinson
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Specific Activity ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Acid Number ◽  
Wild Type ◽  
Amino Terminal ◽  
Er Retention

Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.

scholarly journals The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes.

1986 ◽  
Vol 103 (6) ◽  
pp. 2263-2272 ◽  
Author(s):  
E M Eskridge ◽  
D Shields
Keyword(s):  
Signal Peptide ◽  
Fusion Proteins ◽  
Signal Sequence ◽  
Membrane Vesicles ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Endoplasmic Reticulum Membrane ◽  
Amino Terminal ◽  
Microsomal Membranes

To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.

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