Transmembrane topology of the mammalian KDEL receptor.

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.

Download Full-text

Transmembrane topology of the mammalian KDEL receptor

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6435-6441.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6435-6441

Author(s):

P Singh ◽

B L Tang ◽

S H Wong ◽

W Hong

Keyword(s):

Membrane Protein ◽

Fusion Proteins ◽

Integral Membrane Protein ◽

In Vitro Translation ◽

Translation System ◽

Transmembrane Topology ◽

Amino Terminal ◽

Hydrophilic Region ◽

Kdel Receptor

Download Full-text

Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus.

The Journal of Cell Biology ◽

10.1083/jcb.106.5.1489 ◽

1988 ◽

Vol 106 (5) ◽

pp. 1489-1498 ◽

Cited By ~ 43

Author(s):

J D Hull ◽

R Gilmore ◽

R A Lamb

Keyword(s):

Membrane Protein ◽

Influenza A ◽

Signal Sequence ◽

Integral Membrane Protein ◽

In Vitro Translation ◽

Signal Recognition ◽

M2 Protein ◽

Amino Terminal ◽

Terminal Domain

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.

Download Full-text

Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

10.1055/s-0038-1652739 ◽

1981 ◽

Author(s):

G M Fuller ◽

J M Nickerson

Keyword(s):

Signal Peptide ◽

Hepatoma Cell ◽

Temporal Analysis ◽

In Vitro Translation ◽

Translation System ◽

Hepatoma Cell Line ◽

Amino Terminal ◽

Cellular Processes ◽

Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

Download Full-text

P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

Infection and Immunity ◽

10.1128/iai.69.5.3323-3334.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3323-3334 ◽

Cited By ~ 28

Author(s):

Laila Noppa ◽

Yngve Östberg ◽

Marija Lavrinovicha ◽

Sven Bergström

Keyword(s):

Membrane Protein ◽

Borrelia Burgdorferi ◽

Gene Family ◽

Integral Membrane Protein ◽

Sensu Stricto ◽

In Vitro Translation ◽

Signal Peptidase ◽

In Vitro Cultivation ◽

Type I

ABSTRACT To elucidate antigens present on the bacterial surface ofBorrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis.

Download Full-text

Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits.

The Journal of Cell Biology ◽

10.1083/jcb.116.2.385 ◽

1992 ◽

Vol 116 (2) ◽

pp. 385-393 ◽

Cited By ~ 48

Author(s):

R A Chavez ◽

Z W Hall

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Fusion Proteins ◽

Transmembrane Domains ◽

In Vitro Translation ◽

Translation System ◽

Nicotinic Acetylcholine ◽

Mammalian Muscle ◽

Membrane Spanning

We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2263 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2263-2272 ◽

Cited By ~ 10

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.

Download Full-text

Fibrillin assembly: dimer formation mediated by amino-terminal sequences

Journal of Cell Science ◽

10.1242/jcs.112.20.3549 ◽

1999 ◽

Vol 112 (20) ◽

pp. 3549-3558 ◽

Cited By ~ 1

Author(s):

J.L. Ashworth ◽

V. Kelly ◽

R. Wilson ◽

C.A. Shuttleworth ◽

C.M. Kielty

Keyword(s):

Proteinase K ◽

In Vitro Translation ◽

Translation System ◽

Dimer Formation ◽

Amino Terminal ◽

Reducing Conditions ◽

Sds Page ◽

Fibrillin 1 ◽

The Stability

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9–11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

Download Full-text

Characterization of Two New Structural Glycoproteins, GP3 and GP4, of Equine Arteritis Virus

Journal of Virology ◽

10.1128/jvi.76.21.10829-10840.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10829-10840 ◽

Cited By ~ 34

Author(s):

Roeland Wieringa ◽

Antoine A. F. de Vries ◽

Martin J. B. Raamsman ◽

Peter J. M. Rottier

Keyword(s):

Membrane Protein ◽

Rna Virus ◽

Expression System ◽

Integral Membrane Protein ◽

Envelope Proteins ◽

Equine Arteritis Virus ◽

In Vitro Translation ◽

Infected Cells ◽

Virus Expression

ABSTRACT Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. Four envelope proteins have hitherto been identified in EAV particles: the predominant membrane proteins M and GL, the unglycosylated small envelope protein E, and the nonabundant membrane glycoprotein GS. In this study, we established that the products of EAV open reading frame 3 (ORF3) and ORF4 (designated GP3 and GP4, respectively) are also minor structural glycoproteins. The proteins were first characterized by various analyses after in vitro translation of RNA transcripts in a rabbit reticulocyte lysate in the presence and absence of microsomal membranes. We subsequently expressed ORF3 and -4 in baby hamster kidney cells by using the vaccinia virus expression system and, finally, analyzed the GP3 and GP4 proteins synthesized in EAV-infected cells. The results showed that GP4 is a class I integral membrane protein of 28 kDa with three functional N-glycosylation sites and with little, if any, of its carboxy terminus exposed. Both after independent expression and in EAV-infected cells, the protein localizes in the endoplasmic reticulum (ER), as demonstrated biochemically by analysis of its oligosaccharide side chains and as visualized directly by immunofluorescence studies. GP3, on the other hand, is a heavily glycosylated protein whose hydrophobic amino terminus is not cleaved off. It is an integral membrane protein anchored by either or both of its hydrophobic terminal domains and with no parts detectably exposed cytoplasmically. Also, GP3 localizes in the ER when expressed independently and in the context of an EAV infection. Only a small fraction of the GP3 and GP4 proteins synthesized in infected cells ends up in virions. Most, but not all, of the oligosaccharides of these virion glycoproteins are biochemically mature. Our results bring the number of EAV envelope proteins to six.

Download Full-text