scholarly journals A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains.

1985 ◽  
Vol 101 (6) ◽  
pp. 2292-2301 ◽  
Author(s):  
E Perara ◽  
V R Lingappa
Keyword(s):  
Signal Sequence ◽  
Protein Domains ◽  
Membrane Vesicles ◽  
Glycosylation Site ◽  
Coding Region ◽  
Free System ◽  
Amino Terminal ◽  
Internal Position ◽  
Presence And Absence

To determine whether a functional amino terminal signal sequence can be active at an internal position, a hybrid gene was constructed in which the entire coding region of bovine preprolactin cDNA was inserted into chimpanzee alpha-globin cDNA 109 codons downstream from the initiation codon of globin. When RNA synthesized in vitro from this plasmid (pSPGP1) was translated in the rabbit reticulocyte cell-free system, a 32-kD protein was produced that was both prolactin and globin immunoreactive. When microsomal membranes were present during translation (but not when added posttranslationally), a 26-kD and a 14-kD product were also observed. By immunoreactivity and electrophoretic mobility, the 26-kD protein was identical to mature prolactin, and the 14-kD protein appeared to be the globin domain with the prolactin signal sequence attached at its carboxy terminus. From (a) posttranslational proteolysis in the presence and absence of detergent, (b) sedimentation of vesicles in the presence and absence of sodium carbonate pH 11.5, and (c) N-linked glycosylation of the globin-immunoreactive fragment after insertion of an Asn-X-Ser N-linked glycosylation site into the globin coding region of pSPGP1, it appears that all of the 26-kD and some of the 14-kD products, but none of the 32-kD precursor, have been translocated to the lumen of the membrane vesicles. Thus, when engineered to an internal position, the prolactin signal sequence is able to translocate both flanking protein domains. These data have implications for the understanding of translocation of proteins across the membrane of the endoplasmic reticulum.

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.

Developmental regulation of a novel repetitive protein of Trypanosoma brucei

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

scholarly journals Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

1991 ◽  
Vol 115 (1) ◽  
pp. 267-277 ◽  
Author(s):  
S P Kennedy ◽  
S L Warren ◽  
B G Forget ◽  
J S Morrow
Keyword(s):  
Binding Site ◽  
Binding Capacity ◽  
Repeat Unit ◽  
Expression System ◽  
Membrane Vesicles ◽  
Enzymatic Digestion ◽  
Binding Domain ◽  
Amino Terminal ◽  
Prokaryotic Expression System

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

scholarly journals Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

2001 ◽  
Vol 75 (7) ◽  
pp. 3230-3239 ◽  
Author(s):  
Miran Yoon ◽  
Deborah H. Smith ◽  
Peter Ward ◽  
Francisco J. Medrano ◽  
Aneel K. Aggarwal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Catalytic Domain ◽  
Adeno Associated Virus ◽  
Free System ◽  
Site Specific ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Site Specific Integration ◽  
Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

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 Molecular cloning of the rat analogue of human CD59: structural comparison with human CD59 and identification of a putative active site

1994 ◽  
Vol 304 (2) ◽  
pp. 595-601 ◽  
Author(s):  
N K Rushmere ◽  
R A Harrison ◽  
C W van den Berg ◽  
B P Morgan
Keyword(s):  
Molecular Cloning ◽  
Rat Kidney ◽  
Amino Acid Level ◽  
Three Dimensional ◽  
Glycosylation Site ◽  
Dimensional Structure ◽  
Flanking Sequence ◽  
Coding Region ◽  
Amino Terminal ◽  
Human Cd59

We have previously described the purification and partial characterization of the rat analogue of the human complement regulatory molecule CD59 [Hughes, Piddlesden, Williams, Harrison and Morgan (1992) Biochem. J. 284, 169-176]. We present here the molecular cloning and full sequence analysis of this molecule. A PCR-based approach utilizing primers designed from the amino-terminal protein sequence was used to isolate a full-length cDNA clone from a rat kidney cDNA library. This clone encoded a 92 bp 5′-flanking sequence, a 66 bp signal peptide and a 315 bp coding region containing putative glycosylation and GPI-anchor signals. The 3′ untranslated flanking region was approximately 1.1 kbp long and included the poly-A tail and a CATA repeating sequence. The coding region was 58% identical with the human cDNA at the nucleotide level and 44% identical at the amino acid level. Despite this relatively low overall sequence conservation, several highly conserved stretches were apparent, particularly in the N-terminal portion of the molecule, in the cysteine-rich region immediately preceding the site of glycolipid attachment and in the C-terminal peptide removed during glycolipid attachment. An N-glycosylation site was identified at Asn-16 and a putative glycosylphosphatidylinositol anchor addition site at Asn-79, indicating that the mature processed protein was two residues longer than human CD59. Comparison of the sequences of rat and human CD59, together with consideration of the published three-dimensional structure of human CD59 and functional data, implicates specific regions of the protein in interactions with C-8 and/or C-9.

scholarly journals In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

1999 ◽  
Vol 10 (7) ◽  
pp. 2163-2173 ◽  
Author(s):  
Hans-Georg Koch ◽  
Thomas Hengelage ◽  
Christoph Neumann-Haefelin ◽  
Juan MacFarlane ◽  
Hedda K. Hoffschulte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Free System ◽  
E Coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coliwhich, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

GPI anchor transamidase of Trypanosoma brucei: in vitro assay of the recombinant protein and VSG anchor exchange

2002 ◽  
Vol 115 (12) ◽  
pp. 2529-2539
Author(s):  
Xuedong Kang ◽  
Alexander Szallies ◽  
Marc Rawer ◽  
Hartmut Echner ◽  
Michael Duszenko
Keyword(s):  
Trypanosoma Brucei ◽  
Signal Sequence ◽  
Intracellular Localization ◽  
Glycosylation Site ◽  
Proteolytic Processing ◽  
Gpi Anchor ◽  
Hydrophobic Region ◽  
Western Blots ◽  
Vitro Assay

GPI8 from Trypanosoma brucei was cloned and expressed in Escherichia coli. TbGPI8 encodes a 37 kDa protein (35 kDa after removal of the putative signal sequence) with a pI of 5.5. It contains one potential N-glycosylation site near the N-terminus but no C-terminal hydrophobic region. Enzyme activity assays using trypanosomal lysates or recombinant TbGpi8 exhibited cleavage of the synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine, indicating that TbGpi8 is indeed directly involved in the proteolytic processing of the GPI anchoring signal. Intracellular localization of TbGpi8 within tubular structures, such as the endoplasmic reticulum, was observed by using specific anti-TbGpi8 antibodies. The transamidase mechanism of GPI anchoring was studied in bloodstream forms of Trypanosoma brucei using media containing hydrazine or biotinylated hydrazine. In the presence of the latter nucleophile, part of the newly formed VSG was linked to this instead of the GPI anchor and was not transferred to the cell surface. VSG-hydrazine-biotin was detected by streptavidin in western blots and intracellularly in Golgi-like compartments.

scholarly journals Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

1985 ◽  
Vol 5 (5) ◽  
pp. 916-922 ◽  
Author(s):  
M D Resh ◽  
R L Erikson
Keyword(s):  
Plasma Membrane ◽  
Rous Sarcoma Virus ◽  
Membrane Vesicles ◽  
Terminal Region ◽  
Transformed Cell ◽  
Amino Terminal ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Carboxy Terminal

Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.

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