scholarly journals Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

2007 ◽  
Vol 179 (7) ◽  
pp. 1441-1452 ◽  
Author(s):  
Yuichiro Kida ◽  
Fumiko Morimoto ◽  
Masao Sakaguchi
Keyword(s):  
Type I ◽  
Free System ◽  
Binding Peptide ◽  
Terminal Domain ◽  
N Terminus ◽  
Nascent Chain ◽  
A Cell ◽  
Signal Anchor ◽  
Anchor Sequence ◽  
Streptavidin Binding

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61α. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.

scholarly journals Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences

2013 ◽  
Vol 24 (19) ◽  
pp. 3123-3132 ◽  
Author(s):  
Takaaki Yabuki ◽  
Fumiko Morimoto ◽  
Yuichiro Kida ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Type I ◽  
Surface Plasmon Resonance Analysis ◽  
Binding Peptide ◽  
Resonance Analysis ◽  
N Terminus ◽  
Signal Anchor ◽  
Streptavidin Binding

Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10−8 M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I–like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.

scholarly journals Requirements for the membrane insertion of signal-anchor type proteins.

1991 ◽  
Vol 113 (1) ◽  
pp. 25-34 ◽  
Author(s):  
S High ◽  
N Flint ◽  
B Dobberstein
Keyword(s):  
Signal Recognition Particle ◽  
Membrane Insertion ◽  
Type I ◽  
Type Ii ◽  
Nascent Chain ◽  
Identical Manner ◽  
Signal Anchor ◽  
Protein Components ◽  
Anchor Sequence ◽  
Anchor Proteins

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.

scholarly journals Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2646-2653 ◽  
Author(s):  
Y Liel ◽  
A Rudich ◽  
O Nagauker-Shriker ◽  
T Yermiyahu ◽  
R Levy
Keyword(s):  
Gaucher Disease ◽  
Reticuloendothelial System ◽  
Type I ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Free System ◽  
Nitroblue Tetrazolium Reduction ◽  
Nadph Oxidase Complex ◽  
A Cell ◽  
Nbt Reduction

Abstract Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 529-538 ◽  
Author(s):  
Ana Victoria Bendetowicz ◽  
Jill A. Morris ◽  
Robert J. Wise ◽  
Gary E. Gilbert ◽  
Randal J. Kaufman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Free System ◽  
N Terminus ◽  
A Cell ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

scholarly journals The Streptomyces coelicolor genome encodes a type I ribosome-inactivating protein

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 3021-3030 ◽  
Author(s):  
Ana G. Reyes ◽  
Nick Geukens ◽  
Philip Gutschoven ◽  
Stijn De Graeve ◽  
René De Mot ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Streptomyces Coelicolor ◽  
Protein Translation ◽  
Type I ◽  
Free System ◽  
Ribosome Inactivating Proteins ◽  
E Coli ◽  
Genes Encoding ◽  
A Cell ◽  
A Subunit

Ribosome-inactivating proteins (RIPs) are cytotoxic N-glycosidases identified in numerous plants, but also constitute a subunit of the bacterial Shiga toxin. Classification of plant RIPs is based on the absence (type I) or presence (type II) of an additional lectin module. In Shiga toxin, sugar binding is mediated by a distinct RIP-associated homopentamer. In the genome of two actinomycetes, we identified RIP-like proteins that resemble plant type I RIPs rather than the RIP subunit (StxA) of Shiga toxin. Some representatives of β- and γ-proteobacteria also contain genes encoding RIP-like proteins, but these are homologous to StxA. Here, we describe the isolation and initial characterization of the RIP-like gene product SCO7092 (RIPsc) from the Gram-positive soil bacterium Streptomyces coelicolor. The ripsc gene was expressed in Escherichia coli as a recombinant protein of about 30 kDa, and displayed the characteristic N-glycosidase activity causing specific rRNA depurination. In Streptomyces lividans and E. coli, RIPsc overproduction resulted in a dramatic decrease in the growth rate. In addition, intracellular production was deleterious for Saccharomyces cerevisiae. However, when applied externally to microbial cells, purified RIPsc did not display antibacterial or antifungal activity, suggesting that it cannot enter these cells. In a cell-free system, however, purified S. coelicolor RIPsc protein displayed strong inhibitory activity towards protein translation.

scholarly journals Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2646-2653
Author(s):  
Y Liel ◽  
A Rudich ◽  
O Nagauker-Shriker ◽  
T Yermiyahu ◽  
R Levy
Keyword(s):  
Gaucher Disease ◽  
Reticuloendothelial System ◽  
Type I ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Free System ◽  
Nitroblue Tetrazolium Reduction ◽  
Nadph Oxidase Complex ◽  
A Cell ◽  
Nbt Reduction

Gaucher disease patients are occasionally affected by chronic or fulminant infections. Since Gaucher cells originate from tissue phagocytes, we studied the functional implications of glucocerbroside accumulation on phagocytes in Gaucher disease patients. Circulating monocytes and granulocytes from nine type I Gaucher disease patients, and matched controls, were studied. Evaluation of phagocytic activity included (1) maximal superoxide generation rates following stimulation by phorbol 12-myristate 13-acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucylphenylalanine (FMLP); (2) nitroblue tetrazolium reduction test (NBT); (3) chemotaxis toward FMLP; (4) phagocytosis of OZ particles; and (5) killing activity against Staphylococcus aureus. Superoxide generation in monocytes following PMA, OZ, and FMLP stimulation was significantly suppressed at 52% +/- 15%, 39% +/- 8%, and 51% +/- 11% of control, respectively. Superoxide generation in granulocytes was normal. NBT reduction, staphylococcal killing, and phagocytosis were also markedly decreased in monocytes, and normal in granulocytes. Mean chemotaxis rates were normal in both monocytes and granulocytes; however, decreased chemotactic rates were observed in some patients. The abnormality of superoxide generation could be reproduced in a dose- and time-dependent manner in normal circulating monocytes incubated with glucocerebroside. Superoxide generation in glucocerebroside-conditioned normal monocytes in a cell-free system showed normal superoxide generation, reflecting the integrity of the NADPH oxidase complex itself. These results demonstrate markedly compromised phagocytic functions in circulating monocytes in Gaucher disease patients. These abnormalities can be attributed to accumulation of glucocerebroside, since it could be reproduced in normal monocytes incubated with glucocerebroside. Similar abnormalities in Gaucher cells throughout the reticuloendothelial system could impair host defense, and may be of particular importance in the pathogenesis of osteomyelitis in Gaucher disease patients.

scholarly journals Structural requirements for membrane assembly of proteins spanning the membrane several times.

1989 ◽  
Vol 109 (5) ◽  
pp. 2013-2022 ◽  
Author(s):  
J Lipp ◽  
N Flint ◽  
M T Haeuptle ◽  
B Dobberstein
Keyword(s):  
Amino Acid Sequences ◽  
Membrane Topology ◽  
Translation System ◽  
Opposite Orientation ◽  
Structural Requirements ◽  
The Third ◽  
Free Translation ◽  
A Cell ◽  
Signal Anchor ◽  
Anchor Sequence

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

Translation of type I and type II procollagen messengers in a cell-free system derived from wheat germ

FEBS Letters ◽  
1975 ◽  
Vol 57 (1) ◽  
pp. 47-50 ◽  
Author(s):  
Richard Harwood ◽  
Michael E. Grant ◽  
David S. Jackson
Keyword(s):  
Wheat Germ ◽  
Type I ◽  
Type Ii ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

2020 ◽  
Author(s):  
Mehdi Moustaqil ◽  
Emma Ollivier ◽  
Hsin-Ping Chiu ◽  
Sarah Van Tol ◽  
Paulina Rudolffi-Soto ◽  
...  
Keyword(s):  
Innate Immune ◽  
Type I ◽  
Cleavage Sites ◽  
Free System ◽  
Enhanced Production ◽  
Viral Proteases ◽  
Host Innate Immune Response ◽  
A Cell ◽  
Successful Replication ◽  
Interferon Antagonists

AbstractThe genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type- I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19. Finally, we discovered that one particular species of bats, David’s Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

scholarly journals The N-terminal domain of antithrombin-III is essential for heparin binding and complex-formation with, but not cleavage by, α-thrombin

1992 ◽  
Vol 282 (2) ◽  
pp. 345-351 ◽  
Author(s):  
R C Austin ◽  
W P Sheffield ◽  
R A Rachubinski ◽  
M A Blajchman
Keyword(s):  
Amino Acid ◽  
Complex Formation ◽  
Antithrombin Iii ◽  
Amino Acid Residues ◽  
Heparin Binding ◽  
Free System ◽  
Cell Free System ◽  
Terminal Domain ◽  
At Iii ◽  
A Cell

Normal and mutant forms of human antithrombin-III (AT-III) were synthesized in a cell-free system in order to identify putative functional domains required for heparin binding and complex-formation with alpha-thrombin. Heparin-Sepharose chromatography resulted in the elution of approx. 70% of cell-free-derived normal AT-III-(1-432)-polypeptide as a peak between 0.2 M- and 0.7 M-NaCl. The cell-free-derived normal AT-III also reacted with alpha-thrombin. Approx. 15% of this AT-III formed covalent complexes with alpha-thrombin in 2 min. Unfractionated heparin accelerated the rate of formation of such complexes. Two truncated forms of AT-III (amino acid residues 219-432 and 251-432), containing only the putative thrombin-binding domain, were synthesized independently in this cell-free system. These truncated AT-III polypeptides did not bind heparin and were unable to form stable covalent complexes with alpha-thrombin. However, both of these AT-III polypeptides were cleaved by alpha-thrombin, presumably at the reactive centre Arg-393-Ser-394. The formation of the disulphide bond between Cys-247 and Cys-430 in AT-III-(219-432)-polypeptide had no effect on the results obtained. Mutations in full-length AT-III at Cys-430 had no effect on the ability of AT-III to bind heparin. There was, however, a slight decrease in the formation of stable inhibitory complexes with alpha-thrombin. A cell-free-derived AT-III mutant, devoid of amino acid residues 41-49, which comprise heparin-binding region 1 of AT-III, had slightly decreased heparin binding compared with cell-free-derived normal AT-III-(1-432)-polypeptide. This mutant AT-III polypeptide was unable, however, to form a stable complex with alpha-thrombin. We conclude therefore that the N-terminal domain of AT-III is essential for both heparin binding and complex-formation with alpha-thrombin, but not for the cleavage of AT-III at its reactive centre by alpha-thrombin.

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