180-kD ribosome receptor is essential for both ribosome binding and protein translocation.

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.

Download Full-text

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

The Journal of Cell Biology ◽

10.1083/jcb.103.1.241 ◽

1986 ◽

Vol 103 (1) ◽

pp. 241-253 ◽

Cited By ~ 73

Author(s):

M Hortsch ◽

D Avossa ◽

D I Meyer

Keyword(s):

Endoplasmic Reticulum ◽

Protein Translocation ◽

Signal Recognition Particle ◽

Secretory Protein ◽

Signal Peptidase ◽

Secretory Proteins ◽

Peptidase Activity ◽

Ribosome Binding ◽

Functional Inactivation ◽

Docking Protein

Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Download Full-text

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

The Journal of Cell Biology ◽

10.1083/jcb.117.1.15 ◽

1992 ◽

Vol 117 (1) ◽

pp. 15-25 ◽

Cited By ~ 50

Author(s):

G Migliaccio ◽

CV Nicchitta ◽

G Blobel

Keyword(s):

Membrane Protein ◽

Protein Translocation ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Chain Complex ◽

Secretory Protein ◽

Signal Recognition ◽

Ribosome Binding ◽

Signal Recognition Particle Receptor ◽

Nascent Chain

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.

Download Full-text

Secretory protein translocation in a neurospora crassa in vitro system. Hydrolysis of a nucleoside triphosphate is required for posttranslational translocation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45487-7 ◽

1987 ◽

Vol 262 (35) ◽

pp. 17031-17037

Author(s):

R Addison

Keyword(s):

Neurospora Crassa ◽

Protein Translocation ◽

Secretory Protein ◽

Nucleoside Triphosphate ◽

In Vitro System ◽

Hydrolysis Of

Download Full-text

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1134-1144.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1134-1144 ◽

Cited By ~ 4

Author(s):

F Rozen ◽

I Edery ◽

K Meerovitch ◽

T E Dever ◽

W C Merrick ◽

...

Keyword(s):

Secondary Structure ◽

Translation Initiation ◽

Direct Evidence ◽

Rna Helicase ◽

Initiation Factor ◽

Helicase Activity ◽

Ribosome Binding ◽

Initiation Factors ◽

Translation Initiation Factors ◽

Hydrolysis Of

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.

Download Full-text

Quantitation of the number of molecules of glycophorins C and D on normal red blood cells using radioiodinated Fab fragments of monoclonal antibodies

Blood ◽

10.1182/blood.v83.6.1668.1668 ◽

1994 ◽

Vol 83 (6) ◽

pp. 1668-1672 ◽

Cited By ~ 21

Author(s):

J Smythe ◽

B Gardner ◽

DJ Anstee

Keyword(s):

Monoclonal Antibodies ◽

Blood Cells ◽

Binding Assays ◽

Membrane Glycoproteins ◽

Human Erythrocyte Membrane ◽

Protein 4.1 ◽

Fab Fragments ◽

Attachment Sites ◽

Membrane Attachment ◽

Glycophorin C

Abstract Two rat monoclonal antibodies (BRAC 1 and BRAC 11) have been produced. BRAC 1 recognizes an epitope common to the human erythrocyte membrane glycoproteins glycophorin C (GPC) and glycophorin D (GPD). BRAC 11 is specific for GPC. Fab fragments of these antibodies and BRIC 10, a murine monoclonal anti-GPC, were radioiodinated and used in quantitative binding assays to measure the number of GPC and GPD molecules on normal erythrocytes. Fab fragments of BRAC 11 and BRIC 10 gave values of 143,000 molecules GPC per red blood cell (RBC). Fab fragments of BRAC 1 gave 225,000 molecules of GPC and GPD per RBC. These results indicate that GPC and GPD together are sufficiently abundant to provide membrane attachment sites for all of the protein 4.1 in normal RBCs.

Download Full-text

Phage-displayed Fab fragments against anti-human interleukin-2 receptor α. Detection of antigen-bound phages with anti-cpIII monoclonal antibodies

Apmis ◽

10.1111/j.1699-0463.1997.tb00548.x ◽

1997 ◽

Vol 105 (1-6) ◽

pp. 108-114

Author(s):

EVA RAIVIO ◽

MALIN KRONQVIST ◽

THOMAS N. BRODIN ◽

MATS A. A. PERSSON ◽

CHRISTER LINDQVIST

Keyword(s):

Monoclonal Antibodies ◽

Interleukin 2 ◽

Interleukin 2 Receptor ◽

Fab Fragments

Download Full-text

Functional interplay between DEAD-box RNA helicases Ded1 and Dbp1 in preinitiation complex attachment and scanning on structured mRNAs in vivo

Nucleic Acids Research ◽

10.1093/nar/gkz595 ◽

2019 ◽

Cited By ~ 4

Author(s):

Neelam Dabas Sen ◽

Neha Gupta ◽

Stuart K. Archer ◽

Thomas Preiss ◽

Jon R Lorsch ◽

...

Keyword(s):

Untranslated Region ◽

Double Mutant ◽

Direct Evidence ◽

Translation Efficiency ◽

Rna Structures ◽

Preinitiation Complex ◽

Rna Helicases ◽

Ribosome Binding ◽

Dead Box

Abstract RNA structures that impede ribosome binding or subsequent scanning of the 5′-untranslated region (5′-UTR) for the AUG initiation codon reduce translation efficiency. Yeast DEAD-box RNA helicase Ded1 appears to promote translation by resolving 5′-UTR structures, but whether its paralog, Dbp1, performs similar functions is unknown. Furthermore, direct in vivo evidence was lacking that Ded1 or Dbp1 resolves 5′-UTR structures that impede attachment of the 43S preinitiation complex (PIC) or scanning. Here, profiling of translating 80S ribosomes reveals that the translational efficiencies of many more mRNAs are reduced in a ded1-ts dbp1Δ double mutant versus either single mutant, becoming highly dependent on Dbp1 or Ded1 only when the other helicase is impaired. Such ‘conditionally hyperdependent’ mRNAs contain unusually long 5′-UTRs with heightened propensity for secondary structure and longer transcript lengths. Consistently, overexpressing Dbp1 in ded1 cells improves the translation of many such Ded1-hyperdependent mRNAs. Importantly, Dbp1 mimics Ded1 in conferring greater acceleration of 48S PIC assembly in a purified system on mRNAs harboring structured 5′-UTRs. Profiling 40S initiation complexes in ded1 and dbp1 mutants provides direct evidence that Ded1 and Dbp1 cooperate to stimulate both PIC attachment and scanning on many Ded1/Dbp1-hyperdependent mRNAs in vivo.

Download Full-text

Inhibition of malarial parasite invasion by monoclonal antibodies against glycophorin A correlates with reduction in red cell membrane deformability

Blood ◽

10.1182/blood.v74.5.1836.1836 ◽

1989 ◽

Vol 74 (5) ◽

pp. 1836-1843 ◽

Cited By ~ 23

Author(s):

G Pasvol ◽

JA Chasis ◽

N Mohandas ◽

DJ Anstee ◽

MJ Tanner ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Cell Membrane ◽

Red Cells ◽

Malarial Parasite ◽

Glycophorin A ◽

Red Cell ◽

Red Cell Membrane ◽

Parasite Invasion ◽

Surface Molecules ◽

Fab Fragments

Abstract The effect of well-characterized monoclonal antibodies to red cell surface molecules on the invasion of human red cells by the malarial parasites Plasmodium falciparum and Plasmodium knowlesi was examined. Antibodies to glycophorin A (GP alpha) inhibit invasion for both parasite species, and this is highly correlated with the degree to which they decrease red cell membrane deformability as measured by ektacytometry. This effect on rigidity and invasion was also seen with monovalent Fab fragments. The closer the antibody binding site was to the membrane bilayer, the greater was its effect on inducing membrane rigidity and decreasing parasite invasion. Antibodies to the Wright determinant in particular were the most inhibitory. This differential effect of the various antibodies was not correlated with their binding affinities or the number of sites bound per cell. Antibodies to surface molecules other than GP alpha were without effect. A novel mechanism is described whereby monoclonal antibodies and their Fab fragments directed at determinants on the external surface of red cells might act to inhibit invasion by malarial parasites by altering membrane material properties.

Download Full-text