scholarly journals Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity.

1991 ◽  
Vol 114 (4) ◽  
pp. 639-649 ◽  
Author(s):  
P G Collins ◽  
R Gilmore
Keyword(s):  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Size Exclusion ◽  
Ribosome Binding ◽  
Specific Subset ◽  
Microsomal Membranes ◽  
Membrane Bound

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.

scholarly journals Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate.

1989 ◽  
Vol 109 (6) ◽  
pp. 2617-2622 ◽  
Author(s):  
S L Wolin ◽  
P Walter
Keyword(s):  
Wheat Germ ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Translation Arrest ◽  
Microsomal Membranes ◽  
Reticulocyte Lysate ◽  
Reticulocyte Lysates ◽  
Ribosome Pausing

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.

scholarly journals Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

1994 ◽  
Vol 5 (8) ◽  
pp. 887-897 ◽  
Author(s):  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Beta Subunits ◽  
Signal Recognition ◽  
Binding Assays ◽  
Nascent Polypeptide ◽  
Sequence Recognition ◽  
Gtp Binding ◽  
Microsomal Membranes

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.

scholarly journals Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation

2005 ◽  
Vol 168 (1) ◽  
pp. 67-77 ◽  
Author(s):  
Zhiliang Cheng ◽  
Ying Jiang ◽  
Elisabet C. Mandon ◽  
Reid Gilmore
Keyword(s):  
Signal Recognition Particle ◽  
Point Mutations ◽  
Ribosomal Subunit ◽  
Chain Complex ◽  
Binding Activity ◽  
Initial Contact ◽  
Ribosome Binding ◽  
Cotranslational Translocation ◽  
Translocation Pathway ◽  
Cytoplasmic Loops

The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.

scholarly journals Role of the Cytoplasmic Segments of Sec61α in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex

2000 ◽  
Vol 150 (1) ◽  
pp. 53-64 ◽  
Author(s):  
David Raden ◽  
Weiqun Song ◽  
Reid Gilmore
Keyword(s):  
Protein Translocation ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Dual Function ◽  
Cleavage Sites ◽  
Critical Determinant ◽  
Ribosome Binding ◽  
High Affinity ◽  
Nascent Chain

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH2 and COOH terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.

scholarly journals Signal sequence–independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

2011 ◽  
Vol 22 (13) ◽  
pp. 2309-2323 ◽  
Author(s):  
David Braig ◽  
Miryana Mircheva ◽  
Ilie Sachelaru ◽  
Eli O. van der Sluis ◽  
Lukas Sturm ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Targeting ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Substrate Recognition ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Dependent Protein ◽  
Membrane Bound ◽  
Influence Of Substrate

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence–independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.

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

1992 ◽  
Vol 117 (1) ◽  
pp. 15-25 ◽  
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.

scholarly journals Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

1983 ◽  
Vol 212 (1) ◽  
pp. 161-165 ◽  
Author(s):  
E M Danielsen ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Signal Sequence ◽  
Aminopeptidase N ◽  
Soluble Form ◽  
Translation System ◽  
Translation Product ◽  
Free Translation ◽  
Microsomal Membranes ◽  
A Cell ◽  
Membrane Bound ◽  
Small Intestinal

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

scholarly journals ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein.

1991 ◽  
Vol 114 (1) ◽  
pp. 21-33 ◽  
Author(s):  
K V Kellaris ◽  
S Bowen ◽  
R Gilmore
Keyword(s):  
Membrane Protein ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Microsomal Membrane ◽  
Cross Linking ◽  
Beta Subunits ◽  
Nascent Polypeptide ◽  
Free Translation

We have used the homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER. Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles. Two prominent cross-linked products of 45 and 64 kD were detected. The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles. In agreement with previous investigators, we suggest that the 64-kD product arises by cross-linking of the nascent polypeptide to the 54-kD subunit of SRP. The 45-kD product resists alkaline extraction from the membrane, so we conclude that the 11-kD nascent polypeptide has been crosslinked to an integral membrane protein of approximately 34 kD (imp34). The cross-linked product does not bind to ConA Sepharose, nor is it sensitive to endoglycosidase H digestion; hence imp34 is not identical to the alpha or beta subunits of the signal sequence receptor (SSR). We propose that imp34 functions in concert with SSR to form a translocation site through which nascent polypeptides pass in traversing the membrane bilayer of the rough endoplasmic reticulum.

scholarly journals Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes.

1993 ◽  
Vol 121 (6) ◽  
pp. 1211-1219 ◽  
Author(s):  
S L Wolin ◽  
P Walter
Keyword(s):  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Translation Arrest ◽  
Nascent Chain ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Chain Lengths ◽  
Edta Extraction ◽  
Er Membrane

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane-associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.

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