scholarly journals Signal recognition protein is required for the integration of acetylcholine receptor delta subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membrane.

1982 ◽  
Vol 93 (2) ◽  
pp. 501-506 ◽  
Author(s):  
D J Anderson ◽  
P Walter ◽  
G Blobel
Keyword(s):  
Acetylcholine Receptor ◽  
Proteolytic Enzymes ◽  
Signal Sequence ◽  
Microsomal Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Transmembrane Glycoprotein ◽  
Microsomal Membranes ◽  
Recognition Protein ◽  
Delta Subunit

Purified Signal Recognition Protein (SRP) has previously been shown to be required for the translocation of secretory proteins across the microsomal membrane (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7, 112-7, 116) and to function in the early events of this process (Walter and Blobel, 1981. J. Cell Biol. 91:557-561). We demonstrate here that the delta subunit of acetylcholine receptor (AChR-delta), a transmembrane glycoprotein, likewise requires SRP for its asymmetric integration into microsomal membranes. We further demonstrate by partial sequence analysis that AChR-delta is synthesized with a transient NH2-terminal signal sequence of 21 residues that is cleaved off during integration into microsomal membranes. Integration of AChR-delta into the microsomal membrane vesicles proceeded asymmetrically, yielding a large (44 kdalton) core-glycosylated domain, inaccessible to externally added proteolytic enzymes and a smaller (approximately 16 kdalton) domain exposed on the outside of the vesicles and accessible to externally added proteolytic enzymes. The NH2 terminus of the molecule is contained in the 44-kdalton domain.

scholarly journals Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in-vitro-assembled polysomes synthesizing secretory protein.

1981 ◽  
Vol 91 (2) ◽  
pp. 551-556 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Preceding Paper ◽  
Secretory Protein ◽  
Microsomal Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Lipid Signal ◽  
Recognition Protein

Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin). This selective polysome binding capacity was abolished when the microsomal vesicles were salt-extracted but was restored by an 11S protein (SRP, Signal Recognition Protein) previously purified from the salt-extract of microsomal vesicles (Walter and Blobel, 1980. Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). SRP-dependent polysome recognition and binding to the microsomal membrane was shown to be a prerequisite for chain translocation. Modification of SRP by N-ethyl maleimide abolished its ability to mediate nascent polysome binding to the microsomal vesicles. Likewise, polysome binding to the microsomal membrane was largely abolished when beta-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides. The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the initial events that lead to translocation and provide for its specificity are protein-protein (signal sequence plus ribosome with SRP) and not protein-lipid (signal sequence with lipid bilayer) interactions.

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 The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane.

1995 ◽  
Vol 128 (3) ◽  
pp. 273-282 ◽  
Author(s):  
J D Miller ◽  
S Tajima ◽  
L Lauffer ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Signal Sequence ◽  
Transmembrane Protein ◽  
Signal Recognition Particle ◽  
Endoplasmic Reticulum Membrane ◽  
Protein Chain ◽  
Signal Recognition ◽  
Signal Recognition Particle Receptor ◽  
Er Membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30-kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.

scholarly journals BIP associates with newly synthesized subunits of the mouse muscle nicotinic receptor.

1991 ◽  
Vol 113 (5) ◽  
pp. 1125-1132 ◽  
Author(s):  
P Blount ◽  
J P Merlie
Keyword(s):  
Conformational Change ◽  
Acetylcholine Receptor ◽  
Bond Formation ◽  
Disulfide Bridge ◽  
Alpha Subunit ◽  
Mouse Muscle ◽  
Transmembrane Glycoprotein ◽  
Delta Subunit

A slow conformational change in newly synthesized acetylcholine receptor subunits is thought to be a requisite step in the biogenesis of this multi-subunit transmembrane glycoprotein. Previously, we demonstrated that this early conformational change within the alpha-subunit was inefficient and dependent upon disulfide bond formation (Blount, P. and J.P. Merlie. 1990. J. Cell Biol. 111:2613-2622). Here we show that newly synthesized acetylcholine receptor subunits and subunit complexes in the muscle-like cell line, BC3H-1, are associated with Bip, a ubiquitous binding protein of the endoplasmic reticulum. Characterization of the Bip/alpha-subunit complex in stably transfected fibroblasts revealed that Bip associates with newly synthesized unassembled alpha-subunit and some alpha gamma and alpha delta subunit complexes. Significantly, Bip does not associate well with the more mature form of the alpha-subunit containing an intramolecular disulfide bridge. Hence, Bip may play an important role in the conformational maturation and/or editing of unassembled AChR subunits and subunit complexes in vivo.

scholarly journals Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

1987 ◽  
Vol 104 (2) ◽  
pp. 201-208 ◽  
Author(s):  
M Wiedmann ◽  
T V Kurzchalia ◽  
H Bielka ◽  
T A Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Group 4 ◽  
Lysine Residues ◽  
Nascent Chain

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

scholarly journals An ATP-binding membrane protein is required for protein translocation across the endoplasmic reticulum membrane.

1991 ◽  
Vol 2 (10) ◽  
pp. 851-859 ◽  
Author(s):  
D L Zimmerman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Microsomal Membrane ◽  
Atp Binding ◽  
Endoplasmic Reticulum Membrane ◽  
Receptor Complex

The role of nucleotides in providing energy for polypeptide transfer across the endoplasmic reticulum (ER) membrane is still unknown. To address this question, we treated ER-derived mammalian microsomal vesicles with a photoactivatable analogue of ATP, 8-N3ATP. This treatment resulted in a progressive inhibition of translocation activity. Approximately 20 microsomal membrane proteins were labeled by [alpha 32P]8-N3ATP. Two of these were identified as proteins with putative roles in translocation, alpha signal sequence receptor (SSR), the 35-kDa subunit of the signal sequence receptor complex, and ER-p180, a putative ribosome receptor. We found that there was a positive correlation between inactivation of translocation activity and photolabeling of alpha SSR. In contrast, our data demonstrate that the ATP-binding domain of ER-p180 is dispensable for translocation activity and does not contribute to the observed 8-N3ATP sensitivity of the microsomal vesicles.

scholarly journals The role of topogenic sequences in the movement of proteins through membranes

1987 ◽  
Vol 246 (2) ◽  
pp. 249-261 ◽  
Author(s):  
A Robinson ◽  
B Austen
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Product ◽  
Signal Sequence ◽  
Intact Cell ◽  
Specific Binding ◽  
Endoplasmic Reticulum Membrane ◽  
Inner Membrane ◽  
Cytoplasmic Factors ◽  
Microsomal Membranes

Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table 2). Whereas co-translational translocation and processing were previously considered the norm at the endoplasmic reticulum membrane, several instances of post-translational translocation into endoplasmic reticulum microsomes in vitro have now been described. However, it must be noted that post-translational translocation in vitro is much less efficient than when endoplasmic reticulum membranes are present during translation, and it is possible that in the intact cell translocation occurs during translation. Movement of proteins into chloroplasts and mitochondria occurs after translation. When translocation is post-translational, proteins may perhaps traverse the membrane as folded domains, and the conformational effects of topogenic sequences on these domains may be as envisaged in Wickner's ‘membrane-trigger hypothesis’. Both signal and transit sequences possess amphipathic structures which are capable of interacting with phospholipid bilayers, and these interactions may disturb the bilayer sufficiently to allow entry of the following domains of protein. There is increasing evidence that GTP is required to bind ribosomes and their associated nascent chains to the endoplasmic reticulum membrane. Precisely how the cell's energy is applied to achieve translocation is not clear, but one possibility at the endoplasmic reticulum is that a GTP-hydrolysing transducing mechanism may exist to couple signal sequence receptor binding to movement of the nascent chain across the membrane. Electrochemical gradients are required for protein movement to the mitochondrial inner membrane and across the bacterial inner membrane. Cytoplasmic factors such as SRP, the secA gene product or a 40 kDa protein (for mitochondrial precursors) may act by binding to topogenic sequences and preventing precursor proteins as they are translated from folding into forms which cannot be translocated. Specificity in the cell may be achieved both by targetting interactions between these cytoplasmic factors and their receptors located in target membranes, and also by specific binding of the topogenic sequences to specific proteins integrated into the target membranes. Possible candidates for the latter are the protein of microsomal membranes that reacts with a photoreactive signal peptide to give a 45 kDa complex (Fig. 1), the secY gene product of the bacterial inner membrane, and receptors on the outer membranes of chloroplasts and mitochondria. Whether these aid translocation as well as recognition is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Spontaneous Release of Cytosolic Proteins from Posttranslational Substrates before Their Transport into the Endoplasmic Reticulum

2000 ◽  
Vol 151 (1) ◽  
pp. 167-178 ◽  
Author(s):  
Kathrin Plath ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Active Role ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Cytosolic Proteins ◽  
Protein Substrates ◽  
Ring Complex ◽  
Reticulocyte Lysate

In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide–associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.

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.

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