Translocation of preproinsulin across the endoplasmic reticulum membrane. The relationship between nascent polypeptide size and extent of signal recognition particle-mediated inhibition of protein synthesis.

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.

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Insulin secretory granule biogenesis. Co-ordinate regulation of the biosynthesis of the majority of constituent proteins

Biochemical Journal ◽

10.1042/bj2740073 ◽

1991 ◽

Vol 274 (1) ◽

pp. 73-78 ◽

Cited By ~ 56

Author(s):

P C Guest ◽

E M Bailyes ◽

N G Rutherford ◽

J C Hutton

Keyword(s):

Endoplasmic Reticulum ◽

Secretory Granules ◽

Signal Recognition Particle ◽

Electrophoretic Analysis ◽

Subcellular Fractionation ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition ◽

Translational Initiation ◽

Insulin Secretory Granule ◽

Fold Stimulation

Two-dimensional gel-electrophoretic analysis combined with fluorography and densitometric quantification was used to examine the effects of glucose on the biosynthesis of rat pancreatic islet proteins. An increase in the medium glucose concentration from 2.8 to 16.7 mM produced a 10-20 fold stimulation in the synthesis of 10 out of 260 detected islet proteins, as judged by incorporation of [35S]methionine during a 20 min incubation. The synthetic rates of the majority of the remaining proteins were stimulated by 2-4-fold. Greater resolution achieved by pulse-chase labelling and subcellular fractionation showed that, of 32 major proteins localized to insulin secretory granules, the biosynthesis of 25 were stimulated 15-30-fold by glucose. By contrast, only eight of 160 proteins in the soluble fraction showed a response of similar magnitude. It is concluded that there is a major and co-ordinated activation of the biosyntheses of proteins destined for secretory granules, which most likely occurs at the level of translational initiation and signal-recognition-particle-mediated translocation into the endoplasmic reticulum lumen. However, it is clear that not all granule proteins, or the majority of proteins translocated across the endoplasmic reticulum membrane, are affected in an equivalent manner. In addition, the synthesis of a small number of cytosolic proteins may be increased markedly by insulinotropic stimuli.

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Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Molecular Biology of the Cell ◽

10.1091/mbc.9.1.117 ◽

1998 ◽

Vol 9 (1) ◽

pp. 117-130 ◽

Cited By ~ 39

Author(s):

David Raden ◽

Reid Gilmore

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Chain Complex ◽

Dual Function ◽

Signal Recognition ◽

Nascent Polypeptide ◽

Specific Attachment ◽

Nascent Chain

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.

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

The Journal of Cell Biology ◽

10.1083/jcb.104.2.201 ◽

1987 ◽

Vol 104 (2) ◽

pp. 201-208 ◽

Cited By ~ 77

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.

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Mathematical modeling of the effects of the signal recognition particle on translation and translocation of proteins across the endoplasmic reticulum membrane

Journal of Molecular Biology ◽

10.1016/0022-2836(87)90186-0 ◽

1987 ◽

Vol 195 (3) ◽

pp. 621-636 ◽

Cited By ~ 28

Author(s):

Tom A. Rapoport ◽

Reinhart Heinrich ◽

Peter Walter ◽

Thomas Schulmeister

Keyword(s):

Mathematical Modeling ◽

Endoplasmic Reticulum ◽

Signal Recognition Particle ◽

Endoplasmic Reticulum Membrane ◽

Signal Recognition

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Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model

Biochemistry and Cell Biology ◽

10.1139/o05-147 ◽

2005 ◽

Vol 83 (6) ◽

pp. 687-695 ◽

Cited By ~ 34

Author(s):

Christopher V Nicchitta ◽

Rachel S Lerner ◽

Samuel B Stephens ◽

Rebecca D Dodd ◽

Brook Pyhtila

Keyword(s):

Endoplasmic Reticulum ◽

Protein Synthesis ◽

Signal Sequence ◽

Signal Recognition Particle ◽

Eukaryotic Cell ◽

Signal Recognition ◽

Cytosolic Proteins ◽

Complex Process ◽

Hypothetical Mechanism ◽

Cytosolic Ribosomes

mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER) - bound ribosomes, whereas mRNAs encoding cytosolic proteins are translated on cytosolic ribosomes. The partitioning of mRNAs to the ER occurs by positive selection; cytosolic ribosomes engaged in the translation of signal-sequence-bearing proteins are engaged by the signal-recognition particle (SRP) pathway and subsequently trafficked to the ER. Studies have demonstrated that, in addition to the SRP pathway, mRNAs encoding cytosolic proteins can also be partitioned to the ER, suggesting that RNA partitioning in the eukaryotic cell is a complex process requiring the activity of multiple RNA-partitioning pathways. In this review, key findings on this topic are discussed, and the template-partitioning model, describing a hypothetical mechanism for RNA partitioning in the eukaryotic cell, is proposed.Key words: mRNA, ribosome, endoplasmic reticulum, translation, protein synthesis, signal sequence, RNA localization.

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