Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.

Walter, Peter; Ibrahimi, Ibrahim; Blobel, Günter

doi:10.1083/jcb.91.2.545

Cited by 618 publications

(290 citation statements)

References 14 publications

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“…The fact that SRP14 was cross-linked to ribosomal components in all samples was consistent with the previous observations that SRP could bind directly to ribosomes in a signal sequence-and nascent chain-independent fashion (25,(29)(30)(31). Moreover, it demonstrated that the Alu domain was already in close proximity to the ribosome in the absence of elongation arrest.…”

Section: The Alu Domain Has Conserved Binding Sites On Thesupporting

confidence: 91%

Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

et al. 2003

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The eukaryotic signal recognition particle (SRP) is essential for cotranslational targeting of proteins to the endoplasmic reticulum (ER). The SRP Alu domain is specifically required for delaying nascent chain elongation upon signal sequence recognition by SRP and was therefore proposed to interact directly with ribosomes. Using protein cross-linking, we provide experimental evidence that the Alu binding protein SRP14 is in close physical proximity of several ribosomal proteins in functional complexes. Crosslinking occurs even in the absence of a signal sequence in the nascent chain demonstrating that SRP can bind to all translating ribosomes and that close contacts between the Alu domain and the ribosome are independent of elongation arrest activity. Without a signal sequence, SRP14 cross-links predominantly to a protein of the large subunit. Upon signal sequence recognition, certain cross-linked products become detectable or more abundant revealing a change in the Alu domain-ribosome interface. At this stage, the Alu domain of SRP is located at the ribosomal subunit interface since SRP14 can be cross-linked to proteins from the large and small ribosomal subunits. Hence, these studies reveal differential modes of SRP-ribosome interactions mediated by the Alu domain.Ribosomes translating mRNAs coding for secretory and membrane proteins are specifically targeted to the endoplasmic reticulum (ER) membrane by a cytosolic ribonucleoprotein particle, the signal recognition particle (SRP) (for review, see ref 1). The specificity of this process is ensured by the presence of a signal sequence in the growing peptide chain, which is recognized by SRP when it emerges from the ribosome. Signal sequence recognition by SRP causes a slow down or an arrest in the elongation of the nascent chain (2, 3). The SRP-ribosome-nascent chain complex is then targeted to the ER via the interaction of SRP with its membrane receptor, the SRP receptor (SR), a heterodimeric membrane protein (4,5). SR coordinates the release of SRP from the ribosome with the insertion of the nascent chain into the Sec61 complex, the aqueous translocation pore in the ER membrane (for reviews, see refs 6 and 7). Free SRP can then engage in another targeting round, and membraneassociated ribosomes resume translation at their regular speed, leading to the cotranslational transfer of the nascent chain across or into the ER membrane. As emphasized by

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Section: The Alu Domain Has Conserved Binding Sites On Thesupporting

confidence: 91%

Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

et al. 2003

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“…First, the cytosolic and chloroplast SRPs recognize significantly different forms of "cargo." The cytosolic SRP interacts with ribosome • nascent chain complexes bearing SRP signal sequences (Walter et al, 1981;Schaffitzel et al, 2006), whereas the cpSRP binds to its substrates, LHCPs, as fully translated proteins (Tu et al, 2000;Delille et al, 2000). Second, the cpSRP lacks the SRP RNA, which is otherwise universally conserved in all the other SRP pathways.…”

Section: Discussionmentioning

confidence: 99%

“…Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al, 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al, 1982a).…”

Section: Introductionmentioning

confidence: 99%

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Jaru-Ampornpan

Nguyen

Shan

2009

MBoC

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Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5-to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication. INTRODUCTIONThe signal recognition particle (SRP) and the SRP receptor (SR) comprise the major cellular machineries that cotranslationally deliver newly synthesized proteins from the cytosol to target membranes (Walter and Johnson, 1994;Keenan et al., 2001). Cotranslational protein targeting begins with recognition of the cargo-ribosomes translating nascent polypeptides containing signal sequences-by the SRP (Walter et al., 1981). The cargo is brought to the vicinity of the target membrane via the interaction between the SRP and SRP receptor (FtsY in bacteria) (Gilmore et al., 1982a). On arrival at the membrane, SRP unloads its cargo to the protein-conducting channel, composed of the sec61p complex in eukaryotic cells or secYEG complex in bacteria and archaea (Simon and Blobel, 1991;Gorlich et al., 1992;Halic et al., 2006). The SRP and SRP receptor also reciprocally stimulate each other's GTPase activity (Powers and Walter, 1995). Thus, after cargo unloading, guanosine triphosphate (GTP) hydrolysis drives disassembly of the SRP • SR complex, returning the components into the cytosol for the next round of protein targeting (Connolly et al., 1991).The SRP pathway is conserved throughout all three kingdoms of life. Although the protein components of SRP and SR vary across species, the functional core of SRP is a highly conserved ribonucleoprotein complex, composed of a 54-kDa SRP GTPase (SRP54 in eukaryotes or Ffh in bacteria) and an SRP RNA (Keenan et al., 2001). The SRP receptor also contains a conserved GTPase domain that is highly homologous to the GTPase domain in SRP54, and together the GTPase domains of SRP and SR form a unique subgroup in the GTPase superfamily (Keen...

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“…In eukaryotes, mRNA encoding secreted and membrane proteins can be targeted to the ER co-translationally by the signal recognition particle 1,2 and can be maintained on the ER via the direct interactions between ribosomes and translocons during the translation 3,4 . However, whether mRNAs can be targeted and maintained on the ER independent of either ribosomes or translation was unclear until very recently.…”

Section: Introductionmentioning

confidence: 99%

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Cui

Palazzo

2012

JoVE

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In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions. Video LinkThe video component of this article can be found at

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Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.

Cited by 618 publications

References 14 publications

Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)–SRP Receptor Interaction by the Chloroplast SRP Pathway

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

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