scholarly journals In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host

2002 ◽  
Vol 184 (12) ◽  
pp. 3260-3267 ◽  
Author(s):  
R. Wesley Rose ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Comparisons ◽  
Protein Insertion ◽  
Native Host

ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

scholarly journals SRP19 Is a Dispensable Component of the Signal Recognition Particle in Archaea

2006 ◽  
Vol 189 (1) ◽  
pp. 276-279 ◽  
Author(s):  
Sophie Yurist ◽  
Idit Dahan ◽  
Jerry Eichler
Keyword(s):  
Cell Growth ◽  
Membrane Protein ◽  
Protein Secretion ◽  
Signal Recognition Particle ◽  
Haloferax Volcanii ◽  
Signal Recognition ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Srp Rna

ABSTRACT In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.

scholarly journals Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe.

1983 ◽  
Vol 97 (6) ◽  
pp. 1693-1699 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Protein Translocation ◽  
Solid Phase ◽  
Signal Recognition Particle ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Specific Antibodies ◽  
7Sl Rna ◽  
Radioimmune Assay ◽  
Cell Fractions

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Polypeptide specific antibodies were raised in rabbits against the 72,000-, 68,000-, and 54,000-mol-wt polypeptide of SRP. All three antibodies are shown to neutralize SRP activity in vitro. A solid phase radioimmune assay is described and used to follow SRP in various cell fractions. The partitioning of SRP is shown to be dependent on the ionic conditions of the fractionation. Under conditions approximating physiological ionic strength, SRP is found to be about equally distributed between a membrane associated (38%) and a free (15%) or ribosome associated (47%) state. Furthermore, it is shown that greater than 75% of the total cellular 7SL-RNA is associated with SRP polypeptide in these fractions. Thus it is likely that the major--if not the only--cellular function of 7SL-RNA is as a part of SRP.

scholarly journals Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

2007 ◽  
Vol 178 (4) ◽  
pp. 611-620 ◽  
Author(s):  
Shu-ou Shan ◽  
Sowmya Chandrasekar ◽  
Peter Walter
Keyword(s):  
Conformational Changes ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Gtp Hydrolysis ◽  
Translocation Efficiency ◽  
Cargo Proteins ◽  
Gtpase Activation ◽  
Guanosine Triphosphatase

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP–SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP–SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.

scholarly journals Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

1992 ◽  
Vol 3 (8) ◽  
pp. 895-911 ◽  
Author(s):  
S C Ogg ◽  
M A Poritz ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Mammalian Cells ◽  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Er Membrane

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

scholarly journals The Signal Recognition Particle Pathway Is Required for Virulence in Streptococcus pyogenes

2008 ◽  
Vol 76 (6) ◽  
pp. 2612-2619 ◽  
Author(s):  
Jason W. Rosch ◽  
Luis Alberto Vega ◽  
John M. Beyer ◽  
Ada Lin ◽  
Michael G. Caparon
Keyword(s):  
Streptococcus Pyogenes ◽  
Signal Recognition Particle ◽  
Growth Defect ◽  
Signal Recognition ◽  
Human Pathogens ◽  
Gram Positive ◽  
Zebrafish Model ◽  
Gram Positive Bacteria

ABSTRACT The signal recognition particle (SRP) pathway is a universally conserved pathway for targeting polypeptides for secretion via the cotranslational pathway. In particular, the SRP pathway is thought to be the main mechanism for targeting polypeptides in gram-positive bacteria, including a number of important human pathogens. Though widely considered to be an essential cellular component, recent advances have indicated this pathway may be dispensable in gram-positive bacteria of the genus Streptococcus under in vitro conditions. However, its importance for the pathogenesis of streptococcal disease is unknown. In this study, we investigated the importance of the SRP pathway for virulence factor secretion in the human pathogen Streptococcus pyogenes. While the SRP pathway was not found to be essential for viability in vitro, SRP mutants demonstrated a medium-specific growth defect that could be rescued by the addition of glucose. We also observed that a distinct subset of virulence factors were dependent upon the SRP pathway for secretion, whereas others were completely independent of this pathway. Significantly, deletion of the SRP pathway resulted in mutants that were highly attenuated in both a zebrafish model of necrotic myositis and a murine subcutaneous ulcer model, highlighting the importance of this pathway in vivo. These studies emphasize the importance of the SRP pathway for the in vivo survival and pathogenesis of S. pyogenes.

scholarly journals Emerging View on the Molecular Functions of Sec62 and Sec63 in Protein Translocation

2021 ◽  
Vol 22 (23) ◽  
pp. 12757
Author(s):  
Sung-jun Jung ◽  
Hyun Kim
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Conducting Channel ◽  
Evolutionarily Conserved ◽  
Translocation Pathway ◽  
Molecular Functions ◽  
Sec61 Channel

Most secreted and membrane proteins are targeted to and translocated across the endoplasmic reticulum (ER) membrane through the Sec61 protein-conducting channel. Evolutionarily conserved Sec62 and Sec63 associate with the Sec61 channel, forming the Sec complex and mediating translocation of a subset of proteins. For the last three decades, it has been thought that ER protein targeting and translocation occur via two distinct pathways: signal recognition particle (SRP)-dependent co-translational or SRP-independent, Sec62/Sec63 dependent post-translational translocation pathway. However, recent studies have suggested that ER protein targeting and translocation through the Sec translocon are more intricate than previously thought. This review summarizes the current understanding of the molecular functions of Sec62/Sec63 in ER protein translocation.

scholarly journals Receptor compaction and GTPase movements drive cotranslational protein translocation

2020 ◽  
Author(s):  
Jae Ho Lee ◽  
SangYoon Chung ◽  
Yu-Hsien Hwang Fu ◽  
Ruilin Qian ◽  
Xuemeng Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Biological Regulation ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Cause Of Failure ◽  
Distal Site

AbstractSignal recognition particle (SRP) is a universally conserved targeting machine that couples the synthesis of ~30% of the proteome to their proper membrane localization1,2. In eukaryotic cells, SRP recognizes translating ribosomes bearing hydrophobic signal sequences and, through interaction with SRP receptor (SR), delivers them to the Sec61p translocase on the endoplasmic reticulum (ER) membrane1,2. How SRP ensures efficient and productive initiation of protein translocation at the ER is not well understood. Here, single molecule fluorescence spectroscopy demonstrates that cargo-loaded SRP induces a global compaction of SR, driving a >90 Å movement of the SRP•SR GTPase complex from the vicinity of the ribosome exit, where it initially assembles, to the distal site of SRP. These rearrangements bring translating ribosomes near the membrane, expose conserved Sec61p docking sites on the ribosome and weaken SRP’s interaction with the signal sequence on the nascent polypeptide, thus priming the translating ribosome for engaging the translocation machinery. Disruption of these rearrangements severely impairs cotranslational protein translocation and is the cause of failure in an SRP54 mutant linked to severe congenital neutropenia. Our results demonstrate that multiple largescale molecular motions in the SRP•SR complex are required to drive the transition from protein targeting to translocation; these post-targeting rearrangements provide potential new points for biological regulation as well as disease intervention.

scholarly journals Defining the physiological role of SRP in protein-targeting efficiency and specificity

Science ◽  
2018 ◽  
Vol 359 (6376) ◽  
pp. 689-692 ◽  
Author(s):  
Elizabeth A. Costa ◽  
Kelly Subramanian ◽  
Jodi Nunnari ◽  
Jonathan S. Weissman
Keyword(s):  
Protein Targeting ◽  
Signal Recognition Particle ◽  
Physiological Role ◽  
Ribosome Profiling ◽  
Signal Recognition ◽  
Amino Terminal ◽  
Mitochondrial Defects ◽  
Unanticipated Consequence

The signal recognition particle (SRP) enables cotranslational delivery of proteins for translocation into the endoplasmic reticulum (ER), but its full in vivo role remains incompletely explored. We combined rapid auxin-induced SRP degradation with proximity-specific ribosome profiling to define SRP’s in vivo function in yeast. Despite the classic view that SRP recognizes amino-terminal signal sequences, we show that SRP was generally essential for targeting transmembrane domains regardless of their position relative to the amino terminus. By contrast, many proteins containing cleavable amino-terminal signal peptides were efficiently cotranslationally targeted in SRP’s absence. We also reveal an unanticipated consequence of SRP loss: Transcripts normally targeted to the ER were mistargeted to mitochondria, leading to mitochondrial defects. These results elucidate SRP’s essential roles in maintaining the efficiency and specificity of protein targeting.

scholarly journals Getting on target: The archaeal signal recognition particle

Archaea ◽  
2002 ◽  
Vol 1 (1) ◽  
pp. 27-34 ◽  
Author(s):  
Christian Zwieb ◽  
Jerry Eichler
Keyword(s):  
Membrane Proteins ◽  
Biochemical Analysis ◽  
Protein Targeting ◽  
Polypeptide Chain ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Evolutionary Significance ◽  
Signal Recognition ◽  
Functional Behavior ◽  
Translocation Pathway

Protein translocation begins with the efficient targeting of secreted and membrane proteins to complexes embedded within the membrane. In Eukarya and Bacteria, this is achieved through the interaction of the signal recognition particle (SRP) with the nascent polypeptide chain. In Archaea, homologs of eukaryal and bacterial SRP-mediated translocation pathway components have been identified. Biochemical analysis has revealed that although the archaeal system incorporates various facets of the eukaryal and bacterial targeting systems, numerous aspects of the archaeal system are unique to this domain of life. Moreover, it is becoming increasingly clear that elucidation of the archaeal SRP pathway will provide answers to basic questions about protein targeting that cannot be obtained from examination of eukaryal or bacterial models. In this review, recent data regarding the molecular composition, functional behavior and evolutionary significance of the archaeal signal recognition particle pathway are discussed.

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