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Shu-ou Shan - One of the best experts on this subject based on the ideXlab platform.
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sequential activation of human signal recognition particle by the ribosome and signal sequence drives efficient Protein Targeting
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sowmya Chandrasekar, Sangyoon Chung, Yuhsien Hwang Fu, Shimon Weiss, Shu-ou ShanAbstract:Signal recognition particle (SRP) is a universally conserved Targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective Protein Targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective Protein Targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.
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Regulation of cargo recognition, commitment, and unloading drives cotranslational Protein Targeting
Journal of Cell Biology, 2014Co-Authors: Ishu Saraogi, David Akopian, Shu-ou ShanAbstract:Efficient and accurate Protein localization is essential to cells and requires Protein-Targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their Targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent Protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway. Real-time measurement of cargo transfer from the Targeting to translocation machinery revealed multiple factors that drive this event, including GTPase rearrangement in the SRP–SR complex, stepwise displacement of SRP from the ribosome and signal sequence by SecYEG, and elongation of the nascent polypeptide. Our results elucidate how active and sequential regulation of the SRP–cargo interaction drives efficient and faithful Protein Targeting.
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Co-translational Protein Targeting to the bacterial membrane
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2014Co-Authors: Ishu Saraogi, Shu-ou ShanAbstract:Co-translational Protein Targeting by the Signal Recognition Particle (SRP) is an essential cellular pathway that couples the synthesis of nascent Proteins to their proper cellular localization. The bacterial SRP, which contains the minimal ribonucleoProtein core of this universally conserved Targeting machine, has served as a paradigm for understanding the molecular basis of Protein localization in all cells. In this review, we highlight recent biochemical and structural insights into the molecular mechanisms by which fundamental challenges faced by Protein Targeting machineries are met in the SRP pathway. Collectively, these studies elucidate how an essential SRP RNA and two regulatory GTPases in the SRP and SRP receptor (SR) enable this Targeting machinery to recognize, sense and respond to its biological effectors, i.e. the cargo Protein, the target membrane and the translocation machinery, thus driving efficient and faithful co-translational Protein Targeting. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
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signal recognition particle an essential Protein Targeting machine
Annual Review of Biochemistry, 2013Co-Authors: David Akopian, Xin Zhang, Kuang Shen, Shu-ou ShanAbstract:The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent Proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this Targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo Protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.
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Fingerloop activates cargo delivery and unloading during cotranslational Protein Targeting
Molecular Biology of the Cell, 2013Co-Authors: Aileen Ariosa, Ishu Saraogi, Stacy S. Duncan, April Brown, Gregory J. Phillips, Shu-ou ShanAbstract:During cotranslational Protein Targeting by the signal recognition particle (SRP), information about signal sequence binding in the SRP's M domain must be effectively communicated to its GTPase domain to turn on its interaction with the SRP receptor (SR) and thus deliver the cargo Proteins to the membrane. A universally conserved “fingerloop” lines the signal sequence–binding groove of SRP; the precise role of this fingerloop in Protein Targeting has remained elusive. In this study, we show that the fingerloop plays important roles in SRP function by helping to induce the SRP into a more active conformation that facilitates multiple steps in the pathway, including efficient recruitment of SR, GTPase activation in the SRP•SR complex, and most significantly, the unloading of cargo onto the target membrane. On the basis of these results and recent structural work, we propose that the fingerloop is the first structural element to detect signal sequence binding; this information is relayed to the linker connecting the SRP's M and G domains and thus activates the SRP and SR for carrying out downstream steps in the pathway.
Peter Walter - One of the best experts on this subject based on the ideXlab platform.
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the conserved aaa atpase msp1 confers organelle specificity to tail anchored Proteins
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Voytek Okreglak, Peter WalterAbstract:The accuracy of tail-anchored (TA) Protein Targeting to the endoplasmic reticulum (ER) depends on the Guided Entry of Tail-Anchored (Get) Protein Targeting machinery. The fate of TA Proteins that become inappropriately inserted into other organelles, such as mitochondria, is unknown. Here, we identify Msp1, a conserved, membrane-anchored AAA-ATPase (ATPase associated with a variety of cellular activities) that localizes to mitochondria and peroxisomes, as a critical factor in a quality control pathway that senses and degrades TA Proteins mistargeted to the outer mitochondrial membrane (OMM). Pex15 is normally targeted by the Get pathway to the ER, from where it travels to peroxisomes. Loss of Msp1 or loss of the Get pathway results in the redistribution of Pex15 to mitochondria. Cells lacking both a functional Get pathway and Msp1 accumulate increased amounts of Pex15 on the OMM and display severely dysfunctional mitochondrial morphology. In addition, Msp1 binds and promotes the turnover of a Pex15 mutant that is misdirected to the OMM. Our data suggest that Msp1 functions in local organelle surveillance by extracting mistargeted Proteins, ensuring the fidelity of organelle specific-localization of TA Proteins.
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The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting
Molecular Biology of the Cell, 2007Co-Authors: Niels Bradshaw, Peter WalterAbstract:The RNA component of the signal recognition particle (SRP) is universally required for cotranslational Protein Targeting. Biochemical studies have shown that SRP RNA participates in the central step of Protein Targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP·SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP Protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding Targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the Targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the Protein Targeting machinery.
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signal sequence recognition and Protein Targeting to the endoplasmic reticulum membrane
Annual Review of Cell Biology, 1994Co-Authors: Peter Walter, Arthur E JohnsonAbstract:STRUCTURE OF THE SRP AND SRP RECEPTOR . NOlllelleiatllre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SRP RNA SRP Structllre alld Assembly . SRP Proteill SlIbllllits . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SRP Receptor .
Irmgard Sinning - One of the best experts on this subject based on the ideXlab platform.
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SIMIBI twins in Protein Targeting and localization.
Nature Structural & Molecular Biology, 2013Co-Authors: Gert Bange, Irmgard SinningAbstract:SIMIBI-type NTP-binding Proteins are an ancient subfamily of nucleotide-binding Proteins that comprises both dimeric ATPases and GTPases (SIMIBI 'twins'). This Perspective focuses on a subset of SIMIBI Proteins with a defined function in Protein Targeting and localization, and aims to define common mechanistic principles and differences for these Proteins.
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lipids trigger a conformational switch that regulates signal recognition particle srp mediated Protein Targeting
Journal of Biological Chemistry, 2011Co-Authors: Goran Stjepanovic, Gert Bange, Katja Kapp, Christian Graf, Richard Parlitz, Klemens Wild, Matthias P Mayer, Irmgard SinningAbstract:Co-translational Protein Targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for Protein Targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-Targeting sequence. We show that the membrane-Targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-Protein interactions and their importance in the regulation of Protein Targeting and translocation across biological membranes.
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Protein Targeting by the signal recognition particle
Biological Chemistry, 2009Co-Authors: Przemyslaw Grudnik, Gert Bange, Irmgard SinningAbstract:Protein Targeting by the signal recognition particle (SRP) is universally conserved and starts with the recognition of a signal sequence in the context of a translating ribo- some. SRP54 and FtsY, two multidomain Proteins with guanosine triphosphatase (GTPase) activity, are the cen- tral elements of the SRP system. They have to coordinate the presence of a signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane inser- tion of substrate Proteins. Recent results are integrated in an updated perception of the order of events in SRP- mediated Protein Targeting.
Sergio Grinstein - One of the best experts on this subject based on the ideXlab platform.
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contribution of phosphatidylserine to membrane surface charge and Protein Targeting during phagosome maturation
The Journal of General Physiology, 2009Co-Authors: Tony Yeung, Bryan Heit, Jean Francois Dubuisson, Basil Chiu, Gregory D. Fairn, Andras Kapus, Michele S Swanson, Robert D. Inman, Sergio GrinsteinAbstract:1. 1. Yeung, 2. et al . 2009. J. Cell Biol. doi:[10.1083/jcb.200903020][1] [1]: /lookup/doi/10.1083/jcb.200903020
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contribution of phosphatidylserine to membrane surface charge and Protein Targeting during phagosome maturation
Journal of Cell Biology, 2009Co-Authors: Tony Yeung, Bryan Heit, Jean Francois Dubuisson, Basil Chiu, Gregory D. Fairn, Andras Kapus, Michele S Swanson, Robert D. Inman, Sergio GrinsteinAbstract:During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic Proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic Proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.
Kuang Shen - One of the best experts on this subject based on the ideXlab platform.
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signal recognition particle an essential Protein Targeting machine
Annual Review of Biochemistry, 2013Co-Authors: David Akopian, Xin Zhang, Kuang Shen, Shu-ou ShanAbstract:The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent Proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this Targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo Protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.
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Novel Roles of the SRP RNA in Co-Translational Protein Targeting
2013Co-Authors: Kuang ShenAbstract:The signal recognition particle (SRP) and its receptor (SR) are universally conserved Protein machineries that deliver nascent peptides to their proper destination. The SRP RNA is a universally conserved and essential component of SRP, which serves as the “catalyst” of the Protein Targeting cycle. The SRP RNA accelerates SRP-SR complex formation at the beginning of the Protein Targeting reaction, and triggers GTP hydrolysis and SRP-SR complex disassembly at the end. Here we combined biochemical and biophysical approaches to investigate the molecular mechanism of the functions of the SRP RNA. We found that two functional ends in the SRP RNA mediate distinct functions. The tetraloop end facilitates initial assembly of SRP and SR by mediating an electrostatic interaction with the Lys399 receptor, which ensures efficient and accurate substrate Targeting. At the later stage of the SRP cycle, the SRP-SR complex relocalizes ~ 100 Angstrom to the 5’,3’-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. These results, combined with recent structural work, elucidate the functions of the SRP RNA during the Protein Targeting reaction.
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Activated GTPase Movement on SRP RNA Drives Cotranslational Protein Targeting
Biophysical Journal, 2013Co-Authors: Kuang Shen, David Akopian, Sinan Arslan, Shu-ou ShanAbstract:Signal Recognition Particle (SRP) and its receptor (SR) are co-translational Protein Targeting machineries responsible for delivering ribosome-nascent chain-complexes (RNCs) from cytosol to cellular membranes. Loaded with its cargo (RNC), SRP forms a complex with SR and bring the RNC to the membrane. Extensive rearrangements in this complex activate GTP hydrolysis and unload the RNC onto the translocon. During this process, the SRP RNA plays an essential role in accelerating both SRP-SR complex formation and GTP hydrolysis step. Here we show that the SRP RNA is a bi-functional molecule with its two ends, the tetraloop end and the distal end, stimulating different stages of SRP-SR interaction. using single molecule techniques to direct visualize the global relocalization along the SRP RNA, we demonstrate that the SRP-SR GTPase complex travels over 100A in the Targeting reaction, from the RNA's tetraloop end during initial complex assembly to the distal end during GTPase activation. Moreover, this rearrangement is tightly regulated by the RNC and the translocon. The large-scale movement of the GTPase complex provides an attractive mechanism for coupling GTPase activation to the transfer of RNC from SRP to translocon, thereby ensuring productive Protein Targeting.
