scholarly journals Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon

2018 ◽  
Author(s):  
Michiel J.M. Niesen ◽  
Annika Müller-Lucks ◽  
Rickard Hedman ◽  
Gunnar von Heijne ◽  
Thomas F. Miller
Keyword(s):  
Conformational Changes ◽  
Protein Translocation ◽  
Translation Arrest ◽  
Protein Biogenesis ◽  
Pulling Forces ◽  
Transient Interactions ◽  
Pulling Force ◽  
Electrostatic Coupling ◽  
Polypeptide Chains ◽  
Ribosomal Translation

ABSTRACTDuring ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. Translation arrest peptide (AP) experiments are used to measure the external pulling forces that are exerted on the NC at different lengths during translation. To elucidate the molecular origins of these forces, a recently developed coarsegrained molecular dynamics (CGMD) is used to directly simulate the observed pulling-force profiles, thereby disentangling contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. This combination of experiment and theory reveals mechanistic features of Sec-facilitated membrane integration and protein translocation, including the interplay between transient interactions and conformational changes that occur during ribosomal translation to govern protein biogenesis.

scholarly journals Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing

2020 ◽  
Author(s):  
Eric D. Hoffer ◽  
Samuel Hong ◽  
S. Sunita ◽  
Tatsuya Maehigashi ◽  
Ruben L. Gonzalez ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Data Bank ◽  
Reading Frame ◽  
Translation Fidelity ◽  
Bacterial Ribosome ◽  
Structure Factors ◽  
Sequential Addition ◽  
Polypeptide Chains ◽  
Structural Insights ◽  
Atomic Coordinates

ABSTRACTModifications in the tRNA anticodon, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame.IMPACT STATEMENTChemical modifications near the tRNA anticodon and specific mRNA-tRNA pairs combine to control the ribosomal three-nucleotide mRNA reading frame, essential for the sequential addition of amino acids into polypeptide chains.Data depositionCrystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB codes 6NTA, 6NSH, 6NUO, 6NWY, 6O3M, 6OSI)

scholarly journals Ribosomal protein RPL26 is the principal target of UFMylation

2019 ◽  
Vol 116 (4) ◽  
pp. 1299-1308 ◽  
Author(s):  
Christopher P. Walczak ◽  
Dara E. Leto ◽  
Lichao Zhang ◽  
Celeste Riepe ◽  
Ryan Y. Muller ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Protein Translocation ◽  
Specific Protein ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Targets ◽  
Close Proximity ◽  
Protein Biogenesis ◽  
Membrane Bound ◽  
Enzyme Complexes

Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

scholarly journals Prion Protein Translocation Mechanism Revealed by Pulling Force Studies

2020 ◽  
Vol 432 (16) ◽  
pp. 4447-4465 ◽  
Author(s):  
Theresa Kriegler ◽  
Sven Lang ◽  
Luigi Notari ◽  
Tara Hessa
Keyword(s):  
Prion Protein ◽  
Protein Translocation ◽  
Translocation Mechanism ◽  
Pulling Force

scholarly journals SecA Dimer Cross-Linked at Its Subunit Interface Is Functional for Protein Translocation

2006 ◽  
Vol 188 (1) ◽  
pp. 335-338 ◽  
Author(s):  
Lucia B. Jilaveanu ◽  
Donald Oliver
Keyword(s):  
Conformational Changes ◽  
Protein Transport ◽  
Protein Translocation ◽  
Directed Mutagenesis ◽  
Subunit Interface ◽  
Cargo Proteins ◽  
Considerable Controversy ◽  
The Subject ◽  
Vitro Protein

ABSTRACT SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

scholarly journals Optical Pulling Using Chiral Metalens as a Photonic Probe

Nanomaterials ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3376
Author(s):  
Miao Peng ◽  
Hui Luo ◽  
Zhaojian Zhang ◽  
Tengfang Kuang ◽  
Dingbo Chen ◽  
...  
Keyword(s):  
Incident Light ◽  
Longitudinal Optical ◽  
Optical Force ◽  
Optical Manipulation ◽  
Maximum Value ◽  
Broadband Spectrum ◽  
Course Control ◽  
Pulling Forces ◽  
Potential Applications ◽  
Pulling Force

Optical pulling forces, which can pull objects in the source direction, have emerged as an intensively explored field in recent years. Conventionally, optical pulling forces exerted on objects can be achieved by tailoring the properties of an electromagnetic field, the surrounding environment, or the particles themselves. Recently, the idea of applying conventional lenses or prisms as photonic probes has been proposed to realize an optical pulling force. However, their sizes are far beyond the scope of optical manipulation. Here, we design a chiral metalens as the photonic probe to generate a robust optical pulling force. The induced pulling force exerted on the metalens, characterized by a broadband spectrum over 0.6 μm (from 1.517 to 2.117 μm) bandwidth, reached a maximum value of −83.76 pN/W. Moreover, under the illumination of incident light with different circular polarization states, the longitudinal optical force acting on the metalens showed a circular dichroism response. This means that the longitudinal optical force can be flexibly tuned from a pulling force to a pushing force by controlling the polarization of the incident light. This work could pave the way for a new advanced optical manipulation technique, with potential applications ranging from contactless wafer-scale fabrication to cell assembly and even course control for spacecraft.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Pulling Force

ABSTRACTCurrent model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

scholarly journals Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Safia Omer ◽  
Samuel R Greenberg ◽  
Wei-Lih Lee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spatial Distribution ◽  
Direct Evidence ◽  
Critical Factor ◽  
Spindle Positioning ◽  
Dynein Motor ◽  
Independent Population ◽  
Bud Neck ◽  
Pulling Forces ◽  
Pulling Force

Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

scholarly journals Pullout Capacity Of Cylindrical Block Embedded In Sand

2018 ◽  
Vol 40 (1) ◽  
pp. 30-37
Author(s):  
Krzysztof Sternik ◽  
Katarzyna Dołżyk-Szypcio
Keyword(s):  
3D Model ◽  
Fe Analysis ◽  
Contact Friction ◽  
Embedment Depth ◽  
Pullout Capacity ◽  
Pullout Resistance ◽  
Pulling Forces ◽  
Pulling Force ◽  
Alternative Solutions ◽  
Cylindrical Block

Abstract Calculation of pullout capacity of anchoring concrete cylindrical block by finite element method is carried out. 3D model of the block assumes its free rotation. Alternative solutions with one and two pulling forces attached at different heights of the block are considered. Dependency of the ultimate pulling force on the points of its application, the block’s embedment depth as well as contact friction are investigated. Results of FE analysis and simple engineering estimations are compared. The maximum pullout resistance results from FE analysis when the rotation of the block is prevented.

scholarly journals Voltage Sensing in Bacterial Protein Translocation

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 78 ◽  
Author(s):  
Denis G. Knyazev ◽  
Roland Kuttner ◽  
Ana-Nicoleta Bondar ◽  
Mirjam Zimmerman ◽  
Christine Siligan ◽  
...  
Keyword(s):  
Protein Translocation ◽  
Voltage Sensor ◽  
Voltage Sensitivity ◽  
Bacterial Protein ◽  
Voltage Sensing ◽  
Voltage Dependent ◽  
Membrane Spanning ◽  
Dipolar Orientation ◽  
Polypeptide Chains ◽  
Gating Charges

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

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