scholarly journals Towards the molecular architecture of the peroxisomal receptor docking complex

2020 ◽  
Vol 117 (52) ◽  
pp. 33216-33224
Author(s):  
Pascal Lill ◽  
Tobias Hansen ◽  
Daniel Wendscheck ◽  
Bjoern Udo Klink ◽  
Tomasz Jeziorek ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Single Copy ◽  
Integrative Approach ◽  
Molecular Architecture ◽  
Peroxisomal Membrane ◽  
Structural Support ◽  
Cargo Proteins ◽  
Complete Sequences ◽  
Subunit Organization ◽  
Docking Protein

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles, however, are unclear, mainly because structures of higher-order assemblies are still lacking. Here, using an integrative approach, we provide the structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment, and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20-nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

scholarly journals Towards the molecular architecture of the peroxisomal receptor docking complex

2019 ◽  
Author(s):  
Pascal Lill ◽  
Tobias Hansen ◽  
Daniel Wendscheck ◽  
Bjoern Udo Klink ◽  
Tomasz Jeziorek ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Single Copy ◽  
Integrative Approach ◽  
Molecular Architecture ◽  
Peroxisomal Membrane ◽  
Structural Support ◽  
Cargo Proteins ◽  
Complete Sequences ◽  
Subunit Organization ◽  
Docking Protein

AbstractImport of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles are however unclear, mainly because structures of higher order assemblies are still lacking. Here, using an integrative approach, we provide the first structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20 nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding-sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

scholarly journals cDNA Cloning of the Basement Membrane Chondroitin Sulfate Proteoglycan Core Protein, Bamacan: A Five Domain Structure Including Coiled-Coil Motifs

1997 ◽  
Vol 136 (2) ◽  
pp. 433-444 ◽  
Author(s):  
Rong-Rong Wu ◽  
John R. Couchman
Keyword(s):  
Basement Membrane ◽  
Chondroitin Sulfate ◽  
Core Protein ◽  
Coiled Coil ◽  
Basement Membranes ◽  
Cdna Clones ◽  
Molecular Architecture ◽  
Unusual Structure ◽  
Extracellular Matrix Molecule

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now obtained cDNA clones encoding the entire bamacan core protein of Mr = 138 kD, which reveal a five domain, head-rod-tail configuration. The head and tail are potentially globular, while the central large rod probably forms coiled-coil structures, with one large central and several very short interruptions. This molecular architecture is novel for an extracellular matrix molecule, but it resembles that of a group of intracellular proteins, including some proposed to stabilize the mitotic chromosome scaffold. We have previously proposed a similar stabilizing role for bamacan in the basement membrane matrix. The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser-Gly dipeptides are unfavorable for substitution. Fusion protein antibodies stained basement membranes in a pattern commensurate with bamacan, and they also Western blotted bamacan core protein from rat L2 cell cultures. The antibodies could also specifically immunoprecipitate an in vitro transcription/translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models.

scholarly journals A conserved filamentous assembly underlies the structure of the meiotic chromosome axis

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alan MV West ◽  
Scott C Rosenberg ◽  
Sarah N Ur ◽  
Madison K Lehmer ◽  
Qiaozhen Ye ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Budding Yeast ◽  
Meiotic Chromosome ◽  
Coiled Coil ◽  
Chromosome Organization ◽  
Molecular Architecture ◽  
Master Regulators ◽  
Core Proteins ◽  
Protein Components ◽  
Chromosome Axis

The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that ‘axis core proteins’ from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify ‘closure motifs’ in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control.

scholarly journals Braiding topology and the energy landscape of chromosome organization proteins

2019 ◽  
Vol 117 (3) ◽  
pp. 1468-1477 ◽  
Author(s):  
Dana Krepel ◽  
Aram Davtyan ◽  
Nicholas P. Schafer ◽  
Peter G. Wolynes ◽  
José N. Onuchic
Keyword(s):  
Structural Information ◽  
Protein Complexes ◽  
Energy Landscape ◽  
Critical Role ◽  
Coiled Coil ◽  
Structural Data ◽  
Atomic Scale ◽  
Integrative Approach ◽  
Chromosome Organization ◽  
Conformational Ensemble

Assemblies of structural maintenance of chromosomes (SMC) proteins and kleisin subunits are essential to chromosome organization and segregation across all kingdoms of life. While structural data exist for parts of the SMC−kleisin complexes, complete structures of the entire complexes have yet to be determined, making mechanistic studies difficult. Using an integrative approach that combines crystallographic structural information about the globular subdomains, along with coevolutionary information and an energy landscape optimized force field (AWSEM), we predict atomic-scale structures for several tripartite SMC−kleisin complexes, including prokaryotic condensin, eukaryotic cohesin, and eukaryotic condensin. The molecular dynamics simulations of the SMC−kleisin protein complexes suggest that these complexes exist as a broad conformational ensemble that is made up of different topological isomers. The simulations suggest a critical role for the SMC coiled-coil regions, where the coils intertwine with various linking numbers. The twist and writhe of these braided coils are coupled with the motion of the SMC head domains, suggesting that the complexes may function as topological motors. Opening, closing, and translation along the DNA of the SMC−kleisin protein complexes would allow these motors to couple to the topology of DNA when DNA is entwined with the braided coils.

scholarly journals Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1633 ◽  
Author(s):  
Giel Stalmans ◽  
Anastasia V. Lilina ◽  
Pieter-Jan Vermeire ◽  
Jan Fiala ◽  
Petr Novák ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Molecular Model ◽  
Coiled Coil ◽  
Cross Linking ◽  
Specific Interactions ◽  
Molecular Architecture ◽  
Filament Assembly ◽  
Assembly Mechanism ◽  
Recombinant Fragments ◽  
Chemical Cross Linking

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called ACN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the ACN complex which features a 6.5 nm overlap of the rod ends.

scholarly journals Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

1998 ◽  
Vol 140 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Bettina Huhse ◽  
Peter Rehling ◽  
Markus Albertini ◽  
Lars Blank ◽  
Karl Meller ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Translocation ◽  
Coiled Coil ◽  
Matrix Proteins ◽  
Peroxisomal Membrane ◽  
Trimeric Complex ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Membrane Spanning ◽  
Mutant Cells

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.

scholarly journals Remodeling the zonula adherens in response to tension and the role of afadin in this response

2016 ◽  
Vol 213 (2) ◽  
pp. 243-260 ◽  
Author(s):  
Wangsun Choi ◽  
Bipul R. Acharya ◽  
Grégoire Peyret ◽  
Marc-Antoine Fardin ◽  
René-Marc Mège ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Coiled Coil ◽  
Epithelial Cell Line ◽  
Molecular Architecture ◽  
Superresolution Microscopy ◽  
Tissue Integrity ◽  
Dynamic Coordination ◽  
Altered Cell ◽  
Actin Cables ◽  
Cell Cell

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.

scholarly journals Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

2012 ◽  
Vol 109 (5) ◽  
pp. 1380-1387 ◽  
Author(s):  
K. Lasker ◽  
F. Forster ◽  
S. Bohn ◽  
T. Walzthoeni ◽  
E. Villa ◽  
...  
Keyword(s):  
26S Proteasome ◽  
Integrative Approach ◽  
Molecular Architecture

scholarly journals Bassoon, a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals

1998 ◽  
Vol 142 (2) ◽  
pp. 499-509 ◽  
Author(s):  
Susannetom Dieck ◽  
Lydia Sanmartí-Vila ◽  
Kristina Langnaese ◽  
Karin Richter ◽  
Stefan Kindler ◽  
...  
Keyword(s):  
Active Zone ◽  
Hippocampal Neurons ◽  
Late Onset ◽  
Coiled Coil ◽  
Ultrastructural Level ◽  
Nerve Terminals ◽  
Molecular Architecture ◽  
Coding Region ◽  
Axon Terminals ◽  
Presynaptic Nerve Terminals

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

scholarly journals Molecular ruler of the attachment organelle in Mycoplasma pneumoniae

2021 ◽  
Vol 17 (6) ◽  
pp. e1009621
Author(s):  
Daisuke Nakane ◽  
Kohki Murata ◽  
Tsuyoshi Kenri ◽  
Keigo Shibayama ◽  
Takayuki Nishizaka
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Genetic Manipulation ◽  
Fluorescent Microscopy ◽  
Coiled Coil ◽  
Cell Movement ◽  
Molecular Structures ◽  
Molecular Architecture ◽  
Cell Axis ◽  
Transmission Electron ◽  
Molecular Ruler

Length control is a fundamental requirement for molecular architecture. Even small wall-less bacteria have specially developed macro-molecular structures to support their survival. Mycoplasma pneumoniae, a human pathogen, forms a polar extension called an attachment organelle, which mediates cell division, cytadherence, and cell movement at host cell surface. This characteristic ultrastructure has a constant size of 250–300 nm, but its design principle remains unclear. In this study, we constructed several mutants by genetic manipulation to increase or decrease coiled-coil regions of HMW2, a major component protein of 200 kDa aligned in parallel along the cell axis. HMW2-engineered mutants produced both long and short attachment organelles, which we quantified by transmission electron microscopy and fluorescent microscopy with nano-meter precision. This simple design of HMW2 acting as a molecular ruler for the attachment organelle should provide an insight into bacterial cellular organization and its function for their parasitic lifestyles.

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