Pex17p-dependent assembly of Pex14p/Dyn2p-subcomplexes of the peroxisomal protein import machinery

Chan, Anna; Schummer, Andreas; Fischer, Sabrina; Schröter, Thomas; Cruz-Zaragoza, Luis Daniel; Bender, Julian; Drepper, Friedel; Oeljeklaus, Silke; Kunau, Wolf‐H.; Girzalsky, Wolfgang; Warscheid, Bettina; Erdmann, Ralf

doi:10.1016/j.ejcb.2016.10.004

Cited by 18 publications

(17 citation statements)

References 60 publications

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“…To estimate the proportion of proteins that are modified by DCU at a given site, intensities of DCU-modified peptides identified with a localization probability Ն0.9 were summed up per modified site. For identification of cross-linked peptides, the program pLink (49) was used essentially as described previously (50), however, taking into account zero-length cross-links (Ϫ18.0106 Da) between aspartate or glutamate and lysine or the N-terminal amino group as well as monolink modifications (ϩ206.1783 Da) of aspartate or glutamate. Relative quantification comparing DCCD-treated versus untreated samples for visualization was done by extracted ion chromatograms integrating the first three isotope peaks (mass tolerance 5 ppm) of each precursor ion using Xcalibur Qual Browser software (version 2.2, Thermo Fisher Scientific).…”

Section: Lc-ms/msmentioning

confidence: 99%

Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide

Blümmel

Drepper

Knapp

et al. 2017

Journal of Biological Chemistry

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Twin-arginine translocation (Tat) systems transport folded proteins across cellular membranes with the concerted action of mostly three membrane proteins: TatA, TatB, and TatC. Hetero-oligomers of TatB and TatC form circular substrate-receptor complexes with a central binding cavity for twin-arginine-containing signal peptides. After binding of the substrate, energy from an electro-chemical proton gradient is transduced into the recruitment of TatA oligomers and into the actual translocation event. We previously reported that Tat-dependent protein translocation into membrane vesicles of is blocked by the compound'-dicyclohexylcarbodiimide (DCCD, DCC). We have now identified a highly conserved glutamate residue in the transmembrane region of TatC, which when modified by DCCD interferes with the deep insertion of a Tat signal peptide into the TatBC receptor complex. Our findings are consistent with a hydrophobic binding cavity formed by TatB and TatC inside the lipid bilayer. Moreover, we found that DCCD mediates discrete intramolecular cross-links of TatC involving both its N- and C-tails. These results confirm the close proximity of two distant sequence sections of TatC proposed to concertedly function as the primary docking site for twin-arginine signal peptides.

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Section: Lc-ms/msmentioning

confidence: 99%

Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide

Blümmel

Drepper

Knapp

et al. 2017

Journal of Biological Chemistry

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“…For the former, MS raw data files were first converted to the Mascot generic format (mgf) using msConvert from ProteoWizard, release 3.0.9740 (74). Searches were performed against the forward and reversed amino acid sequences of the recombinant proteins with parameters and filtering criteria as described previously (75). In brief, cross-linked peptide matches were filtered at a false discovery rate (FDR) of 5%, and a minimum E-value of 5 × 10 −2 rejecting adjacent tryptic peptides of the same protein.…”

Section: Cross-linking and Mass Spectrometry Analysesmentioning

confidence: 99%

Molecular basis of F-actin regulation and sarcomere assemblyviamyotilin

Košťan

Pavšič

Puž

et al. 2020

Preprint

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Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain containing protein myotilin provides structural integrity to Z-discs - the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. We used a combination of small angle X-ray scattering, cross-linking mass spectrometry, biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin’s structural role in Z-discs.Significance StatementSarcomeres are the primary structural and functional unit of striated muscles, conferring movement in all animals. The Z-disk is the boundary between adjacent sarcomeres, where actin filaments (F-actin) are anchored. Z-disc protein myotilin, is a scaffold protein, which provides structural integrity to the Z-disc by multiple interactions to its central components, including F-actin and α-actinin-2. Here we provide the structure of myotilin, revealing its structural plasticity in solution and the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond being an interaction hub for Z-disk partners.HighlightsMyotilin is structurally described as a dynamic ensembleFlanking regions enhance F-acting binding to tandem Ig domainsIntegrative structural model of myotilin bound to F-actinMyotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc

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“…However, Pex17p is essential for peroxisomal import of both PTS1 and PTS2 proteins 14 . Strikingly, both import receptors, Pex5p and Pex7p, associate with the docking-complex (Pex13p, Pex14p) in absence of Pex17p, but with decreased efficiency 22 . Pex17p is apparently non-essential for receptor docking, but it increases the efficiency of the docking event.…”

Section: Introductionmentioning

confidence: 98%

“…Pex17p is tightly associated with Pex14p and thus with the docking-complex 21 , but its precise function remains unknown. Although Pex17p is part of the docking complex in yeast, it does not significantly contribute to the assembly of the Pex13p/Pex14p subcomplex 15,21,22 , and its counterpart in higher eukaryotes has not yet been identified. However, Pex17p is essential for peroxisomal import of both PTS1 and PTS2 proteins 14 .…”

Section: Introductionmentioning

confidence: 99%

Towards the molecular architecture of the peroxisomal receptor docking complex

Lill

Hansen

Wendscheck

et al. 2019

Preprint

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Import of yeast peroxisomal matrix proteins is initiated bycytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the membrane docking complex, composed of Pex14p, Pex17p and Pex13p. 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 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.

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Pex17p-dependent assembly of Pex14p/Dyn2p-subcomplexes of the peroxisomal protein import machinery

Cited by 18 publications

References 60 publications

Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide

Structural features of the TatC membrane protein that determine docking and insertion of a twin-arginine signal peptide

Molecular basis of F-actin regulation and sarcomere assemblyviamyotilin

Towards the molecular architecture of the peroxisomal receptor docking complex

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