Isolation of Native Soluble and Membrane-Bound Protein Complexes from Yeast Saccharomyces cerevisiae

Hansen, Tobias; Chan, Anna; Schröter, Thomas; Schwerter, Daniel; Girzalsky, Wolfgang; Erdmann, Ralf

doi:10.1007/978-1-4939-6937-1_4

Cited by 5 publications

(2 citation statements)

References 9 publications

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“…The positive transformants were cultured in SD‐Ura (synthetic dextrose minimal media without uracil) media with 2% w/v galactose overnight at 30°C and harvested in phosphate buffer solution (.2 mM, pH 7.4). Membrane proteins were isolated from the transformed yeast cultures using a previously described protocol (Hansen et al, 2017 ) and analyzed by immunoblotting. Yeast cells were observed between 495 and 520 nm using 488‐nm argon‐ion laser excitation with a Zeiss LSM700 laser scanning confocal microscope.…”

Section: Methodsmentioning

confidence: 99%

Rice aquaporin OsPIP2;2 is a water‐transporting facilitator in relevance to drought‐tolerant responses

et al. 2021

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In rice ( Oryza sativa ), the PLASMA MEMBRANE INTRINSIC PROTEIN (PIP) family of aquaporin has 11 members, OsPIP1;1 to OsPIP1;3, and OsPIP2;1 to OsPIP2;8, which are hypothesized to facilitate the transport of H 2 O and other small compounds across cell membranes. To date, however, only OsPIP1;2, OsPIP2;1, and OsPIP2;4 have been demonstrated for substrate selectivity in their source plant (rice). In this study, OsPIP2;2 was characterized as the most efficient facilitator of H 2 O transport across cell membranes in comparison with the other 10 OsPIPs. In concomitant tests of all OsPIP s, four genes ( OsPIP1;3 , OsPIP2;1 , OsPIP2;2 , and OsPIP2;4 ) were induced to express in leaves of rice plants following a physiological drought stress, while OsPIP2;2 was expressed to the highest level. After de novo expression in frog oocytes and yeast cells, the four OsPIP proteins were localized to the plasma membranes in trimer and tetramer and displayed the activity to increase the membrane permeability to H 2 O. In comparison, OsPIP2;2 was most supportive to H 2 O import to oocytes and yeast cells. After de novo expression in tobacco protoplasts, OsPIP2;2 exceeded OsPIP1;3, OsPIP2;1, and OsPIP2;4 to support H 2 O transport across the plasma membranes. OsPIP2;2‐mediated H 2 O transport was accompanied by drought‐tolerant responses, including increases in concentrations of proline and polyamines, both of which are physiological markers of drought tolerance. In rice protoplasts, H 2 O transport and drought‐tolerant responses, which included expression of marker genes of drought tolerance pathway, were considerably enhanced by OsPIP2;2 overexpression but strongly inhibited by the gene silencing. Furthermore, OsPIP2;2 played a role in maintenance of the cell membrane integrity and effectively protected rice cells from electrolyte leakage caused by the physiological drought stress. These results suggest that OsPIP2;2 is a predominant facilitator of H 2 O transport in relevance to drought tolerance in the plant.

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Section: Methodsmentioning

confidence: 99%

Rice aquaporin OsPIP2;2 is a water‐transporting facilitator in relevance to drought‐tolerant responses

et al. 2021

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“…S3A). We further isolated native complexes from oleic acid-induced cells expressing Pex14p-TPA (tobacco etch virus-proteinA), as previously described (23,37). Using Pex14p-TPA as bait, we isolated the native Pex14p/Pex17p complex from Triton X-100 solubilized whole cell membranes and analyzed again the sample by negative-stain EM (SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Towards the molecular architecture of the peroxisomal receptor docking complex

Lill

Hansen

Wendscheck

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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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.

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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.

show abstract

Isolation of Native Soluble and Membrane-Bound Protein Complexes from Yeast Saccharomyces cerevisiae

Cited by 5 publications

References 9 publications

Rice aquaporin OsPIP2;2 is a water‐transporting facilitator in relevance to drought‐tolerant responses

Rice aquaporin OsPIP2;2 is a water‐transporting facilitator in relevance to drought‐tolerant responses

Towards the molecular architecture of the peroxisomal receptor docking complex

Towards the molecular architecture of the peroxisomal receptor docking complex

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