| 1. |
- Schoebel, Stefan, et al.
(författare)
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Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3
- 2017
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 548:7667
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Tidskriftsartikel (refereegranskat)abstract
- Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome(1-4)-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain(5,6). Hrd1 is the crucial membrane component for retro-translocation(7,8), but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes(9-11). Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.
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| 2. |
- Wang, Hao, et al.
(författare)
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Characterization of aquaporin-driven hydrogen peroxide transport.
- 2020
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Ingår i: Biochimica et biophysica acta. Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1862:2
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are membrane-intrinsic proteins initially defined as water (H2O) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (H2O2). H2O2 is a signaling molecule that partakes in immune responses where its transport is mediated by aquaporins. To shed further light on the molecular basis of the aquaporin function in H2O2 transport, we have characterized an Arabidopsis thaliana aquaporin, AtPIP2;4, recombinantly produced to high yields in Pichia pastoris. Here, we present a newly established assay that allows detection of H2O2 transport by purified aquaporins reconstituted into liposomes, enabling us to compare aquaporin homologues with respect to substrate specificity. To get additional insight into the structural determinants for aquaporin-mediated H2O2 transport, we solved the 3D-structure of AtPIP2;4 to 3.7Å resolution and found structural identity to the water channel from spinach (SoPIP2;1), with the difference that Cd2+ cation is not required to retain the closed conformation. The transport specificities of the two plant aquaporins were compared to a human homologue, AQP1. Overall, we conclude that AtPIP2;4, SoPIP2;1 and hAQP1 are all transporters of both H2O and H2O2, but have different efficiencies for various specificities. Notably, all three homologues expedite H2O transport equally well while the plant aquaporins are more permeable to H2O2 than hAQP1. Comparison of the structures indicates that the observed variations in H2O and H2O2 transport cannot be explained by differences in the monomeric pore. Possibly, the determinants for transport specificities reside in the flexible domains outside the membrane core of these channels.
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| 3. |
- Wang, Hao, et al.
(författare)
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Quantitative analysis of H2O2 transport through purified membrane proteins
- 2020
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Ingår i: MethodsX. - : Elsevier BV. - 2215-0161. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen peroxide (H2O2) is an important signal molecule produced in animal and plant cells. The balance of H2O2 between the intra- and extracellular space is regulated by integral membrane proteins, which thereby modulate signaling. Several methods have been established to analyze aquaporin mediated transport of H2O2 in whole cells with the intrinsic limitation that the amount of protein responsible for a certain activity cannot be standardized. As a consequence, the quantification of the transport and specific activity is difficult to extract making it problematic to compare isoforms and mutated variants of one specific target. Moreover, in cell-based assays, the expression of the target protein may alter the physiological processes of the host cell providing a complication and the risk of misleading results. To improve the measurements of protein based H2O2 transport, we have developed an assay allowing quantitative measurements. • Using purified aquaporin reconstituted in proteoliposomes, transport of H2O2 can be accurately measured. • Inside the liposomes, H2O2 catalyzes the reaction between Amplex Red and horseradish peroxidase (HRP) giving rise to the fluorescent product resorufin. • Analysing pure protein provides direct biochemical evidence of a specific transport excluding putative cellular background. © 2020 The Author(s)
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