National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM052586
米国
引用
ジャーナル: Science / 年: 2020 タイトル: Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex. 著者: Xudong Wu / Marc Siggel / Sergey Ovchinnikov / Wei Mi / Vladimir Svetlov / Evgeny Nudler / Maofu Liao / Gerhard Hummer / Tom A Rapoport / 要旨: Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD- ...Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.
The full sequence of poly-UNK region in HRD3 is PDIGSPFIAQVNGVQMTLQIEPMGRFAFNGNDGNINGDEDDE, UniProt ...The full sequence of poly-UNK region in HRD3 is PDIGSPFIAQVNGVQMTLQIEPMGRFAFNGNDGNINGDEDDE, UniProt reference ID is Q05787.
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実験情報
-
実験
実験
手法: 電子顕微鏡法
EM実験
試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法
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試料調製
構成要素
名称: a complex of Hrd1/Hrd3/Yos9 / タイプ: COMPLEX / Entity ID: all / 由来: RECOMBINANT
分子量
実験値: NO
由来(天然)
生物種: Saccharomyces cerevisiae (パン酵母)
由来(組換発現)
生物種: Saccharomyces cerevisiae (パン酵母)
緩衝液
pH: 7.4
緩衝液成分
ID
濃度
名称
式
Buffer-ID
1
100mM
sodiumchloride
NaCl
1
2
25mM
HEPES
C8H18N2O4S
1
3
0.06 %
digitonin
C56H92O29
1
試料
濃度: 6 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES