TY - JOUR
T1 - Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress
AU - Zhou, Ye
AU - Kastritis, Panagiotis L.
AU - Dougherty, Shannon E.
AU - Bouvette, Jonathan
AU - Hsu, Allen L.
AU - Burbaum, Laura
AU - Mosalaganti, Shyamal
AU - Pfeffer, Stefan
AU - Hagen, Wim J.H.
AU - Förster, Friedrich
AU - Borgnia, Mario J.
AU - Vogel, Christine
AU - Beck, Martin
AU - Bartesaghi, Alberto
AU - Silva, Gustavo M.
N1 - Publisher Copyright:
© 2020 National Academy of Sciences. All rights reserved.
PY - 2020/9/8
Y1 - 2020/9/8
N2 - Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
AB - Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
KW - Cryo-EM
KW - K63 ubiquitin
KW - Oxidative stress
KW - Ribosome
KW - Translation
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U2 - 10.1073/pnas.2005301117
DO - 10.1073/pnas.2005301117
M3 - Article
C2 - 32855298
AN - SCOPUS:85090613674
SN - 0027-8424
VL - 117
SP - 22157
EP - 22166
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 36
ER -