The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. One aspect of this proposal is to clarify how proteins are translocated post-translationally from the cytosol across the bacterial plasma membrane or the eukaryotic endoplasmic reticulum (ER). We have determined X-ray structures of the protein-conducting SecY channel, with and without the ATPase SecA and substrate, and used biochemical experiments to study bacterial and eukaryotic translocation, providing the basis for the present proposal. In eukaryotes, there is a retro-translocation pathway, called ERAD (for ER associated degradation). We have recapitulated ERAD with purified components and have determined a structure of the Hrd1 ubiquitin ligase, which likely forms a protein-conducting channel. Here, we will address central questions of these protein translocation pathways:
Specific aim #1 : What is the mechanism of post-translational translocation? Based on crystal structures and biochemical experiments, we have proposed a ?push-and-slide? model by which SecA mediates post-translational translocation in bacteria. We will perform single-molecule FRET experiments to test this model and clarify how SecA couples its ATPase and mechanical cycles. To address the mechanism of post-translational translocation in eukaryotes, we will determine cryo-EM structures of the yeast Sec complex in the absence and presence of bound substrate and perform biochemical experiments.
Specific aim #2 : How are proteins moved through the membrane during ERAD? We will test whether Hrd1 forms a protein-conducting channel by determining cryo-EM structures of Hrd1 in complex with its partners. We will use reconstituted systems with purified proteins to understand how substrates are selected for ERAD, use crosslinking methods to determine the path of a polypeptide through the retro-translocon, and test the postulated role of auto-ubiquitination of Hrd1 in channel gating.
Specific aim #3 : How are ERAD substrates moved from the ER membrane to the proteasome? We will determine cryo-EM structures of the Cdc48 complex together with substrate, analyze how substrate processing is initiated, how the ATPase complex extracts polypeptides from the ER membrane, and how polypeptides are transferred to the proteasome. We will investigate the role of binding partners of Cdc48 and of shuttling factors in the transfer of substrates to the 26S proteasome, and test whether some substrates can be transferred directly to the 20S proteasome. The mechanism of protein translocation is of great medical importance. Many diseases, including cystic fibrosis, are caused by the misfolding of ER proteins and their degradation. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.

Public Health Relevance

The translocation of proteins across membranes is a fundamental process involved in the biosynthesis and degradation of a large class of proteins. The medical importance of translocation is demonstrated by drugs that inhibit signal sequence binding, which are used for therapeutic intervention in chronic inflammation, as well as by the large number of diseases, including cystic fibrosis and ?1-antitrypsin deficiency, which result from the misfolding of ER proteins and their subsequent degradation in the cytosol. A better understanding of the translocation process both in the forward and backward direction may elucidate the causes of these diseases and result in the development of drugs that allow selective interference.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052586-27
Application #
10149331
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Blatch, Sydella Anne
Project Start
1995-05-01
Project End
2023-02-28
Budget Start
2021-03-01
Budget End
2022-02-28
Support Year
27
Fiscal Year
2021
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Bodnar, Nicholas O; Kim, Kelly H; Ji, Zhejian et al. (2018) Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4. Nat Struct Mol Biol 25:616-622
Wu, Xudong; Rapoport, Tom A (2018) Mechanistic insights into ER-associated protein degradation. Curr Opin Cell Biol 53:22-28
Chen, Yu; Bensing, Barbara A; Seepersaud, Ravin et al. (2018) Unraveling the sequence of cytosolic reactions in the export of GspB adhesin from Streptococcus gordonii. J Biol Chem 293:5360-5373
Schoebel, Stefan; Mi, Wei; Stein, Alexander et al. (2017) Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3. Nature 548:352-355
Bodnar, Nicholas O; Rapoport, Tom A (2017) Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex. Cell 169:722-735.e9
Rapoport, Tom A; Li, Long; Park, Eunyong (2017) Structural and Mechanistic Insights into Protein Translocation. Annu Rev Cell Dev Biol 33:369-390
Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid et al. (2017) Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins. J Biol Chem 292:8007-8018
Baldridge, Ryan D; Rapoport, Tom A (2016) Autoubiquitination of the Hrd1 Ligase Triggers Protein Retrotranslocation in ERAD. Cell 166:394-407
Li, Long; Park, Eunyong; Ling, JingJing et al. (2016) Crystal structure of a substrate-engaged SecY protein-translocation channel. Nature 531:395-399
Tan, Dongyan; Blok, Neil B; Rapoport, Tom A et al. (2016) Structures of the double-ring AAA ATPase Pex1-Pex6 involved in peroxisome biogenesis. FEBS J 283:986-92

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