# Protein Transport Across Membranes

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2021 · $504,790

## Abstract

PROJECT SUMMARY
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.

## Key facts

- **NIH application ID:** 10149331
- **Project number:** 5R01GM052586-27
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Tom A Rapoport
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $504,790
- **Award type:** 5
- **Project period:** 1995-05-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10149331

## Citation

> US National Institutes of Health, RePORTER application 10149331, Protein Transport Across Membranes (5R01GM052586-27). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10149331. Licensed CC0.

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