# BIORAD ChemiDoc MP imaging system

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $40,533

## Abstract

Abstract / Summary
The vast majority of all secreted and plasma membrane proteins are synthesized on membrane-bound
ribosomes at the endoplasmic reticulum (ER). Ribosomes synthesizing this subset of proteins are targeted to
the ER membrane by the signal recognition particle (SRP). SRP binds to signal sequences as they emerge from
the ribosome as part of the growing polypeptide chain. SRP then hands over the nascent polypeptide to the
Sec61 translocon for movement into or across the ER membrane bilayer. Protein folding in the ER lumen
proceeds co-translationally. In the event of protein misfolding, a signaling network, the “unfolded protein
response (UPR)”, is activated that rebalances the ER’s protein folding capacity with the load of proteins entering
the ER. IRE1 is one of the UPR’s main protein misfolding sensors. It is a bifunctional kinase/RNase with a
lumenal domain that is activated by unfolded protein binding. It signals through a non-conventional mRNA
splicing reaction, utilizing its cytoplasmic RNase domain to excise an intron from the mRNA encoding the
transcription factor XBP1. XBP1 drives expression of a host of chaperones, protein folding factors, and
component of the protein transport and degradation machinery to reestablish homeostasis. IRE1 also degrades
select ER-bound mRNAs, thereby reducing the ER protein-folding load in a reaction termed “regulated IRE1
dependent decay (RIDD)”. IRE1 can directly interact with the co-translational translocation machinery, including
the ribosome, SRP, and Sec61 translocon. These data (and supporting evidence) suggest that IRE1, at least in
part, performs its functions in physical contact with ER-bound ribosomes. When activated, however, IRE1 forms
large oligomeric clusters. We recently visualized these clusters in intact cells using correlative light and electron
microscopy. Our high-resolution cryo-tomograms demonstrate that IRE1 clusters are convoluted networks of
narrow, anatomosing ER tubes. Surprisingly, these clusters are entirely devoid of membrane-bound ribosomes.
This poses a paradox, suggesting that active IRE1 must exist in at least two sub-populations, one composed of
small oligomers that can ribosome- and/or SRP-associate, the other composed of large ribosome-free IRE1
clusters. We will map IRE1’s target mRNAs and mechanistic roles to these states. Specifically, we will: (1)
Dissect the mechanistic principles of substrate selection by IRE1. (2) Determine the atomic structure of IRE1
complexed to ribosomes, translocon, and/or SRP using single-particle cryo-EM. (3) Identify the sub-cellular
birthplaces of IRE1-spliced and RIDD-cleaved mRNAs. (4) Assemble IRE1 cluster of defined stoichiometry in
vitro and in cells and assess functional outputs with biochemical and cell-based assays. Defects in proteostasis
lie at the heart of an ever-expanding list of diseases. The proposed experiments aim to define how mammalian
IRE1 selects its mRNA targets and what key molecular players it associat...

## Key facts

- **NIH application ID:** 10799461
- **Project number:** 3R01GM032384-40S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Robert M Stroud
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $40,533
- **Award type:** 3
- **Project period:** 1983-07-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10799461

## Citation

> US National Institutes of Health, RePORTER application 10799461, BIORAD ChemiDoc MP imaging system (3R01GM032384-40S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10799461. Licensed CC0.

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