# Topological regulation of transmembrane proteins through Regulated Alternative Translocation

> **NIH NIH R35** · UT SOUTHWESTERN MEDICAL CENTER · 2021 · $409,583

## Abstract

Summary
 Transmembrane proteins must adopt proper membrane topology to perform their function. In
mammalian cells, the topology of transmembrane proteins is determined by the direction through
which transmembrane helices are inserted into ER during their translation on ER-associated
ribosomes. It has been assumed that protein translocation across the ER membranes is a
constitutive process so that transmembrane proteins must adopt a fixed topology. This assumption
has been challenged by our recent observation that the direction through which transmembrane
helices are inserted into the ER can be reversed under certain physiological conditions. We reported
that ceramide inverted the topology of two polytopic transmembrane proteins, namely TM4SF20 and
CCR5. Since this regulatory mechanism does not flip transmembrane proteins that have already
been synthesized but inverts the topology of newly synthesized proteins by changing the direction
through which transmembrane helices are translocated across membranes, we designated this
process as Regulated Alternative Translocation (RAT).
 This project is initiated to further characterize RAT by delineating the mechanism of this
topological regulation. We will begin by testing the hypothesis that TRAM2 is the ceramide sensor
that interacts with the nascent transmembrane helices subjected to RAT. The approaches developed
for this study can be generalized to identify ceramide interactome, a finding that might reveal more
signaling reactions mediated by the sphingolipid. These approaches may also be applied unbiasedly
to identify proteins interacting with the nascent transmembrane helices subjected to RAT, thereby
providing more mechanistic insights into this novel translocation regulation.
 This project will also identify proteins subject to RAT by a novel proteome-wide approach
capable of measuring topology of transmembrane proteins globally. Achieving this part of the project
will not only reveal the breadth of RAT but also provide essential experimental data for proteome-
wide assembly of topology of transmembrane proteins. Considering that only 10% of mammalian
transmembrane proteins have their topology defined by experimental evidence, accomplishing this
project should greatly improve our understanding of transmembrane proteins.

## Key facts

- **NIH application ID:** 10166533
- **Project number:** 1R35GM140851-01
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** JIN YE
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $409,583
- **Award type:** 1
- **Project period:** 2021-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10166533

## Citation

> US National Institutes of Health, RePORTER application 10166533, Topological regulation of transmembrane proteins through Regulated Alternative Translocation (1R35GM140851-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10166533. Licensed CC0.

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