# Molecular mechanisms of protein glycosylation and trafficking

> **NIH NIH R01** · VAN ANDEL RESEARCH INSTITUTE · 2024 · $451,250

## Abstract

Project Summary
The objective of this proposal is to gain mechanistic and pathological understanding of protein glycosylation
and trafficking. During the previous funding cycle, we have made important contributions to this process. We
have determined the structure of the transmembrane domain insertase EMC complex, revealing an elongated
cavity in the transmembrane region of the structure that can accommodate a weakly hydrophobic
transmembrane helix. We have solved the structures of the protein N-glycosyltransferase (OST) and the
protein O-mannosyltransferase Pmt1-Pmt2, revealing the evolutionarily conserved GT-C folds of their catalytic
subunits. Protein trafficking requires lipid vesicle formation, a process that is dependent on lipid flippase activity
to establish compositional asymmetry between the two leaflets of the bilayer. In this regard, we have
determined the structures of all three classes of yeast lipid flippases. This renewal proposal continues our
overarching goal to understand protein glycosylation and trafficking. We propose to address two specific
knowledge gaps: the structure and mechanism of two protein mannosyltransferases, and how the recently
discovered ternary protein complex Arl1-Gea2-Drs2 couples lipid flipping activity with membrane curvature
formation, thereby facilitating the downstream vesicle budding process for protein and membrane trafficking.
Drs2 is a phosphatidylserine flippase required for the formation of AP-1/clathrin-coated vesicles that travel
back and forth between the trans-Golgi network (TGN) and early endosomes. The small GTPase Arl1 is a
member of the ADP-ribosylation factor (Arf) family that is activated by the Arf guanine nucleotide exchange
factor Gea2. Arl1 operates exclusively in the TGN and is the least well-understood member of the Arf family.
We will reconstitute the ternary complex in vitro and perform a comprehensive structure-function study. Protein
glycosylation and trafficking is intimately linked to tumorigenesis and cancer progression. Our mechanistic
studies will fill these fundamental knowledge gaps, and our derived structures may facilitate the development
of small molecules for cancer treatment.

## Key facts

- **NIH application ID:** 10798253
- **Project number:** 5R01CA231466-07
- **Recipient organization:** VAN ANDEL RESEARCH INSTITUTE
- **Principal Investigator:** Huilin Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $451,250
- **Award type:** 5
- **Project period:** 2018-06-18 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10798253

## Citation

> US National Institutes of Health, RePORTER application 10798253, Molecular mechanisms of protein glycosylation and trafficking (5R01CA231466-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10798253. Licensed CC0.

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