Localization, proteomics, and metabolite profiling reveal a putative vesicular transporter for UDP-glucose.

Yulong Li,Yi Rao,Huasheng Yu,Zhaofa Wu,Rongbo Sun,Cheng Qian,Jianzhi Zeng

doi:10.7554/elife.65417

Abstract

Vesicular neurotransmitter transporters (VNTs) mediate the selective uptake and enrichment of small-molecule neurotransmitters into synaptic vesicles (SVs) and are therefore a major determinant of the synaptic output of specific neurons. To identify novel VNTs expressed on SVs (thus identifying new neurotransmitters and/or neuromodulators), we conducted localization profiling of 361 solute carrier (SLC) transporters tagging with a fluorescent protein in neurons, which revealed 40 possible candidates through comparison with a known SV marker. We parallelly performed proteomics analysis of immunoisolated SVs and identified seven transporters in overlap. Ultrastructural analysis further supported that one of the transporters, SLC35D3, localized to SVs. Finally, by combining metabolite profiling with a radiolabeled substrate transport assay, we identified UDP-glucose as the principal substrate for SLC35D3. These results provide new insights into the functional role of SLC transporters in neurotransmission and improve our understanding of the molecular diversity of chemical transmitters.

Highlights

The release of extracellular signaling molecules by secretory vesicles is a strategy used by a wide range of cell types and tissues and plays an essential role under both physiological and pathological conditions (Burgoyne and Morgan, 2003)
Dark gray bars represent known vesicular transporters, magenta bars represent SLC35 transporters, light gray bars represent non-vesicular organelle markers, and white bars represent the Solute Carrier (SLC) transporters screened in this study
We found that cells expressing the SLC35A2 transporter had significantly increased uptake of both the previously known substrate UDP-galactose and the newly identified substrate UDP-glucose compared to control cells (Figure 5B), validating our deorphanization strategy combining metabolite profiling and the radiolabeled transport assay

Summary

Introduction

The release of extracellular signaling molecules by secretory vesicles is a strategy used by a wide range of cell types and tissues and plays an essential role under both physiological and pathological conditions (Burgoyne and Morgan, 2003). Vesicular neurotransmitter transporters (VNTs) such as VGLUT and VGAT (which transport glutamate and GABA, respectively) are essential for the transport of small-molecule neurotransmitters into synaptic vesicles (SVs) These selective transporters determine the category, amount, and transport kinetics of neurotransmitters, thereby establishing the molecular basis of the underlying chemical neurotransmission (Blakely and Edwards, 2012). The physiological role of transporter proteins is closely coupled with their subcellular localization; to date, localization profiling of transporters – SLC transporters, including which are expressed on secretory organelles in primary cells – has not been systematically studied. Further ultrastructural analysis using APEX2-based EM supported that the SLC35D3 is capable of trafficking to SVs. we combined metabolite analysis and the radiolabeled substrate transport assay in subcellular organelles and identified UDP-glucose as the principal substrate of SLC35D3

Results

Discussion

Materials and methods