Golgi-IP, a tool for multimodal analysis of Golgi molecular content

Rotimi Fasimoye, Wentao Dong, Raja S. Nirujogi, Eshaan S. Rawat, Miharu Iguchi, Kwamina Nyame, Toan K. Phung, Enrico Bagnoli, Alan R. Prescott, Dario R Alessi (Lead / Corresponding author), Monther Abu-Remaileh (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)
63 Downloads (Pure)

Abstract

The Golgi is a membrane-bound organelle that is essential for protein and lipid biosynthesis. It represents a central trafficking hub that sorts proteins and lipids to various destinations or for secretion from the cell. The Golgi has emerged as a docking platform for cellular signaling pathways including LRRK2 kinase whose deregulation leads to Parkinson disease. Golgi dysfunction is associated with a broad spectrum of diseases including cancer, neurodegeneration, and cardiovascular diseases. To allow the study of the Golgi at high resolution, we report a rapid Golgi immunoprecipitation technique (Golgi-IP) to isolate intact Golgi mini-stacks for subsequent analysis of their content. By fusing the Golgi-resident protein TMEM115 to three tandem HA epitopes (GolgiTAG), we purified the Golgi using Golgi-IP with minimal contamination from other compartments. We then established an analysis pipeline using liquid chromatography coupled with mass spectrometry to characterize the human Golgi proteome, metabolome, and lipidome. Subcellular proteomics confirmed known Golgi proteins and identified proteins not previously associated with the Golgi. Metabolite profiling established the human Golgi metabolome and revealed the enrichment of uridine-diphosphate (UDP) sugars and their derivatives, which is consistent with their roles in protein and lipid glycosylation. Furthermore, targeted metabolomics validated SLC35A2 as the subcellular transporter for UDP-hexose. Finally, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol, and phosphatidylserine are the most abundant Golgi lipids and that glycosphingolipids are enriched in this compartment. Altogether, our work establishes a comprehensive molecular map of the human Golgi and provides a powerful method to study the Golgi with high precision in health and disease.

Original languageEnglish
Article numbere2219953120
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume120
Issue number20
Early online date8 May 2023
DOIs
Publication statusPublished - 16 May 2023

Keywords

  • Humans
  • Golgi Apparatus/metabolism
  • Chromatography, Liquid
  • Proteome/metabolism
  • Lipids
  • Uridine Diphosphate/metabolism
  • proteomics
  • metabolomics
  • organelle
  • Golgi
  • lipidomics

ASJC Scopus subject areas

  • General

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