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

ABSTRACT: The Golgi plays an essential role in protein and lipid glycosylation. Golgi dysfunction is associated with a variety of diseases including cancer, neurodegeneration and cardiovascular disease. We developed a powerful immunoprecipitation technique to isolate the Golgi by tagging TMEM115. Golgi-immunoprecipitation enriched protein markers specific for the organelle with minimal contamination from other subcellular compartments. Using this technique, we characterised the Golgi proteome, metabolome and lipidome. Proteomics analysis confirmed capture of known Golgi proteins and identified additional candidates enriched in the organelle. Metabolomics analysis revealed selective enrichment of uridine-diphosphate (UDP) sugars and their derivatives and validated SLC35A2 as the subcellular transporter for UDP-hexose. Lastly, lipidomics analysis showed that phospholipids including phosphatidylcholine, phosphatidylinositol and phosphatidylserine are the top enriched Golgi lipids. Our Golgi-IP workflow provides a paradigm for multimodal analysis of Golgi molecular content with enhanced subcellular precision.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Kidney

DISEASE(S): Parkinson's Disease

SUBMITTER: Raja Sekhar Nirujogi

LAB HEAD: Dario R Alessi

PROVIDER: PXD038046 | Pride | 2023-04-24

REPOSITORIES: Pride

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			Action	DRS
	20201013_013830_GolgiPool_DDA.kit	Other
	GT-DDA_dDIA_SPlib_Nov2021_28F10E6F534820211127_160542.psar	Other
	Golgi-IP.sne	Other
	Golgi-IP_DDA_Phospho__STY_Sites.txt	Txt
	Golgi-IP_DDA_allPeptides.txt	Txt

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

Project description: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.

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