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

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.

Parkinson’s VPS35[D620N] mutation induces LRRK2 mediated lysosomal association of RILPL1 and TMEM55B

“…1E). To investigate this further, we performed LysoTag (42) and GolgiTag (45) immunoprecipitations in parallel from littermate-matched wild type and homozygous VPS35[D620N] knock-in MEFs (Fig. 1F).…”

“…Wild type and homozygous VPS35[D620N] knock-in MEFs stably expressing the LysoTag (42) or GolgiTag (45) were generated using the protocol described in dx.doi.org/10.17504/protocols.io.6qpvr456bgmk/v1. Briefly, littermate matched primary MEFs were first immortalized by SV40 Large T Antigen viral transduction prior to the stable expression of target proteins (LysoTag, GolgiTag or HA-Empty (vector only encoding a HA tag) by a subsequent viral transduction.…”

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

“…Multiple studies have established that whole-organelle immunoprecipitation (IP) enables the analysis of native organellar proteomes, metabolomes and lipidomes by LC-MS 19–24 . In a native workflow, cells are mechanically homogenized under gentle conditions, exposing organelles that maintain their structural integrity and internal composition 20 .…”

“…In a native workflow, cells are mechanically homogenized under gentle conditions, exposing organelles that maintain their structural integrity and internal composition 20 . So far, pioneering studies have each primarily focused on a single membrane-bound organelle including mitochondrion 19,20 , lysosome 21 , peroxisome 22 , early-endosome 23 and Golgi complex 24 . We reasoned that this strategy could be globally applied to all the different compartments of a given cell type to obtain a comprehensive map of the subcellular human proteome (Figures 1A-D).…”

“…Local protein neighborhoods can be defined by proximity ligation using organelle-specific markers tagged with labeling enzymes such as APEX 15 or BioID 16–18 . The protein composition of entire cellular compartments can be further measured following immunoprecipitation 19–24 , biochemical fractionation or sequential solubilization methods 25,26 . Fractionation can be used to purify a specific organelle for in-depth analysis 27 , or to characterize all cellular compartments from a given sample using protein correlation profiling 28–35 .…”