Golgi-IP, a novel 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 signalling 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 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 novel ones. Metabolite profiling established the first known human Golgi metabolome and revealed the selective 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 states.

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

“…1D). To investigate this further, we performed LysoTag ( 43 ) and GolgiTag ( 47 ) immunoprecipitations in parallel from littermate-matched wild type and homozygous VPS35[D620N] knock-in MEFs (Fig. 1E).…”

“…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.…”

VPS13B is localized at the cis-trans Golgi complex interface and is a functional partner of FAM177A1

“…We demonstrate that loss of VPS13B in HeLa cells delays Golgi complex recovery after its Brefeldin A (BFA)-induced dispersion, raising the possibility that VPS13B’s lipid transfer function may have a role in Golgi complex reformation. We further provide evidence in mammalian cells and in zebrafish for a functional partnership between VPS13B and FAM177A1, a recently identified Golgi complex protein (Fasimoye et al, 2023; Hickey et al, 2023)(Legro et al, in revision) of unknown function implicated in a human neurodevelopmental disorder (Alazami et al, 2015)(Legro et al, in revision).…”

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

“…1D). To investigate this further, we performed LysoTag (43) and GolgiTag (47) IPs in parallel from littermatematched WT and homozygous VPS35[D620N] knock-in MEFs (Fig. 1E).…”

“…WT and homozygous VPS35[D620N] knock-in MEFs stably expressing the LysoTag (43) or GolgiTag (47) were generated using the protocol described in (66). Briefly, littermate-matched primary MEFs were first immortalized by SV40 large T antigen viral transduction before the stable expression of target proteins [LysoTag, GolgiTag, or HA-Empty (vector only encoding a HA tag)] by a subsequent viral transduction.…”