Spatial proteomics defines the content of trafficking vesicles captured by golgin tethers

Abstract

Intracellular traffic between compartments of the secretory and endocytic pathways is mediated by vesicle-based carriers. The proteomes of carriers destined for many organelles are ill-defined because the vesicular intermediates are transient, low-abundance and difficult to purify. Here, we combine vesicle relocalisation with organelle proteomics and Bayesian analysis to define the content of different endosome-derived vesicles destined for the trans-Golgi network (TGN). The golgin coiled-coil proteins golgin-97 and GCC88, shown previously to capture endosome-derived vesicles at the TGN, were individually relocalised to mitochondria and the content of the subsequently re-routed vesicles was determined by organelle proteomics. Our findings reveal 45 integral and 51 peripheral membrane proteins re-routed by golgin-97, evidence for a distinct class of vesicles shared by golgin-97 and GCC88, and various cargoes specific to individual golgins. These results illustrate a general strategy for analysing intracellular sub-proteomes by combining acute cellular re-wiring with high-resolution spatial proteomics.

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line

“…13,14 ). The hyperLOPIT platform and its derivative LOPIT-DC have now been applied to multiple biological contexts and questions 21,22,24,25,27,28 . We present here the first iteration of dynamic-hyperLOPIT for characterisation of protein relocalisation events during cellular perturbation using the THP-1 monocytic cell line which is notoriously difficult to use for microscopy imaging.…”

“…These methods provide a global overview of a cell population by accurately pinpointing thousands of proteins to distinct subcellular niches, enabling molecular annotation of whole organelles without the need for organelle purification [17][18][19] . One of the forerunners in this field is LOPIT-Localisation of Organelle Proteins by Isotope Tagging 20 , and its more recent iterations, hyperLOPIT (hyperplexed LOPIT) [21][22][23][24][25][26][27] and LOPIT-DC (LOPIT after Differential ultraCentrifugation) 24,28 . The hyperLOPIT method combines extensive biochemical cell fractionation with multiplexed high-resolution MS-based proteomics for the simultaneous analysis of the steady-state distribution of thousands of native proteins within a sample.…”

Combinatorial deployment of F-actin regulators to build complex 3D actin structures in vivo

“…Although, as discussed above, the ANRPs have been implicated in several different mechanisms of actin-regulation, many of the ANRPs have been shown to either activate or stabilize Arp2/3 complex function ( Uruno et al, 2001 ; Weaver et al, 2002 ; Uetrecht and Bear, 2006 ; Pollitt and Insall, 2009 ; Bhattacharya et al, 2016 ). We therefore adapted a mitochondrial-tagging assay ( Shin et al, 2020 ) to recruit ANRPs to the mitochondria and then tested the degree to which Arp3 and F-actin become ectopically localized. Since DPod1, Cortactin, Coronin, and Scar had the strongest effects on actin cap formation, we fused each of these ANRPs to mCherry and an outer mitochondrial membrane mito-tag (Tom70-HA, 58 amino acids).…”

Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE

“…In addition, CRISPR-Cas9 knockouts coupled with spatial proteomics has given insights into AP-4 vesicles (Davies et al ., 2018), as well as AP-5 cargo (Hirst et al ., 2018). In a study by Shin et al . (2020), the golgin long coiled-coil proteins that selectively capture vesicles destined for the Golgi were re-located to the mitochondria by replacing their Golgi targeting domains with a mitochondrial transmembrane domain (Shin et al ., 2020).…”

“…In a study by Shin et al . (2020), the golgin long coiled-coil proteins that selectively capture vesicles destined for the Golgi were re-located to the mitochondria by replacing their Golgi targeting domains with a mitochondrial transmembrane domain (Shin et al ., 2020). This allowed the authors to readily observe the vesicle cargo and regulatory proteins that are redirected to the mitochondria, whilst avoiding technical issues that arise because of the redundancy of the gol-gins and their transient interaction with vesicles.…”