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The ADP ribosylation factors interactome (ARFome) in human cell lines

ABSTRACT: We performed a 24h proximity labeling (BioID) of 28 constitutively active human ARFs to define the global ARFome, captured in native cellular micro-environments. This approach allowed us to reveal the landscape of ARF effectors and to define the specificity of ARF GAPs. Because our BioID provides a 24h history of protein interactions, we interrogated the repertoire of candidate localizations of these proteins to localize the ARFs functions at better resolution. Finally, we discovered a potentially new coat complex recruited via the newly identified PLD1 activator ARL14 on endosomes for retrograde traffic.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jean-Francois Cote

PROVIDER: MSV000090627 | MassIVE | Tue Nov 01 13:12:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and gene ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first quantitative proteomic analysis of signaling endosomes isolated from motor neurons and allows the assembly of a functional map of these axonal carriers involved in long-range neuronal signaling.

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The ADP ribosylation factors interactome (ARFome) in human cell lines

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