Mechanistic basis for SNX27-Retromer coupling to ESCPE-1 in promoting endosomal cargo recycling

Simonetti, Boris; Guo, Qian; Giménez-Andrés, Manuel; Chen, Kai‐En; Moody, Edmund R.R.; Evans, Ashley J.; Danson, Chris M.; Williams, Tom A.; Collins, Brett M.; Cullen, Peter J.

doi:10.1101/2021.08.28.457928

Cited by 4 publications

(4 citation statements)

References 87 publications

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“…Despite attempts to crystallise Munc18-1 in complex with various Mint1 peptides we were unable determine a high-resolution structure of this complex, including with stabilized Munc18-1 variants (Peleg et al, 2021). The machine-learning structure-prediction algorithm AlphaFold2 (Evans et al, 2022; Jumper et al, 2021) has been successfully used to predict structures of protein-peptide complexes by ourselves and others (Ko and Lee, 2021; Poetz et al, 2021; Simonetti et al, 2021; Tsaban et al, 2021). We performed a series of modelling experiments to map the complex between Munc18-1 and Mint1 using the ColabFold implementation of AlphaFold2 (Mirdita et al, 2022).…”

Section: Resultsmentioning

confidence: 99%

“…The machine-learning structure-prediction algorithm AlphaFold2 (Evans et al , 2022; Jumper et al , 2021) has been successfully used to predict structures of protein-peptide complexes by ourselves and others (Ko & Lee, 2021; Poetz et al , 2021; Simonetti et al , 2021; Tsaban et al , 2021). We began our analyses of Mint interactions with a series of modelling experiments to map the complex between Munc18-1 and Mint1 using the ColabFold implementation of AlphaFold2 (Mirdita et al , 2022).…”

Section: Resultsmentioning

confidence: 99%

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Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Weeratunga

Gormal

Liu³

et al. 2023

Preprint

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Munc18-interacting proteins (Mints) are multi-domain adaptors that regulate neuronal membrane trafficking, signalling and neurotransmission. Mint1 and Mint2 are highly expressed in the brain and play overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. The mechanism by which Mint1 mediates the interaction of Munc18-1 with Syntaxin1a (Sx1a) to control formation of the fusogenic SNARE complex and facilitate neurotransmission remains unclear. Here, we have studied both the specific interactions of the neuronal Mint proteins with Munc18-1 as well as their wider interactome. Using biochemical and biophysical binding approaches we identify a short acidic [alpha];-helical motif (AHM) within Mint1 and Mint2 that is necessary and sufficient for specific binding to Munc18-1. In silico modelling of this interaction with AlphaFold2 and extensive validation by structure-based mutagenesis in vitro, showed that the AHM in Mint forms an [alpha];-helical structure, binds a conserved surface on Munc18-1 domain3b. Although Munc18-1 can interact with Mint and Sx1a proteins simultaneously we find they have mutually reduced affinities, suggesting an allosteric coupling between the two proteins. Finally, using AlphaFold2 we probed the wider network of interactions governed by the Mint scaffolds, and provide a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ARF3/ARF4 small GTPases, neurexins, and the AP3 clathrin adaptor complex. Overall, our data provides insights into the diversity of interactions mediated by the Mint family and suggests that Mints may help to control a key trigger point in SNARE complex assembly and vesicle fusion.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Weeratunga

Gormal

Liu³

et al. 2023

Preprint

Self Cite

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“…Rab and Arf family proteins are marked in blue, SNXs in black, and cargo molecules in green. Based on references [ 43 , 44 , 46 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 ].…”

Section: Figurementioning

confidence: 99%

Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions

Lučin

Zagorac

Marcelić

et al. 2022

IJMS

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Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captured along with the capsids and incorporated into the virions as host cell signatures. In this report, we reviewed the existing information on host cell signatures in human cytomegalovirus (HCMV) virions. We analyzed the published proteomes of the HCMV virion preparations that identified a large number of host cell proteins. Virion purification methods are not yet advanced enough to separate all of the components of the rich extracellular material, including the large amounts of non-vesicular extracellular particles (NVEPs). Therefore, we used the proteomic data from large and small extracellular vesicles (lEVs and sEVs) and NVEPs to filter out the host cell proteins identified in the viral proteomes. Using these filters, we were able to narrow down the analysis of the host cell signatures within the virions and determine that envelopment likely occurs at the membranes derived from the tubular recycling endosomes. Many of these signatures were also found at the autophagosomes, suggesting that the CMV-infected cell forms membrane organelles with phagophore growth properties using early endosomal host cell machinery that coordinates endosomal recycling.

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“…Such functional interaction relies on SNX27-mediated binding of the disordered amino-termini of the SNX1/2 subunits. This event ensures cargo protein retrieval from lysosomal degradation by SNX27-Retromer into ESCPE-1 tubules [88,98,99].…”

Section: Escpe-1mentioning

confidence: 99%

Deliver on Time or Pay the Fine: Scheduling in Membrane Trafficking

Placidi

Campa

2021

IJMS

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Membrane trafficking is all about time. Automation in such a biological process is crucial to ensure management and delivery of cellular cargoes with spatiotemporal precision. Shared molecular regulators and differential engagement of trafficking components improve robustness of molecular sorting. Sequential recruitment of low affinity protein complexes ensures directionality of the process and, concomitantly, serves as a kinetic proofreading mechanism to discriminate cargoes from the whole endocytosed material. This strategy helps cells to minimize losses and operating errors in membrane trafficking, thereby matching the appealed deadline. Here, we summarize the molecular pathways of molecular sorting, focusing on their timing and efficacy. We also highlight experimental procedures and genetic approaches to robustly probe these pathways, in order to guide mechanistic studies at the interface between biochemistry and quantitative biology.

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Mechanistic basis for SNX27-Retromer coupling to ESCPE-1 in promoting endosomal cargo recycling

Cited by 4 publications

References 87 publications

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Interrogation and validation of the interactome of neuronal Munc18-interacting Mint proteins with AlphaFold2

Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions

Deliver on Time or Pay the Fine: Scheduling in Membrane Trafficking

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