Biology subjects

Cattin-Ortola, J.

Publications and source records attributed to Cattin-Ortola, J..

8 recordsLinked to original sources

bioRxiv2024

RABGAP1 acts as a sensor to facilitate sorting and processing of amyloid precursor protein

A key hallmark of Alzheimers disease (AD) is the accumulation of extracellular amyloid-{beta} plaques in the brains of patients. Amyloid-{beta} is a 40-42 amino acid peptide produced by the proteolytic processing of amyloid precursor protein (APP) by a series of membrane-bound proteases. APP is a type-I transmembrane protein and thus its trafficking to encounter the proteases represents a rate-limiting step in the progression of AD. Although there has been a focused research effort to understand APP processing, its trafficking itinerary and machinery is incompletely understood. To address this we have performed an unbiased interaction screen for interactors of the cytosolic tail of APP. We identified previously characterised APP binders, as well as novel interactors. We have mapped the binding of APP to multiple new machineries, including RABGAP1. We have demonstrated that RAB-GAP1 partially co-localises with APP and directly interacts with a YENPTY motif in the APP cytosolic tail. Depletion or overexpression of RABGAP1 caused mistrafficking and misprocessing of endogenous APP in both human and rodent neurons. Interestingly, this effect was dependent on the GAP activity of RABGAP1, demonstrating that RABGAP1 affects the trafficking of APP by modulating the RAB activity on endosomal subdomains. This novel trafficking mechanism has implications for other NPXY cargoes and also presents a possible therapeutic avenue to explore.

cell biology↗

bioRxiv2023

Cargo selective vesicle tethering: the structural basis for binding of specific cargo proteins by the Golgi tether component TBC1D23

For accurate membrane traffic it is essential that vesicles and other carriers tether and fuse to only the correct compartment. The TGN-localised golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23 that forms a bridge between the golgins and endosome-derived vesicles. The C-terminal domain of TBC1D23 is responsible for vesicle capture, but how it recognises a specific type of vesicle was unclear. A search for binding partners of the C-terminal domain surprisingly revealed direct binding to carboxypeptidase D (CPD) and syntaxin-16, both known cargo proteins of the captured vesicles. Binding is via a TLY-containing sequence present in both proteins. A crystal structure reveals how this "acidic TLY motif" binds to the C-terminal domain of TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the C-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle. One sentence summaryA class of transport vesicle destined for the Golgi is recognized by a tether binding directly to the cargo it is carrying.

cell biology↗

bioRxiv2020

Sequences in the cytoplasmic tail of SARS-CoV-2 spike facilitate syncytia formation

The Spike (S) protein of SARS-CoV-2 binds ACE2 to direct fusion with host cells. S comprises a large external domain, a transmembrane domain (TMD) and a short cytoplasmic tail. Understanding the intracellular trafficking of S is relevant to SARS-CoV-2 infection, and to vaccines expressing full-length S from mRNA or adenovirus vectors. We have applied proteomics to identify cellular factors that interact with the cytoplasmic tail of S. We confirmed interactions with the COPI and COPII vesicle coats, ERM family actin regulators, and the WIPI3 autophagy component. The COPII binding-site promotes exit from the endoplasmic reticulum (ER), and although COPI-binding should retain S in the early Golgi where viral budding occurs, there is a suboptimal histidine residue in the recognition motif. As a result, S leaks to the surface where it accumulates and can direct the formation of multinucleate syncytia. Thus, the trafficking signals in the tail of S indicate that syncytia play a role in the SARS-CoV-2 lifecycle.

cell biology↗

bioRxiv2020

Furin cleavage of SARS-CoV-2 Spike promotes but is not essential for infection and cell-cell fusion

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells by binding to the host cell receptor Ace2 and undergoing virus-host membrane fusion. Fusion is triggered by the protease TMPRSS2, which processes the viral Spike (S) protein to reveal the fusion peptide. SARS-CoV-2 has evolved a multibasic site at the S1-S2 boundary, which is thought to be cleaved by furin in order to prime S protein for TMPRSS2 processing. Here we show that CRISPR-Cas9 knockout of furin reduces, but does not prevent, the production of infectious SARS-CoV-2 virus. Comparing S processing in furin knockout cells to multibasic site mutants reveals that while loss of furin substantially reduces S1-S2 cleavage it does not prevent it. SARS-CoV-2 S protein also mediates cell-cell fusion, potentially allowing virus to spread virion-independently. We show that loss of furin in either donor or acceptor cells reduces, but does not prevent, TMPRSS2-dependent cell-cell fusion, unlike mutation of the multibasic site that completely prevents syncytia formation. Our results show that while furin promotes both SARS-CoV-2 infectivity and cell-cell spread it is not essential, suggesting furin inhibitors will not prevent viral spread.

microbiology↗

bioRxiv2019

Determining the content of vesicles captured by golgin tethers using LOPIT-DC

The internal organisation of the cell depends on tethers at destination organelles to selectively capture incoming transport vesicles to facilitate SNARE-mediated fusion. The golgin long coiled-coil proteins function as tethers that contributes to this specificity at the Golgi (1). Golgin-97, golgin-245 and GCC88 golgins of the trans-Golgi capture vesicles derived from endosomes, which serve to recycle the critical Golgi machinery required to deliver lysosomal hydrolases and to maintain exocytosis. Retrograde trafficking from endosomes to the trans-Golgi network (TGN) is a complex process that involves the sorting of transmembrane cargo proteins into distinct transport vesicles by adaptors from multiple pathways. The content of these distinct vesicles, which golgin they target and the factors that mediate this targeting are not well understood. The major challenges that have limited advances in these areas is the transient nature of vesicle tethering, and the redundancies in their mechanisms that confound experimental dissection. To gain better insight into these problems, we performed organelle proteomics using the Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) method on a system in which an ectopic golgin causes vesicles to accumulate in a tethered state (2). By incorporating Bayesian statistical modelling into our analysis (3), we determined that over 45 transmembrane proteins and 51 peripheral membrane proteins of the endosomal network are on vesicles captured by golgin-97, including known cargo and components of the clathrin/AP-1, retromer-dependent and -independent transport pathways. We also determined a distinct class of vesicles shared by golgin-97, golgin-245 and GCC88 that is enriched in TMEM87A, a multi-pass transmembrane protein of unknown function that has previously been implicated in endosome-to-Golgi retrograde transport (4). Finally, we categorically demonstrate that the vesicles that these golgins capture are retrograde transport vesicles based on the lack of enrichment of lysosomal hydrolases in our LOPIT-DC data, and from correlative light electron tomography images of spherical vesicles captured by golgin-97. Together, our study demonstrates the power of combining LOPIT-DC with Bayesian statistical analysis in interrogating the dynamic spatial movement of proteins in transport vesicles.

cell biology↗

bioRxiv2019

CCDC186 recruits EARP and controls dense-core vesicle cargo sorting by exit

The regulated release of peptide hormones depends on their packaging into dense-core vesicles (DCVs). Two models have been proposed for DCV cargo sorting. The \"sorting by entry\" model proposes that DCV cargos selectively enter nascent DCVs at the trans-Golgi network (TGN). The \"sorting by exit\" model proposes that sorting occurs by the post-TGN removal of non-DCV cargos and retention of mature DCV cargos. Here we show that the coiled-coil protein CCDC186 controls sorting by exit. Ccdc186 KO insulinoma cells secrete less insulin, fail to retain insulin and carboxypeptidase E in mature DCVs at the cell periphery, and fail to remove carboxypeptidase D from immature DCVs. A mutation affecting the endosome-associated recycling protein (EARP) complex causes similar defects in DCV cargo retention and removal. CCDC186 and EARP may act together to control the post-Golgi retention of cargos in mature DCVs.

cell biology↗

bioRxiv2018

EIPR1 controls dense-core vesicle cargo sorting and EARP complex localization in insulinoma cells

Dense-core vesicles (DCVs) are secretory vesicles found in neurons and endocrine cells. DCVs package and release cargos including neuropeptides, biogenic amines, and peptide hormones. We recently identified the endosome-associated recycling protein (EARP) complex and the EARP-interacting protein EIPR-1 as proteins important for controlling levels of DCV cargos in C. elegans neurons. Here we determine the role of mammalian EIPR1 in insulinoma cells. We find that in Eipr1 KO cells, there is reduced insulin secretion, and mature DCV cargos such as insulin and carboxypeptidase E (CPE) accumulate near the trans-Golgi network and are not retained in mature DCVs in the cell periphery. In addition, we find that EIPR1 is required for the stability of the EARP complex subunits and for the localization of EARP and its association with membranes, but EIPR1 does not affect localization or function of the related Golgi-associated retrograde protein (GARP) complex. EARP is localized to two distinct compartments related to its function: an endosomal compartment and a DCV biogenesis-related compartment. We propose that EIPR1 functions with EARP to control both endocytic recycling and DCV maturation.

cell biology↗

bioRxiv2017

The Dense-Core Vesicle Maturation Protein CCCP-1 Binds both RAB-2 and Membranes through a Conserved C-terminal Coiled-coil Domain

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.\n\nSynopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

biochemistry↗