Biology subjects

Munro, S.

Publications and source records attributed to Munro, S..

13 recordsLinked to original sources

bioRxiv2026

The rhomboid-like pseudoprotease TMEM115 defines a two-factor mechanism for Rab6A effector recruitment

Protein trafficking and organelle identity depends on precise localisation of membrane proteins, anchored by small GTPases that bind to specific cellular compartments. Here we show that the rhomboid-like pseudoprotease TMEM115 defines an unexpected two-factor system with Rab6A to target the golgin TMF1. In the absence of TMEM115, TMF1 is delocalised from the rim of the Golgi apparatus; instead, it is retained in Golgi-targeting vesicles. TMEM115, TMF1, and Rab6A assemble into a ternary complex which, in striking contrast to canonical rhomboid-like mechanisms, is independent of transmembrane domain interactions. Disruption of the TMEM115-Rab6A-TMF1 complex affects the efficiency of trafficking of proteins from the Golgi; moreover, human disease-associated mutations target the interfaces of this ternary complex, thereby delocalising TMF1 from the Golgi. Finally, loss of TMEM115 in Drosophila causes abnormal Golgi morphology, and mouse knockouts undergo perinatal death. We propose that two-factor effector recruitment defines a mechanism for precise Rab GTPase-mediated membrane targeting.

cell biology↗

bioRxiv2025

The Golgi vesicle tether p115/USO1 can bind directly to the ER exit site organiser Sec16A

Newly-made secretory and membrane proteins exit the endoplasmic reticulum (ER) in COPII vesicles that form at specialised ER exit sites. These exit sites are typically near to the early Golgi compartments that receive these vesicles. A key player in the delivery of vesicles to the early Golgi is p115 (USO1), a homodimer with a folded head domain and a coiled-coil tail that is anchored to Golgi membranes. p115 has been shown to capture vesicles and to bind to SNARE proteins to promote membrane fusion. Here we report that the head domain of human p115 can bind directly to Sec16A, a large scaffolding protein that organises ER sites and promotes COPII vesicle formation. Structural prediction and deletion mapping define the region of interaction to a conserved motif in the unstructured N-terminal region of Sec16A, and mutations in p115 that block motif binding reduce the efficiency of secretion. This interaction could potentially allow a subset of p115 molecules to reach across from the early Golgi to the ER exit sites to contribute to the large-scale organisation of the early secretory pathway.

cell biology↗

bioRxiv2025

The mechanistic basis of cargo selection during Golgi maturation

The multiple cisternae of the Golgi apparatus contain resident membrane proteins crucial for lipid and protein glycosylation. How Golgi residents remain in their designated compartments despite a constant flow of secretory cargo is incompletely understood. Here, we determined the structure of the COPI vesicle coat containing GOLPH3, an adaptor protein that binds the cytosolic tails of many Golgi residents. The structure, together with structure-guided mutagenesis and functional assays, reveals how GOLPH3 uses coincidence detection of COPI and lipid to engage Golgi residents preferentially at late cisternae. Our findings rationalize the logic of cisternal maturation and explain how COPI can engage different types of substrates in different Golgi cisternae to retrieve some proteins back to the ER while retaining others within the Golgi apparatus.

cell biology↗

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↗

bioRxiv2024

The FAM114A proteins are adaptors for the recycling of Golgi enzymes

The Golgi apparatus contains many resident enzymes that must remain in place whilst their substrates flow through on their journey from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the rims of the Golgi stack to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and at least one COPI adaptor protein, GOLPH3, has been shown to recruit enzymes into vesicles in a specific part of the stack. We have used proximity biotinylation to identify further components of intra-Golgi transport vesicles and found FAM114A2, an uncharacterised cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1 showed that they bind to numerous Golgi resident proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not result in substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Generation of Drosophila mutants lacking FAM114A revealed defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins are COPI vesicle resident proteins that bind to Golgi enzymes and so are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

cell biology↗

bioRxiv2024

Tracking exocytic vesicle movements with MSP-tracker/viewer reveals the spatial control of secretion in epithelial cells.

Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analysing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1{micro}m/sec. This is reduced to 0.85 {micro}m/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerisation cause lateral Cad99C secretion. Thus, microtubule organisation plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarised transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organised than previously thought.

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↗

bioRxiv2022

Functional unknomics: closing the knowledge gap to accelerate biomedical research

The human genome encodes [~]20,000 proteins, many still uncharacterised. Scientific and social factors have resulted in a focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected. To address this, we have developed an "Unknome database" that ranks proteins based on how little is known about them. We applied RNAi in Drosophila to 260 unknown genes that are conserved between flies and humans. About a quarter are required for viability, and functional screening of the rest revealed hits for fertility, development, locomotion, protein quality control and resilience to stress. CRISPR/Cas9 gene disruption validated a component of Notch signalling and two genes contributing to male fertility. Our work demonstrates the importance of poorly understood genes, provides a resource for future research acceleration, and highlights a need for our awareness of ignorance to be protected from erosion by automated database annotation.

molecular biology↗

bioRxiv2021

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

cell biology↗

bioRxiv2020

A cryo-EM structure of metazoan TRAPPIII, the multisubunit complex that activates the GTPase Rab1

The TRAPP complexes are highly conserved nucleotide exchange factors, with TRAPPIII activating Rab1 and TRAPPII acting primarily on Rab11. The two complexes share a core of small subunits that affect nucleotide exchange, but are distinguished by additional large subunits that are essential for activity in vivo, and are mutated in a range of human disorders. Crystal structures of the core subunits have revealed the mechanism of Rab activation, but how and why the large subunits associate with the core remains unclear. We report here a cryo-EM structure of the entire TRAPPIII complex from Drosophila. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact bound Rab1, indicating how the arms could alter the Rab specificity of the core. A lower resolution structure of TRAPPII shows a similar architecture, and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

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↗

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

In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5 and Rab9. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

cell biology↗