Biology subjects

Jackson, L. P.

Publications and source records attributed to Jackson, L. P..

9 recordsLinked to original sources

bioRxiv2024

VARP binds SNX27 to promote endosomal supercomplex formation on membranes

Multiple essential membrane trafficking pathways converge at endosomes to maintain cellular homeostasis by sorting critical transmembrane cargo proteins to the plasma membrane or the trans-Golgi network (TGN). The Retromer heterotrimer (VPS26/VPS35/VPS29 subunits) binds multiple sorting nexin (SNX) proteins on endosomal membranes, but molecular mechanisms regarding formation and regulation of metazoan SNX/Retromer complexes have been elusive. Here, we combine biochemical and biophysical approaches with AlphaFold2 Multimer modeling to identify a direct interaction between the VARP N-terminus and SNX27 PDZ domain. VARP and SNX27 interact with high nanomolar affinity using the binding pocket established for PDZ binding motif (PDZbm) cargo. Specific point mutations in VARP abrogate the interaction in vitro. We further establish a full biochemical reconstitution system using purified mammalian proteins to directly and systematically test whether multiple endosomal coat complexes are recruited to membranes to generate tubules. We successfully use purified coat components to demonstrate which combinations of Retromer with SNX27, ESCPE-1 (SNX2/SNX6), or both complexes can remodel membranes containing physiological cargo motifs and phospholipid composition. SNX27, alone and with Retromer, induces tubule formation in the presence of PI(3)P and PDZ cargo motifs. ESCPE-1 deforms membranes enriched with Folch I and CI-MPR cargo motifs, but surprisingly does not recruit Retromer. Finally, we find VARP is required to reconstitute a proposed endosomal "supercomplex" containing SNX27, ESCPE-1, and Retromer on PI(3)P-enriched membranes. These data suggest VARP functions as a key regulator in metazoans to promote cargo sorting out of endosomes.

biochemistry↗

bioRxiv2023

Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure.

cell biology↗

bioRxiv2022

An interaction between betaprime-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling

The essential COPI vesicular coat mediates retrieval of key transmembrane proteins at the Golgi and endosomes following recruitment by the small GTPase, Arf1. ArfGAP proteins regulate COPI coats, but molecular details for COPI recognition by ArfGAP proteins remain elusive. Biochemical and biophysical data reveal how {beta}-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity (KD ~1 {micro}M). Calorimetry data demonstrate both {beta}-COP propeller domains are required to bind Glo3 using electrostatic interactions. An acidic patch on {beta}-COP (D437/D450) interacts with Glo3 lysine residues located within the BoCCS (Binding of Coatomer, Cargo, and SNAREs) region. Targeted point mutations in either Glo3 BoCCS or {beta}-COP abrogate the interaction in vitro, and loss of the {beta}-COP/Glo3 interaction drives Ste2 mis-sorting to the vacuole and aberrant Golgi morphology in budding yeast. Together, these data suggest cells require the {beta}-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where {beta}-COP may serve as a molecular platform to coordinate binding to multiple protein partners, including Glo3, Arf1, and the COPI F-subcomplex.

cell biology↗

bioRxiv2022

Improved mammalian retromer cryo-EM structures reveal a new assembly interface

Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin (SNX) proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in other cellular events, including mitochondrial homeostasis, nutrient sensing, autophagy, and fission events. Mechanisms for mammalian retromer assembly remain undefined, and retromer engages multiple sorting nexin proteins to sort cargoes to different destinations. Published structures demonstrate mammalian retromer forms oligomers in vitro, but several structures were poorly resolved. We report here improved retromer oligomer structures using single particle cryo-electron microscopy (cryo-EM) by combining data collected from tilted specimens with multiple improvements in data processing, including using a three-dimensional (3D) starting model for improved automated particle picking in RELION. A retromer mutant (3KE retromer) that breaks VPS35-mediated interfaces was used to determine a structure of a new assembly interface formed by the VPS26A and VPS35 N-termini. The interface reveals how an N-terminal VPS26A arrestin saddle can link retromer chains by engaging a neighboring VPS35 N-terminus, on the opposite side from the well-characterized C-VPS26/N-VPS35 interaction observed within heterotrimers. The new interaction interface exhibits substantial buried surface area ([~]7,000 [A]2) and further suggests metazoan retromer may serve as an adaptable scaffold.

biochemistry↗

bioRxiv2021

Ubiquitination drives COPI priming and Golgi SNARE localization

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime COPI assembly to ensure incorporation of these essential cargoes into vesicles but the regulation of these events is poorly understood. Here we report a roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs. The ability of COPI to bind ubiquitin through its N-terminal WD repeat domain of {beta}COP or through an unrelated ubiquitin-binding domain (UBD) is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3 and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is thought to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

cell biology↗

bioRxiv2020

De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinsons disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.

biochemistry↗

bioRxiv2019

Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes

In metazoans, retromer (VPS26/VPS35/VPS29) associates with sorting nexin (SNX) proteins to form coats on endosomal tubules and sort cargo proteins to the trans-Golgi network (TGN) or plasma membrane. This core complex is highly conserved from yeast to humans, but molecular mechanisms of metazoan retromer assembly remain undefined. Here we combine single particle cryo-electron microscopy with biophysical methods to uncover multiple oligomer structures formed by mammalian retromer. Two-dimensional class averages in ice reveal the retromer heterotrimer; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical studies in solution. We provide cryo-EM reconstructions of all species, including pseudo-atomic resolution detail for key sub-structures. Multi-body refinement demonstrates how retromer heterotrimers and dimers adopt a range of conformations. Our structures identify a flexible yet highly conserved electrostatic interface in dimers formed by interactions between VPS35 subunits. We generate a structure-based mutant to disrupt this key interface in vitro and introduce equivalent mutations into S. cerevisiae to demonstrate the mutant exhibits a cargo sorting defect. Together, structures and complementary functional data in budding yeast imply a conserved assembly interface across eukaryotes. These data further suggest mammalian retromer acts as an adaptable and plastic scaffold that accommodates interactions with different SNXs to sort multiple cargoes from endosomes their final destinations.

cell biology↗

bioRxiv2017

AP-4 vesicles unmasked by organellar proteomics to reveal their cargo and machinery

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including Dynamic Organellar Maps, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the \"ATG9A reservoir\" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

cell biology↗

bioRxiv2017

COPI Mediates Recycling Of An Exocytic SNARE From Endosomes By Recognition Of A Ubiquitin Sorting Signal

ABSTRACTThe COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI mediates delivery of a budding yeast SNARE (Snc1) from early endosomes to the Golgi complex through recognition of a polyubiquitin sorting signal. Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through early endosomes back to the Golgi for reuse. Removal of ubiquitin from Snc1, or deletion of a {beta}-COP subunit propeller domain that binds K63-linked polyubiquitin, causes aberrant accumulation of Snc1 in early endosomes. Moreover, replacement of the {beta}-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles at early endosomes for Golgi delivery.

cell biology↗