Tepsin binds LC3B to promote ATG9A trafficking and delivery.

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 preferentially over 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. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

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 L C3-Interacting R egion (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.

An interaction between ß'-COP and the ArfGAP, Glo3, maintains post-Golgi cargo recycling.

The essential COPI coat mediates retrieval of 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 ArfGAPs remain elusive. Biochemical and biophysical data reveal how ß'-COP propeller domains directly engage the yeast ArfGAP, Glo3, with a low micromolar binding affinity. Calorimetry data demonstrate that both ß'-COP propeller domains are required to bind Glo3. An acidic patch on ß'-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 ß'-COP abrogate the interaction in vitro, and loss of the ß'-COP/Glo3 interaction drives Ste2 missorting to the vacuole and aberrant Golgi morphology in budding yeast. These data suggest that cells require the ß'-COP/Glo3 interaction for cargo recycling via endosomes and the TGN, where ß'-COP serves as a molecular platform to coordinate binding to multiple proteins, including Glo3, Arf1, and the COPI F-subcomplex.

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

Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in other cellular processes, 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-EM by combining data collected from tilted specimens with multiple advancements in data processing, including using a 3D starting model for enhanced automated particle picking in RELION. We used a retromer mutant (3KE retromer) that breaks VPS35-mediated interfaces 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 (∼7000 Å2) and further suggests that metazoan retromer may serve as an adaptable scaffold.

De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex.

The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson's 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 that most of the cyclic peptides bind to Vps29 via a Pro-Leu­containing 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. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

Toward Understanding the Molecular Role of SNX27/Retromer in Human Health and Disease.

Aberrations in membrane trafficking pathways have profound effects in cellular dynamics of cellular sorting processes and can drive severe physiological outcomes. Sorting nexin 27 (SNX27) is a metazoan-specific sorting nexin protein from the PX-FERM domain family and is required for endosomal recycling of many important transmembrane receptors. Multiple studies have shown SNX27-mediated recycling requires association with retromer, one of the best-known regulators of endosomal trafficking. SNX27/retromer downregulation is strongly linked to Down's Syndrome (DS) via glutamate receptor dysfunction and to Alzheimer's Disease (AD) through increased intracellular production of amyloid peptides from amyloid precursor protein (APP) breakdown. SNX27 is further linked to addiction via its role in potassium channel trafficking, and its over-expression is linked to tumorigenesis, cancer progression, and metastasis. Thus, the correct sorting of multiple receptors by SNX27/retromer is vital for normal cellular function to prevent human diseases. The role of SNX27 in regulating cargo recycling from endosomes to the cell surface is firmly established, but how SNX27 assembles with retromer to generate tubulovesicular carriers remains elusive. Whether SNX27/retromer may be a putative therapeutic target to prevent neurodegenerative disease is now an emerging area of study. This review will provide an update on our molecular understanding of endosomal trafficking events mediated by the SNX27/retromer complex on endosomes.

X-ray Fiber Diffraction and Computational Analyses of Stacked Hexads in Supramolecular Polymers: Insight into Self-Assembly in Water by Prospective Prebiotic Nucleobases.

Aqueous solutions of equimolar mixtures of 2,4,6-triaminopyrimidine (TAP) and carboxylic acid substituted cyanuric acid (CyCo6 or R-4MeCyCo6) monomers self-assemble into gel-forming supramolecular polymers. Macroscopic fibers drawn from these mixtures were analyzed by X-ray diffraction to determine their molecular structures. Computational methods were used to explore the intrinsic intermolecular interactions that contribute to the structure and stability of these assemblies. Both polymers are formed by the stacking of hexameric rosettes, (TAP/CyCo6)3 or (TAP/R-4MeCyCo6)3, respectively, into long, stiff, twisted stacks of essentially planar rosettes. Chiral, left-handed supramolecular polymers with a helical twist angle of -26.7° per hexad are formed when the pure enantiomer R-4MeCyCo6 is used. These hexad stacks pack into bundles with a hexagonal crystalline lattice organization perpendicular to the axis of the macroscopic fiber. Polymers formed from TAP and CyCo6, both of which are achiral, assemble into macroscopic domains that are packed as a centered rectangular lattice. Within these domains, the individual polymers exist as either right-handed or left-handed helical stacks, with twist angles of +15° or -15° per hexad, respectively. The remarkable ability of TAP and cyanuric acid derivatives to self-assemble in water, and the structural features of their supramolecular polymers reported here, provide additional support for the proposal that these heterocycles could have served as recognition units for an early form of nucleic acids, before the emergence of RNA.

The Glo3 GAP crystal structure supports the molecular niche model for ArfGAPs in COPI coats.

Arf GTPase activating (ArfGAP) proteins are critical regulatory and effector proteins in membrane trafficking pathways. Budding yeast contain two ArfGAP proteins (Gcs1 and Glo3) implicated in COPI coat function at the Golgi, and yeast require Glo3 catalytic function for viability. A new X-ray crystal structure of the Glo3 GAP domain was determined at 2.1 Å resolution using molecular replacement methods. The structure reveals a Cys4-family zinc finger motif with an invariant residue (R59) positioned to act as an "arginine finger" during catalysis. Comparisons among eukaryotic GAP domains show a key difference between ArfGAP1 and ArfGAP2/3 family members in the final helix located within the domain. Conservation at both the sequence and structural levels suggest the Glo3 GAP domain interacts with yeast Arf1 switch I and II regions to promote catalysis. Together, the structural data presented here provide additional evidence for placing Glo3 near Arf1 triads within membrane-assembled COPI coats and further support the molecular niche model for COPI coat regulation by ArfGAPs.

Unveiling the cryo-EM structure of retromer.

Retromer (VPS26/VPS35/VPS29) is a highly conserved eukaryotic protein complex that localizes to endosomes to sort transmembrane protein cargoes into vesicles and elongated tubules. Retromer mediates retrieval pathways from endosomes to the trans-Golgi network in all eukaryotes and further facilitates recycling pathways to the plasma membrane in metazoans. In cells, retromer engages multiple partners to orchestrate the formation of tubulovesicular structures, including sorting nexin (SNX) proteins, cargo adaptors, GTPases, regulators, and actin remodeling proteins. Retromer-mediated pathways are especially important for sorting cargoes required for neuronal maintenance, which links retromer loss or mutations to multiple human brain diseases and disorders. Structural and biochemical studies have long contributed to the understanding of retromer biology, but recent advances in cryo-electron microscopy and cryo-electron tomography have further uncovered exciting new snapshots of reconstituted retromer structures. These new structures reveal retromer assembles into an arch-shaped scaffold and suggest the scaffold may be flexible and adaptable in cells. Interactions with cargo adaptors, particularly SNXs, likely orient the scaffold with respect to phosphatidylinositol-3-phosphate (PtdIns3P)-enriched membranes. Pharmacological small molecule chaperones have further been shown to stabilize retromer in cultured cell and mouse models, but mechanisms by which these molecules bind remain unknown. This review will emphasize recent structural and biophysical advances in understanding retromer structure as the field moves towards a molecular view of retromer assembly and regulation on membranes.

Mammalian Retromer Is an Adaptable Scaffold for Cargo Sorting from Endosomes.

Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (∼5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations.

Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin.

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

Molecular Basis for the Interaction Between AP4 ß4 and its Accessory Protein, Tepsin.

The adaptor protein 4 (AP4) complex (ϵ/ß4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 ß4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal ß4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the ß4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on ß4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.