Sorting Motif Research Articles

AP-1 contributes to endosomal targeting of ubiquitin ligase RNF13 via a secondary and novel non-canonical binding motif.

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. Ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis, and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability of RNF13 to interact with AP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosine-based motif within RNF13's C-terminal region that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular, and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses an AP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by variant of RNF13's dileucine sorting motif.

Revising pathogenesis of AP1S1-related MEDNIK syndrome: a missense variant in the AP1S1 gene as a causal genetic lesion.

MEDNIK syndrome is a rare autosomal recessive disease characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratoderma, and caused by variants in the adaptor-related protein complex 1 subunit sigma 1 (AP1S1) gene. This gene encodes the σ1A protein, which is a subunit of the adaptor protein complex 1 (AP-1), a key component of the intracellular protein trafficking machinery. Previous work identified three AP1S1 nonsense, frameshift and splice-site variants in MEDNIK patients predicted to encode truncated σ1A proteins, with consequent AP-1 dysfunction. However, two AP1S1 missense variants (c.269T > C and c.346G > A) were recently reported in patients who presented with severe enteropathy but no additional symptoms of MEDNIK. This condition was described as a novel non-syndromic form of congenital diarrhea caused specifically by the AP1S1 missense variants. In this study, we report two patients with the same c.269T > C variant, who, contrary to the previous cases, presented as complete MEDNIK syndrome. These data substantially revise the presentation of disorders associated with AP1S1 gene variants and indicate that all the identified pathogenic AP1S1 variants result in MEDNIK syndrome. We also provide a series of functional analyses that elucidate the impact of the c.269T > C variant on σ1A function, contributing to a better understanding of the molecular pathogenesis of MEDNIK syndrome. KEY MESSAGES: A missense AP1S1 c.269T > C (σ1A L90P) variant causes full MEDNIK syndrome. The σ1A L90P variant is largely unable to assemble into the AP-1 complex. The σ1A L90P variant fails to bind [DE]XXXL[LI] sorting motifs. The σ1A L90P variant results in loss-of-function of the protein.

Robust and Irreversible Sortase-Mediated Ligation by Empolyment of Sarkosyl.

Sortase-mediated ligation (SML) is a widely used method for peptide and protein ligation due to ease of substrate preparation and fast enzymatic kinetics. But there are drawbacks that limit broader applications. Sorting motif in substrates may not be exposed to sortase efficiently due to folding or aggregation. In addition, the ligation is reversible under transpeptidation equilibrium that restricts ligation yield. Here we report a simple but robust method to overcome such limitations. By employment of sarkosyl, the detergent alters substrate conformation to raise sorting motif accessibility for sortase catalysis. Moreover, transpeptidation becomes irreversible presumably by formation of micelle to shield ligation products from sortase. In consequence, excellent yields were achieved from sortase variants with different substrate specificity. Notably, this method is compatible with peptides or proteins capable of forming liquid-liquid phase separation (LLPS), presenting a powerful approach for the conjugation of aggregation-prone substrates. Therefore, we believe the sarkosyl-enhanced SML could be widely applied in peptide and protein chemistry and the unique irreversible transpeptidation mechanism offers an insight to detergent-driven equilibrium.

Sequence and structural insights of monoleucine-based sorting motifs contained within the cytoplasmic domains of basolateral proteins.

Delivery to the correct membrane domain in polarized epithelial cells is a critical regulatory mechanism for transmembrane proteins. The trafficking of these proteins is directed by short amino acid sequences known as sorting motifs. In six basolaterally-localized proteins lacking the canonical tyrosine- and dileucine-based basolateral sorting motifs, a monoleucine-based sorting motif has been identified. This review will discuss these proteins with an identified monoleucine-based sorting motif, their conserved structural features, as well as the future directions of study for this non-canonical basolateral sorting motif.

A Group of Highly Secretory miRNAs Correlates with Lymph Node Metastasis and Poor Prognosis in Oral Squamous Cell Carcinoma.

MicroRNAs (miRNAs) in oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) play a pivotal role in modulating intercellular communications between tumor cells and other cells in the microenvironment, thereby influencing tumor progression and the efficacy of therapeutic interventions. However, a comprehensive inventory of these secretory miRNAs in sEVs and their biological and clinical implications remains elusive. This study aims to profile the miRNA content of OSCC cell line sEVs and computationally elucidate their biological and clinical relevance. We conducted miRNA sequencing to compare the miRNA profiles of OSCC cells and their corresponding sEVs. Our motif enrichment analysis identified specific sorting motifs that are implicated in either cellular retention or preferential sEV secretion. Target cell analysis suggested that the sEV miRNAs potentially interact with various immune cell types, including natural killer cells and dendritic cells. Additionally, we explored the clinical relevance of these miRNAs by correlating their expression levels with TNM stages and patient survival outcomes. Intriguingly, our findings revealed that a distinct sEV miRNA signature is associated with lymph node metastasis and poorer survival in patients in TCGA-HNSC dataset. Collectively, this research furthers our understanding of the miRNA sorting mechanisms in OSCC and underscores their clinical implications.

Crystal structure of the pilus-specific sortase from early colonizing oral Streptococcus sanguinis captures an active open-lid conformation

Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection.

Out of the ESCPE room: Emerging roles of endosomal SNX-BARs in receptor transport and host-pathogen interaction.

Several functions of the human cell, such as sensing nutrients, cell movement and interaction with the surrounding environment, depend on a myriad of transmembrane proteins and their associated proteins and lipids (collectively termed "cargoes"). To successfully perform their tasks, cargo must be sorted and delivered to the right place, at the right time, and in the right amount. To achieve this, eukaryotic cells have evolved a highly organized sorting platform, the endosomal network. Here, a variety of specialized multiprotein complexes sort cargo into itineraries leading to either their degradation or their recycling to various organelles for further rounds of reuse. A key sorting complex is the Endosomal SNX-BAR Sorting Complex for Promoting Exit (ESCPE-1) that promotes the recycling of an array of cargos to the plasma membrane and/or the trans-Golgi network. ESCPE-1 recognizes a hydrophobic-based sorting motif in numerous cargoes and orchestrates their packaging into tubular carriers that pinch off from the endosome and travel to the target organelle. A wide range of pathogens mimic this sorting motif to hijack ESCPE-1 transport to promote their invasion and survival within infected cells. In other instances, ESCPE-1 exerts restrictive functions against pathogens by limiting their replication and infection. In this review, we discuss ESCPE-1 assembly and functions, with a particular focus on recent advances in the understanding of its role in membrane trafficking, cellular homeostasis and host-pathogen interaction.

Polarized cell plasma membrane composition and protein sorting.

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between plasma membrane domains in epithelial cells have been resolved. Particularly, the lipid profile of the basolateral membrane remains unstudied, leaving doubts about the differentiation and lipid separation of the apical and basolateral membrane. We use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We observe that the apical membrane is enriched in highly saturated lipids and glycolipids relative to the basolateral membrane, with apical membrane biophysical properties reflecting a raft-enriched environment. Similar to lipids, the determinants of protein sorting between apical and basolateral domains are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical membrane hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. We systematically evaluate the structural determinants of apical versus basolateral sorting with a focus on transmembrane domain features, protein raft affinity, and glycosylation. We find that these protein features cooperatively direct protein sorting to the apical membrane.

In silico and in vitro analysis of the impact of single substitutions within EXO-motifs on Hsa-MiR-1246 intercellular transfer in breast cancer cell

MiR-1246 has recently gained much attention and many studies have shown its oncogenic role in colorectal, breast, lung, and ovarian cancers. However, miR-1246 processing, stability, and mechanisms directing miR-1246 into neighbor cells remain still unclear. In this study, we aimed to determine the role of single-nucleotide substitutions within short exosome sorting motifs — so-called EXO-motifs: GGAG and GCAG present in miR-1246 sequence on its intracellular stability and extracellular transfer. We applied in silico methods such as 2D and 3D structure analysis and modeling of protein interactions. We also performed in vitro validation through the transfection of fluorescently labeled miRNA to MDA-MB-231 cells, which we analyzed by flow cytometry and fluorescent microscopy. Our results suggest that nucleotides alterations that disturbed miR-1246 EXO-motifs were able to modulate miRNA-1246 stability and its transfer level to the neighboring cells, suggesting that the molecular mechanism of RNA stability and intercellular transfer can be closely related.

Sorting motifs target the movement protein of ourmia melon virus to the trans-Golgi network and plasmodesmata

Plants have a highly sophisticated endomembrane system targeted by plant viruses for cell-to-cell movement. The movement protein (MP) of ourmia melon virus (OuMV) is delivered to plasmodesmata (PD) and forms tubules to facilitate cell-to-cell movement. In our previous work, we identified the sites of OuMV for correct subcellular localization; however, the pathways involved in PD-targeting of OuMV MP are largely unclear, and their identification was the aim of this work. In this study, we demonstrate that OuMV MP localizes to the trans-Golgi network (TGN) but not to the multivesicular body/prevacuolar compartment or Golgi, and carries two putative sorting motifs, a tyrosine (Y) and a dileucine (LL) motif, near its N-terminus. Substitutions in these motifs result in loss of OuMV infectivity in Nicotiana benthamiana and Arabidopsis. Live cell imaging of GFP-labeled sorting motif mutants reveals that both motifs are required for targeting OuMV MP to PD and for efficient systemic infection but show differences in functionality. Moreover, co-immunoprecipitation assays coupled with mass spectrometry identified a series of host factors that could interact with the OuMV MP and link the MP with various pathways, in particular vesicle trafficking and membrane lipids. Our findings delineate the trafficking route of OuMV MP for PD-targeting and elucidate host factors potentially recruited by OuMV MP that could be modified to impair OuMV infection.

Acidic dileucine motifs in the cylindrical inclusion protein of turnip mosaic virus are crucial for endosomal targeting and viral replication

Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2β), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2β. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2β. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2β. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic‐tracking styryl‐stained endosomes. The mutations were then introduced into a full‐length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2β for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.

Axon-dendrite and apical-basolateral sorting in a single neuron.

Cells are highly organized machines with functionally specialized compartments. For example, membrane proteins are localized to axons or dendrites in neurons and to apical or basolateral surfaces in epithelial cells. Interestingly, many sensory cells—including vertebrate photoreceptors and olfactory neurons—exhibit both neuronal and epithelial features. Here, we show that Caenorhabditis elegans amphid neurons simultaneously exhibit axon-dendrite sorting like a neuron and apical-basolateral sorting like an epithelial cell. The distal ∼5–10 µm of the dendrite is apical, while the remainder of the dendrite, soma, and axon are basolateral. To determine how proteins are sorted among these compartments, we studied the localization of the conserved adhesion molecule SAX-7/L1CAM. Using minimal synthetic transmembrane proteins, we found that the 91-aa cytoplasmic tail of SAX-7 is necessary and sufficient to direct basolateral localization. Basolateral localization can be fully recapitulated using either of 2 short (10-aa or 19-aa) tail sequences that, respectively, resemble dileucine and Tyr-based motifs known to mediate sorting in mammalian epithelia. The Tyr-based motif is conserved in human L1CAM but had not previously been assigned a function. Disrupting key residues in either sequence leads to apical localization, while “improving” them to match epithelial sorting motifs leads to axon-only localization. Indeed, changing only 2 residues in a short motif is sufficient to redirect the protein between apical, basolateral, and axonal localization. Our results demonstrate that axon-dendrite and apical-basolateral sorting pathways can coexist in a single cell, and suggest that subtle changes to short sequence motifs are sufficient to redirect proteins between these pathways.

Composition and sorting of polarized cell plasma membrane compartments

Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane include self-assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting.

Examination of Galectin-3 Recruitment into Multivesicular Bodies for Exosomal Secretion.

Cells use unconventional secretion to deliver the β-galactoside binding lectin galectin-3 from the cell interior into the extracellular milieu. This process starts with galectin-3 recruitment into intraluminal vesicles (ILVs), which are later released at the plasma membrane as exosomes. Electron microscopy is utilized to determine the location of GFP-tagged galectin-3 in pelleted exosomes. We also describe how these vesicles are harvested from cell culture media to determine their composition. The fluorescent protein GFP was fused with the exosomal sorting motif of galectin-3 to direct GFP into exosomes. Recruitment of this fusion construct into the lumen of exosomes can be assessed by proteinase K accessibility analysis.

Sortase-Mediated Multi-Fragment Assemblies by Ligation Site Switching.

Sortase‐mediated ligation (SML) is a powerful tool of protein chemistry allowing the ligation of peptides containing LPxTG sorting motifs and N‐terminal glycine nucleophiles. The installation of a sorting motif into the product prohibits the assembly of multiple fragments by SML. Here we report multi‐fragment SML based on switchable sortase substrates. Substitution of the Leu residue by disulfide‐containing Cys(StBu) results in active sorting motifs, which are inactivatable by reduction. In combination with a photo‐protected N‐Gly nucleophile, multi‐fragment SML is enabled by repetitive cycles of SML and ligation site switching. The feasibility of this approach was demonstrated by a proof‐of‐concept four‐fragment ligation, the assembly of peptide probes for bivalent chromatin binding proteins and oligomerization of peptide antigens. Biochemical and immuno‐assays demonstrated functionality of these probes rendering them promising tools for immunology and chromatin biochemistry.

A systematic review of Sec24 cargo interactome.

Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII-mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. We also discuss how the Sec24 secretion system is hijacked by viral (eg, VSV-G, Hepatitis B envelope protein) and bacterial (eg, the enteropathogenic Escherichia coli type III secretion system effector NleA/EspI) pathogens.

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling.

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans.

S-palmitoylation determines TMEM55B-dependent positioning of lysosomes.

The spatiotemporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo. TMEM55B (also known as PIP4P1), as an integral lysosomal membrane protein, is a component of such a complex that mediates the retrograde transport of lysosomes by establishing interactions with the cytosolic scaffold protein JIP4 (also known as SPAG9) and dynein-dynactin. Here, we show that TMEM55B and its paralog TMEM55A (PIP4P2) are S-palmitoylated proteins that are lipidated at multiple cysteine residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes retention of the mutated protein in the Golgi. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor protein-dependent sorting of TMEM55B. Our data suggest a critical role for S-palmitoylation in the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

Analysis of the SNARE Stx8 recycling reveals that the retromer-sorting motif has undergone evolutionary divergence.

Fsv1/Stx8 is a Schizosaccharomyces pombe protein similar to mammalian syntaxin 8. stx8Δ cells are sensitive to salts, and the prevacuolar endosome (PVE) is altered in stx8Δ cells. These defects depend on the SNARE domain, data that confirm the conserved function of syntaxin8 and Stx8 in vesicle fusion at the PVE. Stx8 localizes at the trans-Golgi network (TGN) and the prevacuolar endosome (PVE), and its recycling depends on the retromer component Vps35, and on the sorting nexins Vps5, Vps17, and Snx3. Several experimental approaches demonstrate that Stx8 is a cargo of the Snx3-retromer. Using extensive truncation and alanine scanning mutagenesis, we identified the Stx8 sorting signal. This signal is an IEMeaM sequence that is located in an unstructured protein region, must be distant from the transmembrane (TM) helix, and where the 133I, 134E, 135M, and 138M residues are all essential for recycling. This sorting motif is different from those described for most retromer cargoes, which include aromatic residues, and resembles the sorting motif of mammalian polycystin-2 (PC2). Comparison of Stx8 and PC2 motifs leads to an IEMxx(I/M) consensus. Computer-assisted screening for this and for a loose Ψ(E/D)ΨXXΨ motif (where Ψ is a hydrophobic residue with large aliphatic chain) shows that syntaxin 8 and PC2 homologues from other organisms bear variation of this motif. The phylogeny of the Stx8 sorting motifs from the Schizosaccharomyces species shows that their divergence is similar to that of the genus, showing that they have undergone evolutionary divergence. A preliminary analysis of the motifs in syntaxin 8 and PC2 sequences from various organisms suggests that they might have also undergone evolutionary divergence, what suggests that the presence of almost-identical motifs in Stx8 and PC2 might be a case of convergent evolution.

X-ray structures of Clostridium perfringens sortase C with C-terminal cell wall sorting motif of LPST demonstrate role of subsite for substrate-binding and structural variations of catalytic site

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.