Putative Cargo Research Articles

Ex vivo nanoscale abluminal mapping of putative cargo receptors at the blood-brain barrier of expanded brain capillaries

Receptor mediated transport of therapeutic antibodies through the blood-brain barrier (BBB) give promise for drug delivery to alleviate brain diseases. We developed a low-cost method to obtain nanoscale localization data of putative cargo receptors. We combine existing ex vivo isolation methods with expansion microscopy (ExM) to analyze receptor localizations in brain microcapillaries. Using this approach, we show how to analyze receptor localizations in endothelial cells of brain microcapillaries in relation to the abluminal marker collagen IV. By choosing the thinnest capillaries, microcapillaries for analysis, we ensure the validity of collagen IV as an abluminal marker. With this tool, we confirm transferrin receptors as well as sortilin to be both luminally and abluminally localized. Furthermore, we identify basigin to be an abluminal receptor. Our methodology can be adapted to analyze different types of isolated brain capillaries and we anticipate that this approach will be very useful for the research community to gain new insight into cargo receptor trafficking in the slim brain endothelial cells to elucidate novel paths for future drug design.

ERV14 receptor impacts mycelial growth via its interactions with cell wall synthase and transporters in Aspergillus niger.

Efficient protein secretion is closely correlated with vesicle sorting and packaging, especially with cargo receptor-mediated selective transport for ER exit. Even though Aspergillus niger is considered an industrially natural host for protein production due to its exceptional secretion capacity, the trafficking mechanism in the early secretory pathway remains a black box for us to explore. Here, we identified and characterized all putative ER cargo receptors of the three families in A. niger. We successfully constructed overexpression and deletion strains of each receptor and compared the colony morphology and protein secretion status of each strain. Among them, the deletion of Erv14 severely inhibited mycelial growth and secretion of extracellular proteins such as glucoamylase. To gain a comprehensive understanding of the proteins associated with Erv14, we developed a high-throughput method by combining yeast two-hybrid (Y2H) with next-generation sequencing (NGS) technology. We found Erv14 specifically interacted with transporters. Following further validation of the quantitative membrane proteome, we determined that Erv14 was associated with the transport of proteins involved in processes such as cell wall synthesis, lipid metabolism, and organic substrate metabolism.

Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex.

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. We show that aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that aTm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.

Modification of the pH Dependence of Assembly of Yeast Cargo Receptor Emp47p Coiled-Coil Domains: Computational Design and Experimental Mutagenesis.

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46pcc and Emp47pcc) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46pcc serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. To examine this hypothesis, we constructed two structural models in which the side chains of E303 were negatively charged or protonated and analyzed the effects of these charged states on the structure of the heterocomplex using molecular dynamics (MD) simulations. The calculated structures suggested the side chains of E303 to be protonated in the heterocomplex, even at neutral pH. Based on these computational results, the pH dependence of Emp47pcc homo-oligomer assembly was experimentally modified by a glutamate mutation on its hydrophobic surface. The Q306E mutant of Emp47pcc underwent a structural transition at physiological pH. Our results suggest a method for modifying pH-dependent protein-protein interactions.

Atomic Structure of the Francisella T6SS Central Spike Reveals a Unique α-Helical Lid and a Putative Cargo

Francisella bacteria rely on a phylogenetically distinct type VI secretion system (T6SS) to escape host phagosomes and cause the fatal disease tularemia, but the structural and molecular mechanisms involved are unknown. Here we report the atomic structure of the Francisella T6SS central spike complex, obtained by cryo-electron microscopy. Our structural and functional studies demonstrate that, unlike the single-protein spike composition of other T6SS subtypes, Francisella T6SS's central spike is formed by two proteins, PdpA and VgrG, akin to T4-bacteriophage gp27 and gp5, respectively, and that PdpA has unique characteristics, including a putative cargo within its cavity and an N-terminal helical lid. Structure-guided mutagenesis demonstrates that the PdpA N-terminal lid and C-terminal spike are essential to Francisella T6SS function. PdpA isthus both an adaptor, connecting VgrG to the tube, and a likely carrier of secreted cargo. These findings are important to understanding Francisella pathogenicity and designing therapeutics to combat tularemia.

In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1

Iron is universally important to cellular metabolism, and mitoferrin-1 and -2 have been proposed to be the iron importers of mitochondria, the cell's assembly plant of heme and iron-sulfur clusters. These iron-containing prosthetic groups are critical for a host of physiological processes ranging from oxygen transport and energy consumption to maintaining protein structural integrity. Mitoferrin-1 (Mfrn1) belongs to the mitochondrial carrier (MC) family and is atypical given its putative metallic cargo; most MCs transport nucleotides, amino acids, or other small- to medium-size metabolites. Despite the clear importance of Mfrn1 in iron utilization, its transport activity has not been demonstrated unambiguously. To bridge this knowledge gap, we have purified recombinant Mfrn1 under non-denaturing conditions and probed its metal ion-binding and transport functions. Isothermal titration calorimetry indicates that Mfrn1 has micromolar affinity for Fe(II), Mn(II), Co(II), and Ni(II). Mfrn1 was incorporated into defined liposomes, and iron transport was reconstituted in vitro, demonstrating that Mfrn1 can transport iron. Mfrn1 can also transport manganese, cobalt, copper, and zinc but discriminates against nickel. Experiments with candidate ligands for cellular labile iron reveal that Mfrn1 transports free iron and not a chelated iron complex and selects against alkali divalent ions. Extensive mutagenesis identified multiple residues that are crucial for metal binding, transport activity, or both. There is a clear abundance of residues with side chains that can coordinate first-row transition metal ions, suggesting that these could form primary or auxiliary metal-binding sites during the transport process.

The assembly mechanism of coiled-coil domains of the yeast cargo receptors Emp46p/47p and the mutational alteration of pH-dependency of complex formation.

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p are responsible for their complex-formation in the Endoplasmic Reticulum. In vitro experiments using coiled-coil domains (Emp46pcc/47pcc) have indicated that formation of the hetero-complex is pH-dependent and that amino acid Glu303 of Emp46pcc is a key residue in this process. In this study, we investigated the effects of various mutations on complex formation and discovered the mechanism for its pH-dependency, which is that dissociation of the complex at low pH arises mainly from stabilization of Emp46pcc itself. Moreover, destabilization by the introduction of a histidine residue in Emp46pcc to repel a lysine residue in Emp47pcc, caused an upward shift in the pH profile of complex formation. Another mutation in Emp46pcc, a proline to an alanine (P291A), increased the stability of the helical structure, especially at low pH and shifted the transition pH upward. Combination of these pH-shifting mutations had an additive effect on the pH profile of complex formation. Thus, we successfully constructed coiled-coils that can react to a wide range of pH, encompassing more appropriate values for use in sensing physiological pH changes in the cell.

Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin.

Soluble cytosolic proteins vital to axonal and presynaptic function are synthesized in the neuronal soma and conveyed via slow axonal transport. Our previous studies suggest that the overall slow transport of synapsin is mediated by dynamic assembly/disassembly of cargo complexes followed by short-range vectorial transit (the "dynamic recruitment" model). However, neither the composition of these complexes nor the mechanistic basis for the dynamic behavior is understood. In this study, we first examined putative cargo complexes associated with synapsin using coimmunoprecipitation and multidimensional protein identification technology mass spectrometry (MS). MS data indicate that synapsin is part of a multiprotein complex enriched in chaperones/cochaperones including Hsc70. Axonal synapsin-Hsc70 coclusters are also visualized by two-color superresolution microscopy. Inhibition of Hsc70 ATPase activity blocked the slow transport of synapsin, disrupted axonal synapsin organization, and attenuated Hsc70-synapsin associations, advocating a model where Hsc70 activity dynamically clusters cytosolic proteins into cargo complexes, allowing transport. Collectively, our study offers insight into the molecular organization of cytosolic transport complexes and identifies a novel regulator of slow transport.

Search for KPNA7 cargo proteins in human cells reveals MVP and ZNF414 as novel regulators of cancer cell growth

Karyopherin alpha 7 (KPNA7) belongs to a family of nuclear import proteins that recognize and bind nuclear localization signals (NLSs) in proteins to be transported to the nucleus. Previously we found that KPNA7 is overexpressed in a subset of pancreatic cancer cell lines and acts as a critical regulator of growth in these cells. This characteristic of KPNA7 is likely to be mediated by its cargo proteins that are still mainly unknown. Here, we used protein affinity chromatography in Hs700T and MIA PaCa-2 pancreatic cancer cell lines and identified 377 putative KPNA7 cargo proteins, most of which were known or predicted to localize to the nucleus. The interaction was confirmed for two of the candidates, MVP and ZNF414, using co-immunoprecipitation, and their transport to the nucleus was hindered by siRNA based KPNA7 silencing. Most importantly, silencing of MVP and ZNF414 resulted in marked reduction in Hs700T cell growth. In conclusion, these data uncover two previously unknown human KPNA7 cargo proteins with distinct roles as novel regulators of pancreatic cancer cell growth, thus deepening our understanding on the contribution of nuclear transport in cancer pathogenesis.

Erv14 cargo receptor participates in yeast salt tolerance via its interaction with the plasma-membrane Nha1 cation/proton antiporter

The yeast Nha1p Na+, K+/H+ antiporter has a house-keeping role in pH and cation homeostasis. It is also needed to alleviate excess Na+ or K+ from the cytoplasm under high external concentrations of these cations. Erv14p, a putative cargo receptor for transmembrane proteins is required for trafficking of Nha1p from the endoplasmic reticulum to the plasma membrane. Sensitivity to high Na+ concentrations of the erv14 mutant associated to the intracellular mislocalization of Nha1p-GFP, together with a lower Na+ efflux, indicate the involvement of this mutual association to accomplish the survival of the yeast cell upon sodium stress. This observation is supported by the protein–protein interaction between Erv14p and Nha1p detected by the mating-based Split Ubiquitin System and co-immunoprecipitation assays. Our results indicate that even though Erv14p interacts with Nha1p through the TMD, the C-terminal is important not only for the efficient delivery of Nha1p to the plasma membrane but also for its dimerization to accomplish its role in yeast salt tolerance.

PH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p

Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions in vitro solely with their coiled-coil segments, which form stable heterotetramers in the neutral condition but segregate at lower pH. Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems.

Modulating endoplasmic reticulum-Golgi cargo receptors for improving secretion of carrier-fused heterologous proteins in the filamentous fungus Aspergillus oryzae.

Filamentous fungi are excellent hosts for industrial protein production due to their superior secretory capacity; however, the yield of heterologous eukaryotic proteins is generally lower than that of fungal or endogenous proteins. Although activating protein folding machinery in the endoplasmic reticulum (ER) improves the yield, the importance of intracellular transport machinery for heterologous protein secretion is poorly understood. Here, using Aspergillus oryzae as a model filamentous fungus, we studied the involvement of two putative lectin-like cargo receptors, A. oryzae Vip36 (AoVip36) and AoEmp47, in the secretion of heterologous proteins expressed in fusion with the endogenous enzyme α-amylase as the carrier. Fluorescence microscopy revealed that mDsRed-tagged AoVip36 localized in the Golgi compartment, whereas AoEmp47 showed localization in both the ER and the Golgi compartment. Deletion of AoVip36 and AoEmp47 improved heterologous protein secretion, but only AoVip36 deletion had a negative effect on the secretion of α-amylase. Analysis of ER-enriched cell fractions revealed that AoVip36 and AoEmp47 were involved in the retention of heterologous proteins in the ER. However, the overexpression of each cargo receptor had a different effect on heterologous protein secretion: AoVip36 enhanced the secretion, whereas AoEmp47 promoted the intracellular retention. Taken together, our data suggest that AoVip36 and AoEmp47 hinder the secretion of heterologous proteins by promoting their retention in the ER but that AoVip36 also promotes the secretion of heterologous proteins. Moreover, we found that genetic deletion of these putative ER-Golgi cargo receptors significantly improves heterologous protein production. The present study is the first to propose that ER-Golgi transport is a bottleneck for heterologous protein production in filamentous fungi.

The puzzle of chloroplast vesicle transport - involvement of GTPases.

In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum network, Golgi bodies, secretory granules, endosome, and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence indicates presence of a vesicle transport system in chloroplasts. Little is known about the protein components of this system. However, as chloroplasts harbor the photosynthetic apparatus that ultimately supports most organisms on the planet, close attention to their pathways is warranted. This may also reveal novel diversification and/or distinct solutions to the problems posed by the targeted intra-cellular trafficking of important molecules. To date two homologs to well-known yeast cytosolic vesicle transport proteins, CPSAR1 and CPRabA5e (CP, chloroplast localized), have been shown to have roles in chloroplast vesicle transport, both being GTPases. Bioinformatic data indicate that several homologs of cytosolic vesicle transport system components are putatively chloroplast-localized and in addition other proteins have been implicated to participate in chloroplast vesicle transport, including vesicle-inducing protein in plastids 1, thylakoid formation 1, snowy cotyledon 2/cotyledon chloroplast biogenesis factor, curvature thylakoid 1 proteins, and a dynamin like GTPase FZO-like protein. Several putative potential cargo proteins have also been identified, including building blocks of the photosynthetic apparatus. Here we discuss details of the largely unknown putative chloroplast vesicle transport system, focusing on GTPase-related components.

An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein-protein interaction in vivo.

New Putative Chloroplast Vesicle Transport Components and Cargo Proteins Revealed Using a Bioinformatics Approach: An Arabidopsis Model

Proteins and lipids are known to be transported to targeted cytosolic compartments in vesicles. A similar system in chloroplasts is suggested to transfer lipids from the inner envelope to the thylakoids. However, little is known about both possible cargo proteins and the proteins required to build a functional vesicle transport system in chloroplasts. A few components have been suggested, but only one (CPSAR1) has a verified location in chloroplast vesicles. This protein is localized in the donor membrane (envelope) and vesicles, but not in the target membrane (thylakoids) suggesting it plays a similar role to a cytosolic homologue, Sar1, in the secretory pathway. Thus, we hypothesized that there may be more similarities, in addition to lipid transport, between the vesicle transport systems in the cytosol and chloroplast, i.e. similar vesicle transport components, possible cargo proteins and receptors. Therefore, using a bioinformatics approach we searched for putative chloroplast components in the model plant Arabidopsis thaliana, corresponding mainly to components of the cytosolic vesicle transport system that may act in coordination with previously proposed COPII chloroplast homologues. We found several additional possible components, supporting the notion of a fully functional vesicle transport system in chloroplasts. Moreover, we found motifs in thylakoid-located proteins similar to those of COPII vesicle cargo proteins, supporting the hypothesis that chloroplast vesicles may transport thylakoid proteins from the envelope to the thylakoid membrane. Several putative cargo proteins are involved in photosynthesis, thus we propose the existence of a novel thylakoid protein pathway that is important for construction and maintenance of the photosynthetic machinery.

Structural basis for endosomal trafficking of diverse transmembrane cargos by PX-FERM proteins.

Transit of proteins through the endosomal organelle following endocytosis is critical for regulating the homeostasis of cell-surface proteins and controlling signal transduction pathways. However, the mechanisms that control these membrane-transport processes are poorly understood. The Phox-homology (PX) domain-containing proteins sorting nexin (SNX) 17, SNX27, and SNX31 have emerged recently as key regulators of endosomal recycling and bind conserved Asn-Pro-Xaa-Tyr-sorting signals in transmembrane cargos via an atypical band, 4.1/ezrin/radixin/moesin (FERM) domain. Here we present the crystal structure of the SNX17 FERM domain bound to the sorting motif of the P-selectin adhesion protein, revealing both the architecture of the atypical FERM domain and the molecular basis for recognition of these essential sorting sequences. We further show that the PX-FERM proteins share a promiscuous ability to bind a wide array of putative cargo molecules, including receptor tyrosine kinases, and propose a model for their coordinated molecular interactions with membrane, cargo, and regulatory proteins.

PAIRing Up Cargo Proteins

​AllAll newly made proteins destined to be secreted from our cells or to function at the cell surface—and many of those that function in membranes within the cell—start their lives in a convoluted heap of membranes within cells called the endoplasmic reticulum (ER). From there, they begin a journey that will take them through the stacked membrane compartments of the Golgi apparatus to their eventual destinations, transported from one compartment to another in small membrane vesicles. Proteins known as cargo receptors often perform the key function of selecting and tethering cargo proteins within the transport vesicles, but only a few such receptors have been identified to date, and which cargo proteins they pair with was mostly unknown. Now, Maya Schuldiner and her colleagues have devised a large-scale approach to discover these pairings between cargo proteins and cargo receptors, laying the groundwork to uncover the mechanisms that underlie this indispensable machinery. Towards a cellular “Traffickome.” Systematic analysis of cargo receptors (CRs) demonstrates the rules governing specificity versus promiscuity in ER exit. The scientists call their approach “pairing analysis of cargo receptors”, or PAIRS. They start with yeast strains that have mutations in candidate cargo receptors—proteins that previous studies found were involved in traffic between the ER and the Golgi apparatus. A robotic system then systematically crosses these mutant yeast strains with a collection of other strains, each of which contains a potential cargo protein tagged with green fluorescent protein (GFP). The researchers then use an automated high-throughput microscope to help see which of the resulting strains have retained green fluorescence in the ER due to the cargo protein accumulating in the absence of its functional receptor. This strategy can be used to uncover the receptor for a specific cargo of interest. They also show that this same methodology can be used to identify new cargo proteins for a known cargo receptor. Schuldiner and colleagues mutated nine putative cargo receptors and followed the fates of 157 cargo proteins with their PAIRS technique. Knocking out these receptors affected 31 cargo proteins, 27 of which had not previously been linked to a particular cargo receptor. The spectrum of cargo uncovered for the cargo receptors helped define what specific cargo each receptor recognized. For example, past research hinted that all cargo for the receptor Erv26 were mannosyltransferases that function in the Golgi apparatus, but PAIRS suggests that Erv26 is specific for a subset of this group because the transport of several types of mannosyltransferase out of the ER was not affected by knocking out Erv26. Perhaps the most striking finding was that the cargo receptor Erv14 interacts with a large number of cargo proteins. PAIRS showed that Erv14 is required for the trafficking of about a third of all plasma membrane proteins tested, including proteins with a wide array of functions and structures (permeases, transporters, multidrug transporters, lipid flippases, eisosome components, and proteins linked to cell polarity or cell wall regulation). The numerous cargo proteins identified for Erv14 are all membrane-spanning proteins that reside late in the secretory pathway. Such proteins typically have longer transmembrane domains (TMDs) than other membrane-spanning proteins. When the researchers varied the length of the TMD of one of the cargoes bound by Erv14—Mid2—they found that the variants with the shortest TMDs had the most difficulty leaving the ER; in other words, Erv14 recognizes TMD length rather than sequence. Such a mechanism might permit a few receptors to transport a diverse range of cargo proteins without the need for multiple sequence-specific receptor motifs. When Mid2 was fused to the tail of Sys1, a Golgi protein, it exited the ER rapidly, regardless of the length of its TMD, indicating that cargo proteins with a short TMD can be “rescued” by adding a different trafficking signal. Intriguingly, Mid2 variants with medium-length TMDs appeared to accumulate in the Golgi apparatus, hinting that TMD length might affect exit from the Golgi as well as the ER. This finding suggests the PAIRS approach might be used to shed light on the cargo sorting rules that apply to membrane protein traffic from the Golgi, as well as between other compartments. Of the 157 cargo proteins the researchers analyzed, 126 were still able to exit the ER when any one of the nine candidate receptors was mutated, suggesting they may bind non-specifically to the proteins that coat the vesicles leaving the ER, or that they bind to cargo receptors that remain to be discovered. One of the next steps will be to uncover the many more cargo receptors that may exist in the more than 50 percent of yeast proteins that still have unknown function. Herzig Y, Sharpe HJ, Elbaz Y, Munro S, Schuldiner M (2012) A Systematic Approach to Pair Secretory Cargo Receptors with Their Cargo Suggests a Mechanism for Cargo Selection by Erv14. doi:10.1371/journal.pbio.1001329

A Systematic Approach to Pair Secretory Cargo Receptors with Their Cargo Suggests a Mechanism for Cargo Selection by Erv14

Author SummaryAll cells sense their environment, respond to it, and communicate with neighboring cells. To perform these functions, cells use an impressive array of proteins that they display on their surface membranes and secrete into their external environment. Newly synthesized proteins destined for the surface of nucleated cells, or to be secreted into the environment must enter the secretory pathway through the endoplasmic reticulum. Those that reside there remain behind, but most leave for their next destination as cargo proteins in lipid vesicles. To be packaged into vesicles, many of them require a “cargo receptor,” which recognizes and tethers specific cargo proteins in the vesicles. Our study takes a systematic approach to identify the range of cargo proteins that bind to each of the known receptors in yeast. By using this approach, we both discover new cargo for known cargo receptors and delineate the rule that governs cargo selection for one cargo receptor, Erv14. Thus, our study demonstrates a novel approach to identify the cargo for any receptor or to discover new cargo receptors.

Sec1/Munc18 Protein Stabilizes Fusion-Competent Syntaxin for Membrane Fusion in Arabidopsis Cytokinesis

Intracellular membrane fusion requires complexes of syntaxins with other SNARE proteins and regulatory Sec1/Munc18 (SM) proteins. In membrane fusion mediating, e.g., neurotransmitter release or glucose-stimulated insulin secretion in mammals, SM proteins preferentially interact with the inactive closed, rather than the active open, conformation of syntaxin or with the assembled SNARE complex. Other membrane fusion processes such as vacuolar fusion in yeast involve like membranes carrying cis-SNARE complexes, and the role of SM protein is unknown. We investigated syntaxin-SM protein interaction in membrane fusion of Arabidopsis cytokinesis, which involves cytokinesis-specific syntaxin KNOLLE and SM protein KEULE. KEULE interacted with an open conformation of KNOLLE that complemented both knolle and keule mutants. This interaction occurred at the cell division plane and required the KNOLLE linker sequence between helix Hc and SNARE domain. Our results suggest that in cytokinesis, SM protein stabilizes the fusion-competent open form of syntaxin, thereby promoting trans-SNARE complex formation.