Cnidarian-Symbiodiniaceae symbiosis establishment is independent of photosynthesis.

Photosynthesis shapes the symbiotic relationships between cnidarians and Symbiodiniaceae algae-with many cnidarian hosts requiring symbiont photosynthate for survival-but little is known about how photosynthesis impacts symbiosis establishment. Here, we show that during symbiosis establishment, infection, proliferation, and maintenance can proceed without photosynthesis, but the ability to do so is dependent on specific cnidarian-Symbiodiniaceae relationships. The evaluation of 31 pairs of symbiotic relationships (five species of Symbiodiniaceae in sea anemone, coral, and jellyfish hosts) revealed that infection can occur without photosynthesis. A UV mutagenesis method for Symbiodiniaceae was established and used to generate six photosynthetic mutants that can infect these hosts. Without photosynthesis, Symbiodiniaceae cannot proliferate in the sea anemone Aiptasia or jellyfish Cassiopea but can proliferate in the juvenile polyps of the coral Acropora. After 6 months of darkness, Breviolum minutum is maintained within Aiptasia, indicating that Symbiodiniaceae maintenance can be independent of photosynthesis. Manipulating photosynthesis provides insights into cnidarian-Symbiodiniaceae symbiosis.

Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.

The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function. This article is protected by copyright. All rights reserved.

Recombinant MBP-pσ1 expressed in soybean seeds delays onset and reduces developing disease in an animal model of multiple sclerosis.

It is estimated that multiple sclerosis (MS) affects over 2.8 million people worldwide, with a prevalence that is expected to continue growing over time. Unfortunately, there is no cure for this autoimmune disease. For several decades, antigen-specific treatments have been used in animal models of experimental autoimmune encephalomyelitis (EAE) to demonstrate their potential for suppressing autoimmune responses. Successes with preventing and limiting ongoing MS disease have been documented using a wide variety of myelin proteins, peptides, autoantigen-conjugates, and mimics when administered by various routes. While those successes were not translatable in the clinic, we have learned a great deal about the roadblocks and hurdles that must be addressed if such therapies are to be useful. Reovirus sigma1 protein (pσ1) is an attachment protein that allows the virus to target M cells with high affinity. Previous studies showed that autoantigens tethered to pσ1 delivered potent tolerogenic signals and diminished autoimmunity following therapeutic intervention. In this proof-of-concept study, we expressed a model multi-epitope autoantigen (human myelin basic protein, MBP) fused to pσ1 in soybean seeds. The expression of chimeric MBP-pσ1 was stable over multiple generations and formed the necessary multimeric structures required for binding to target cells. When administered to SJL mice prophylactically as an oral therapeutic, soymilk formulations containing MBP-pσ1 delayed the onset of clinical EAE and significantly reduced developing disease. These results demonstrate the practicality of soybean as a host for producing and formulating immune-modulating therapies to treat autoimmune diseases.

New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System.

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Influence of Cationic meso-Substituted Porphyrins on the Antimicrobial Photodynamic Efficacy and Cell Membrane Interaction in Escherichia coli.

Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges (1⁻4) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, Escherichia coli (E. coli), and their interaction with the cell membrane. The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1⁻3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1⁻3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.

Lipid trafficking by yeast Snx4 family SNX-BAR proteins promotes autophagy and vacuole membrane fusion.

Cargo-selective and nonselective autophagy pathways employ a common core autophagy machinery that directs biogenesis of an autophagosome that eventually fuses with the lysosome to mediate turnover of macromolecules. In yeast ( Saccharomyces cerevisiae) cells, several selective autophagy pathways fail in cells lacking the dimeric Snx4/Atg24 and Atg20/Snx42 sorting nexins containing a BAR domain (SNX-BARs), which function as coat proteins of endosome-derived retrograde transport carriers. It is unclear whether endosomal sorting by Snx4 proteins contributes to autophagy. Cells lacking Snx4 display a deficiency in starvation induced, nonselective autophagy that is severely exacerbated by ablation of mitochondrial phosphatidylethanolamine synthesis. Under these conditions, phosphatidylserine accumulates in the membranes of the endosome and vacuole, autophagy intermediates accumulate within the cytoplasm, and homotypic vacuole fusion is impaired. The Snx4-Atg20 dimer displays preference for binding and remodeling of phosphatidylserine-containing membrane in vitro, suggesting that Snx4-Atg20-coated carriers export phosphatidylserine-rich membrane from the endosome. Autophagy and vacuole fusion are restored by increasing phosphatidylethanolamine biosynthesis via alternative pathways, indicating that retrograde sorting by the Snx4 family sorting nexins maintains glycerophospholipid homeostasis required for autophagy and fusion competence of the vacuole membrane.

Distinct complexes of yeast Snx4 family SNX-BARs mediate retrograde trafficking of Snc1 and Atg27.

The yeast SNX4 sub-family of sorting nexin containing a Bin-Amphiphysin-Rvs domain (SNX-BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4-Atg20 and Snx4-Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat 3 distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.

Biogenesis of endosome-derived transport carriers.

Sorting of macromolecules within the endosomal system is vital for physiological control of nutrient homeostasis, cell motility, and proteostasis. Trafficking routes that export macromolecules from the endosome via vesicle and tubule transport carriers constitute plasma membrane recycling and retrograde endosome-to-Golgi pathways. Proteins of the sorting nexin family have been discovered to function at nearly every step of endosomal transport carrier biogenesis and it is becoming increasingly clear that they form the core machineries of cargo-specific transport pathways that are closely integrated with cellular physiology. Here, we summarize recent progress in elucidating the pathways that mediate the biogenesis of endosome-derived transport carriers.

Fission of SNX-BAR-coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1.

Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR-coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.

Structural insights into assembly and regulation of the plasma membrane phosphatidylinositol 4-kinase complex.

Plasma membrane PI4P helps determine the identity of this membrane and plays a key role in signal transduction as the precursor of PI(4,5)P2 and its metabolites. Here, we report the atomic structure of the protein scaffold that is required for the plasma membrane localization and function of Stt4/PI4KIIIα, the PI 4-kinase responsible for this PI4P pool. Both proteins of the scaffold, Efr3 and YPP1/TTC7, are composed of α-helical repeats, which are arranged into a rod in Efr3 and a superhelix in Ypp1. A conserved basic patch in Efr3, which binds acidic phospholipids, anchors the complex to the plasma membrane. Stt4/PI4KIIIα is recruited by interacting with the Ypp1 C-terminal lobe, which also binds to unstructured regions in the Efr3 C terminus. Phosphorylation of this Efr3 region counteracts Ypp1 binding, thus providing a mechanism through which Stt4/PI4KIIIα recruitment, and thus a metabolic reaction of fundamental importance in cell physiology, can be regulated.

Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport.

Vesicle-mediated protein transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, protein transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for protein transport in this pathway are unknown. Here, we show that the Arf GTPase-activating protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular transport between the EE and the TGN.

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis.

Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis. In this study we analyzed scd5-PP1Δ2, a mutation causing impaired PP1 binding. scd5-PP1Δ2 caused hyperphosphorylation of several Prk1 endocytic targets. Live-cell imaging of 15 endocytic components in scd5-PP1Δ2 revealed that most factors arriving before the invagination/actin phase of endocytosis had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homolog), whose lifetime was extended nearly fourfold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than twofold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1Δ2 were suppressed by SLA1 overexpression. This was dependent on the LxxQxTG repeats (SR) of Sla1, which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the dephosphorylated state. Without the SR, Sla1ΔSR was still recruited to the cell surface, but was less concentrated in cortical patches than Pan1. sla1ΔSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region the SH3 region of Sla1 causes constitutive negative regulation of Las17 (WASp). These results demonstrate that Scd5/PP1 is important for recycling Prk1 targets to initiate new rounds of endocytosis and provide new mechanistic information on the role of the Sla1 SR domain in regulating progression to the invagination/actin phase of endocytosis.

Lessons from yeast for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is the major pathway for internalization of membrane proteins from the cell surface. Half a century of studies have uncovered tremendous insights into how a clathrin-coated vesicle is formed. More recently, the advent of live-cell imaging has provided a dynamic view of this process. As CME is highly conserved from yeast to humans, budding yeast provides an evolutionary template for this process and has been a valuable system for dissecting the underlying molecular mechanisms. In this review we trace the formation of a clathrin-coated vesicle from initiation to uncoating, focusing on key findings from the yeast system.

Clathrin functions in the absence of the terminal domain binding site for adaptor-associated clathrin-box motifs.

Clathrin is involved in vesicle formation in the trans-Golgi network (TGN)/endosomal system and during endocytosis. Clathrin recruitment to membranes is mediated by the clathrin heavy chain (HC) N-terminal domain (TD), which forms a seven-bladed beta-propeller. TD binds membrane-associated adaptors, which have short peptide motifs, either the clathrin-box (CBM) and/or the W-box; however, the importance of the TD binding sites for these motifs has not been tested in vivo. We investigated the importance of the TD in clathrin function by generating 1) mutations in the yeast HC gene (CHC1) to disrupt the binding sites for the CBM and W-box (chc1-box), and 2) four TD-specific temperature-sensitive alleles of CHC1. We found that TD is important for the retention of resident TGN enzymes and endocytosis of alpha-factor; however, the known adaptor binding sites are not necessary, because chc1-box caused little to no effect on trafficking pathways involving clathrin. The Chc1-box TD was able to interact with the endocytic adaptor Ent2 in a CBM-dependent manner, and HCs encoded by chc1-box formed clathrin-coated vesicles. These data suggest that additional or alternative binding sites exist on the TD propeller to help facilitate the recruitment of clathrin to sites of vesicle formation.

Smooth muscle titin Zq domain interaction with the smooth muscle alpha-actinin central rod.

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein alpha-actinin. In striated muscle Z-disks, alpha-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in alpha-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the alpha-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to alpha-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in alpha-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for alpha-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the alpha-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the alpha-actinin central rod.

Smooth muscle alpha-actinin interaction with smitin.

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells also contain the actin filament-crosslinking protein alpha-actinin. In striated muscle sarcomeres, interactions between the myosin-binding protein titin and alpha-actinin in the Z-line provide an important structural linkage. We previously discovered a titin-like protein, smitin, associated with the contractile apparatus of smooth muscle cells. Purified native smooth muscle alpha-actinin binds with nanomolar affinity to smitin in smitin-myosin coassemblies in vitro. Smooth muscle alpha-actinin also interacts with striated muscle titin. In contrast to striated muscle alpha-actinin interaction with titin and smitin, which is significantly enhanced by PIP2, smooth muscle alpha-actinin interacts with smitin and titin equally well in the presence and absence of PIP2. Using expressed regions of smooth muscle alpha-actinin, we have demonstrated smitin-binding sites in the smooth muscle alpha-actinin R2-R3 spectrin-like repeat rod domain and a C-terminal domain formed by cryptic EF-hand structures. These smitin-binding sites are highly homologous to the titin-binding sites of striated muscle alpha-actinin. Our results suggest that direct interaction between alpha-actinin and titin or titin-like proteins is a common feature of actin-myosin II contractile structures in striated muscle and smooth muscle cells and that the molecular bases for alpha-actinin interaction with these proteins are similar, although regulation of these interactions may differ according to tissue.