The Sec1/Munc18-like proteins TgSec1 and TgVps45 play pivotal roles in assembly of the pellicle and sub-pellicle network in Toxoplasma gondii

Post-Golgi vesicle trafficking is indispensable for precise movement of proteins to the pellicle, the sub-pellicle network and apical secretory organelles in Apicomplexa. However, only a small number of molecular complexes involved in trafficking, tethering and fusion of vesicles have been identified in Toxoplasma gondii. Consequently, it is unclear how complicated vesicle trafficking is accomplished in this parasite. Sec1/Munc18-like (SM) proteins are essential components of protein complexes involved in vesicle fusion. Here, we found that depletion of the SM protein TgSec1 using an auxin-inducible degron-based conditional knockout strategy led to mislocalization of plasma membrane proteins. By contrast, conditional depletion of the SM protein TgVps45 led to morphological changes, asymmetrical loss of the inner membrane complex and defects in nucleation of sub-pellicular microtubules, polarization and symmetrical assembly of daughter parasites during repeated endodyogeny. TgVps45 interacts with the SNARE protein TgStx16 and TgVAMP4-1. Conditional ablation of TgStx16 causes the similar growth defect like TgVps45 deficiency suggested they work together for the vesicle fusion at TGN. These findings indicate that these two SM proteins are crucial for assembly of pellicle and sub-pellicle network in T. gondii respectively.     

Cystinosin, MPDU1, SWEETs and KDELR belong to a well-defined protein family with putative function of cargo receptors involved in vesicle trafficking

Classification of proteins into families based on remote homology often helps prediction of their biological function. Here we describe prediction of protein cargo receptors involved in vesicle formation and protein trafficking. Hidden Markov model profile-to-profile searches in protein databases using endoplasmic reticulum lumen protein retaining receptors (KDEL, Erd2) as query reveal a large and diverse family of proteins with seven transmembrane helices and common topology and, most likely, similar function. Their coding genes exist in all eukaryota and in several prokaryota. Some are responsible for metabolic diseases (cystinosis, congenital disorder of glycosylation), others are candidate genes for genetic disorders (cleft lip and palate, certain forms of cancer) or solute uptake and efflux (SWEETs) and many have not yet been assigned a function. Comparison with the properties of KDEL receptors suggests that the family members could be involved in protein trafficking and serve as cargo receptors. This prediction sheds new light on a range of biologically, medically and agronomically important proteins and could open the way to discovering the function of many genes not yet annotated. Experimental testing is suggested.     

Suppression of coatomer mutants by a new protein family with COPI and COPII binding motifs in Saccharomyces cerevisiae

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (gamma-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.     

GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus

The organelles within secretory and endocytotic pathways in mammalian cells have acidified lumens, and regulation of their acidic pH is critical for the trafficking, processing and glycosylation of cargo proteins and lipids, as well as the morphological integrity of the organelles. How organelle lumen acidification is regulated, and how luminal pH elevation disturbs these fundamental cellular processes, is largely unknown. Here, we describe a novel molecule involved in Golgi acidification. First, mutant cells defective in Golgi acidification were established that exhibited delayed protein transport, impaired glycosylation and Golgi disorganization. Using expression cloning, a novel Golgi-resident multi-transmembrane protein, named Golgi pH regulator (GPHR), was identified as being responsible for the mutant cells. After reconstitution in planar lipid bilayers, GPHR exhibited a voltage-dependent anion-channel activity that may function in counterion conductance. Thus, GPHR modulates Golgi functions through regulation of acidification.     

Rab proteins in gastric parietal cells: evidence for the membrane recycling hypothesis.

The gastric parietal cell secretes large quantities of HCl into the lumen of the gastric gland in response to secretagogues such as histamine. In the membrane recycling hypothesis, this secretory activity requires the trafficking of the gastric H+/K(+)-ATPase to the cell surface from intracellular tubulovesicles. The Rab subclass of small GTP-binding proteins is thought to confer specificity to vesicle transport throughout the secretory pathway, and previous investigations established that Rab11 is highly expressed in gastric parietal cells. Recent discoveries in intra-Golgi transport and neuronal synaptic vesicle fusion have fortuitously converged on an evolutionarily conserved protein complex involved in vesicle docking and fusion. Recent results indicate that Rab11 is involved in the apical targeting of vesicles in parietal cells and other epithelial cells throughout the gastrointestinal tract. In support of the membrane recycling hypothesis, Rab co-segregates with H+/K(+)-ATPase in parietal cells. The presence of Rab11 on tubulovesicles supports a role for this Rab protein in recycling vesicle trafficking.     

Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway

Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Myelin biogenesis: vesicle transport in oligodendrocytes

Intracellular trafficking of membranes plays an essential role in the biogenesis and maintenance of myelin. The requisite proteins and lipids are transported from their sites of synthesis to myelin via vesicles. Vesicle transport is tightly coordinated with synthesis of lipids and proteins. To maintain the structural and functional organization of oligodendrocytes it is essential synchronize the various pathways of vesicle transport and to coordinate vesicle transport with reorganization of cytoskeleton. The systems that regulate the targeting of protein to myelin by vesicle transport are now being described. Here we review the current knowledge of these systems including those involved in (a) protein folding, (b) protein sorting and formation of carrier vesicles, (c) vesicle transport along elements of the cytoskeleton, and (d) vesicle targeting/fusion.     

TgDrpC,an atypical dynamin‐related protein in Toxoplasma gondii,is associated with vesicular transport factors and parasite division

Dynamin‐related proteins (Drps) are involved in diverse processes such as organelle division and vesicle trafficking. The intracellular parasite Toxoplasma gondii possesses three distinct Drps. TgDrpC, whose function remains unresolved, is unusual in that it lacks a conserved GTPase Effector Domain, which is typically required for function. Here, we show that TgDrpC localizes to cytoplasmic puncta; however, in dividing parasites, TgDrpC redistributes to the growing edge of the daughter cells. By conditional knockdown, we determined that loss of TgDrpC stalls division and leads to rapid deterioration of multiple organelles and the IMC. We also show that TgDrpC interacts with proteins that exhibit homology to those involved in vesicle transport, including members of the adaptor complex 2. Two of these proteins, a homolog of the adaptor protein 2 (AP‐2) complex subunit alpha‐1 and a homolog of the ezrin–radixin–moesin (ERM) family proteins, localize to puncta and associate with the daughter cells. Consistent with the association with vesicle transport proteins, re‐distribution of TgDrpC to the IMC during division is dependent on post‐Golgi trafficking. Together, these results support that TgDrpC contributes to vesicle trafficking and is critical for stability of parasite organelles and division.     

Quantitative proteomics identified 3 oxidative phosphorylation genes with clinical prognostic significance in gastric cancer

The aim of the present study was to explore the underlying mechanisms involved in gastric cancer (GC) formation using data‐independent acquisition (DIA) quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in biological metabolic processes in GC. Totally, 745 differentially expressed proteins (DEPs) were found in GC tissues vs. gastric normal tissues. Despite enormous complexity in the details of the underlying regulatory network, we find that clusters of proteins from the DEPs were mainly involved in 38 pathways. All of the identified DEPs involved in oxidative phosphorylation were down‐regulated. Moreover, GC possesses significantly altered biological metabolic processes, such as NADH dehydrogenase complex assembly and tricarboxylic acid cycle, which is mostly consistent with that in KEGG analysis. Furthermore the higher expression of UQCRQ, NDUFB7 and UQCRC2 were positively correlated with a better prognosis, implicating these proteins may as novel candidate diagnostic and prognostic biomarkers.     

Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.     

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila

The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.     

In Vivo Neuron‐Wide Analysis of Synaptic Vesicle Precursor Trafficking

During synapse development, synaptic proteins must be targeted to sites of presynaptic release. Directed transport as well as local sequestration of synaptic vesicle precursors (SVPs), membranous organelles containing many synaptic proteins, might contribute to this process. Using neuron‐wide time‐lapse microscopy, we studied SVP dynamics in the DA9 motor neuron in Caenorhabditis elegans. SVP transport was highly dynamic and bi‐directional throughout the entire neuron, including the dendrite. While SVP trafficking was anterogradely biased in axonal segments prior to the synaptic domain, directionality of SVP movement was stochastic in the dendrite and distal axon. Furthermore, frequency of movement and speed were variable between different compartments. These data provide evidence that SVP transport is differentially regulated in distinct neuronal domains. It also suggests that polarized SVP transport in concert with local vesicle capturing is necessary for accurate presynapse formation and maintenance. SVP trafficking analysis of two hypomorphs for UNC‐104/KIF1A in combination with mathematical modeling identified directionality of movement, entry of SVPs into the axon as well as axonal speeds as the important determinants of steady‐state SVP distributions. Furthermore, detailed dissection of speed distributions for wild‐type and unc‐104/kif1a mutant animals revealed an unexpected role for UNC‐104/KIF1A in dendritic SVP trafficking.      

Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport

Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P₄-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P₄-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P₄-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.     

SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana [corrected

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22 degrees C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.     

Early steps in plastid evolution: current ideas and controversies

Some nuclear‐encoded proteins are imported into higher plant plastids via the endomembrane (EM) system. Compared with multi‐protein Toc and Tic translocons required for most plastid protein import, the relatively uncomplicated nature of EM trafficking led to suggestions that it was the original transport mechanism for nuclear‐encoded endosymbiont proteins, and critical for the early stages of plastid evolution. Its apparent simplicity disappears, however, when EM transport is considered in light of selective constraints likely encountered during the conversion of stable endosymbionts into fully integrated organelles. From this perspective it is more parsimonious to presume the early evolution of post‐translational protein import via simpler, ancestral forms of modern Toc and Tic plastid translocons, with EM trafficking arising later to accommodate glycosylation and/or protein targeting to multiple cellular locations. This hypothesis is supported by both empirical and comparative data, and is consistent with the relative paucity of EM‐based transport to modern primary plastids.     

Analysis of COPII Vesicles Indicates a Role for the Emp47–Ssp120 Complex in Transport of Cell Surface Glycoproteins

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium‐binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47–Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.      

Ras subfamily GTPases regulate development,aflatoxin biosynthesis and pathogenicity in the fungus Aspergillus flavus

Ras subfamily proteins are molecular switches in signal transduction pathways of many eukaryotes that regulate a variety of cellular processes. Here, the Ras subfamily, encoded by six genes, was identified in Aspergillus flavus: rasA, rasB, rasC, rab-33, rheb and rsr1. The rsr1 deletion mutant (∆rsr1), rheb deletion mutant (∆rheb) and double deletion mutant (∆rheb/rsr1) displayed significantly decreased growth and sporulation. Sclerotia formation was significantly decreased for ∆rheb or ∆rheb/rsr1 but increased for ∆rsr1. Aflatoxin production was significantly increased in ∆rheb but decreased in ∆rsr1 and ∆rheb/rsr1. We found that rsr1 and rheb are crucial for the pathogenicity of A. flavus. Quantitative proteomics identified 520 differentially expressed proteins (DEPs) for the ∆rsr1 mutant and 133 DEPs for the ∆rheb mutant. These DEPs were annotated in multiple biological processes and KEGG pathways in A. flavus. Importantly, we identified the cytokinesis protein SepA in the protein–protein interaction network of rsr1, and deletion mutants showed that SepA has pleiotropic effects on growth and AF biosynthesis, which may depend on Rsr1 for regulation in A. flavus. Our results indicated that these Ras subfamily proteins exhibited functional redundancy with each other but there were also differences in A. flavus.     

GFS9/TT9 contributes to intracellular membrane trafficking and flavonoid accumulation in Arabidopsis thaliana

Flavonoids are the most important pigments for the coloration of flowers and seeds. In plant cells, flavonoids are synthesized by a multi‐enzyme complex located on the cytosolic surface of the endoplasmic reticulum, and they accumulate in vacuoles. Two non‐exclusive pathways have been proposed to mediate flavonoid transport to vacuoles: the membrane transporter‐mediated pathway and the vesicle trafficking‐mediated pathway. No molecules involved in the vesicle trafficking‐mediated pathway have been identified, however. Here, we show that a membrane trafficking factor, GFS9, has a role in flavonoid accumulation in the vacuole. We screened a library of Arabidopsis thaliana mutants with defects in vesicle trafficking, and isolated the gfs9 mutant with abnormal pale tan‐colored seeds caused by low flavonoid accumulation levels. gfs9 is allelic to the unidentified transparent testa mutant tt9. The responsible gene for these phenotypes encodes a previously uncharacterized protein containing a region that is conserved among eukaryotes. GFS9 is a peripheral membrane protein localized at the Golgi apparatus. GFS9 deficiency causes several membrane trafficking defects, including the mis‐sorting of vacuolar proteins, vacuole fragmentation, the aggregation of enlarged vesicles, and the proliferation of autophagosome‐like structures. These results suggest that GFS9 is required for vacuolar development through membrane fusion at vacuoles. Our findings introduce a concept that plants use GFS9‐mediated membrane trafficking machinery for delivery of not only proteins but also phytochemicals, such as flavonoids, to vacuoles.