Membrane trafficking mediated by OsDRP2B is specific for cellulose biosynthesis

Increasing evidence has revealed that membrane trafficking is highly associated with cell wall metabolism. Factors involved in vesicle delivery, e.g., cytoskeleton and motor proteins, have showed regulatory effects on cell wall structure and components. However, little is known about the involvement of other trafficking components in distribution of cell wall-related compartments. Dynamins are important proteins functioning in membrane tubulation and vesiculation. Recently, we have reported characterization of the rice dynamin-related protein 2B (OsDRP2B). Mutation in OsDRP2B causes a significant reduction in cellulose content. Its association with the trans-Golgi network (TGN) and clathrin-coated vesicles and the reduced CESA4 abundance at the bc3 plasma membrane suggest that BC3/OsDRP2B is involved in the transport of essential elements for cellulose synthesis. Here, we provide additional evidence for BC3 subcellular localization via observing OsDRP2B-GFP in living root hairs of transgenic plants. Uronic acid and fractional composition analyses further confirm that the amount of arabinoxylan and other noncellulosic polysaccharides is increased in bc3. However, three putative xylan synthesis genes are downregulated in mutant plant revealed by real-time PCR analysis. These results imply that compartments delivered by OsDRP2B are specifically responsible for cellulose biosynthesis.Key words: OsDRP2B, cellulose biosynthesis, membrane trafficking, brittleness, ricePlant cell wall is an extracellular matrix enriched in polysaccharides. Except for cellulose that is produced at the plasma membrane by cellulose synthase (CESA) complexes, most of the cell wall products are assumed being synthesized inside cells, e.g., in the Golgi apparatus and secreted outside through complex membrane trafficking. Besides the cell wall-localized products, some proteins essential for cellulose biosynthesis need to be translocated onto the plasma membrane to facilitate cellulose formation.^1,2 Intracellular trafficking is therefore a key level for regulating cell wall composition and architecture, which are highly dynamic during cellular development.³ This notion is substantiated by the fact that wall architecture within the same cell is heterogeneity, indicating the presence of cell wall specific deposition domains.^4,5 For example, pectins are often located at the cell corners.³ Different de-esterified homogalacturonan (HG) are present along the growing pollen tubes or root hairs: tips have highly esterified HG; the de-esterified degree is increased after tips.⁶ Although it is believed that these specific patterns could be the result of the targeted secretion of polysaccharides,³ our knowledge about the polysaccharide secretion is still very few. Currently, in vivo viewing CESA-containing compartments and the movement inside living cells have provided direct evidence for the trafficking action of CESA compartments.^2,7,8 The delivery and removal of CESA complexes to/from the plasma membrane are very complicated, which require the involvement of many components, such as cytoskeleton and syntaxins.^7,9,10 Syntaxins, part of SNARE complexes, function as docking factor of cell wall-related compartments during cell plate formation.¹⁰ Dynamin and dynamin-related proteins (DRPs) are involved in diverse events of cellular membrane remodeling.¹¹ It remains unknown about whether DRPs are responsible for CESA trafficking. Recently, we have reported that BC3, the rice DRP2B protein, plays a role in complex membrane trafficking and affects the biosynthesis of secondary walls. Here, we provide additional cellular and wall chemical data to confirm that BC3/OsDRP2B is specifically involved in the secondary cell wall cellulose synthesis.     

RAB2A controls MT1‐MMP endocytic and E‐cadherin polarized Golgi trafficking to promote invasive breast cancer programs

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER‐to‐Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post‐endocytic trafficking of membrane‐bound MT1‐MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E‐cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.     

A Novel Fluorescent Labeling Method Enables Monitoring of Spatio‐Temporal Dynamics of Developing Microsporidia

The microsporidium, Anncaliia algerae (Brachiola algerae), is a eukaryotic obligate intracellular parasite first isolated from mosquitoes and is an important opportunistic human pathogen that can cause morbidity and mortality among immune‐compromised individuals including patients with AIDS and those undergoing chemotherapy. There is little known about the Microsporidia–host cell interface in living host cells, due to current approaches being limited by the lack of fluorescent reporters for detecting the parasite lifecycle. Here, we have developed and applied novel vital fluorescent parasite labeling methodologies in conjunction with fluorescent protein‐tagged reporters to track simultaneously the dynamics of both parasite and host cell specific components, including the secretory and endocytic trafficking pathways, during the entire infection time period. We have found dramatic changes in the dynamics of host secretory trafficking organelles during the course of infection. The Golgi compartment is gradually disassembled and regenerated into mini‐Golgi structures in parallel with cellular microtubule depolymerization. Importantly, we find that Microsporidia progeny are associated with these de novo formed mini‐Golgi structures. These host structures appear to create a membrane bound niche environment for parasite development. Our studies presented here provide novel imaging tools and methodologies that will facilitate in understanding the biology of microsporidial parasites in the living host.     

Ferlins Show Tissue‐Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans‐Golgi/Recycling Ferlins

Ferlins are a family of transmembrane‐anchored vesicle fusion proteins uniquely characterized by 5–7 tandem cytoplasmic C2 domains, Ca²⁺‐regulated phospholipid‐binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb‐girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca²⁺‐dependent, vesicle‐mediated membrane repair and otoferlin mutations cause non‐syndromic deafness due to defective Ca²⁺‐triggered auditory neurotransmission. In this study, we describe the tissue‐specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D‐structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7‐positive late endosomes, supporting potential roles in the late‐endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans‐Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans‐Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type‐I and type‐II ferlins segregate as PM/late‐endosomal or trans‐Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.      

The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus

Membrane trafficking is vital to plant development and adaptation to the environment. It is suggested that post‐Golgi vesicles and multivesicular bodies are essential for plant defence against directly penetrating fungal parasites at the cell wall. However, the actual plant proteins involved in membrane transport for defence are largely unidentified. We applied a candidate gene approach and single cell transient‐induced gene silencing for the identification of membrane trafficking proteins of barley involved in the response to the fungal pathogen Blumeria graminis f.sp. hordei. This revealed potential components of vesicle tethering complexes [putative exocyst subunit HvEXO70F‐like and subunits of the conserved oligomeric Golgi (COG) complex] and Golgi membrane trafficking (COPIγ coatomer and HvYPT1‐like RAB GTPase) as essential for resistance to fungal penetration into the host cell.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

Aluminium effects on mechanical properties of cell wall analogues

Aluminium (Al) toxicity adversely impacts plant productivity in acid soils by restricting root growth and although several mechanisms are involved the physiological basis of decreased root elongation remains unclear. Understanding the primary mechanisms of Al rhizotoxicity is hindered due to the rapid effects of soluble Al on root growth and the close proximity of many cellular components within the cell wall, plasma membrane, cytosol and nucleus with which Al may react. To overcome some of these difficulties, we report on a novel method for investigating Al interactions with Komagataeibacter xylinus bacterial cellulose (BC)‐pectin composites as cell wall analogues. The growth of K. xylinus in the presence of various plant cell wall polysaccharides, such as pectin, has provided a unique in vitro model system with which to investigate the interactions of Al with plant cell wall polysaccharides. The BC‐pectin composites reacted in a similar way with Al as do plant cell walls, providing insights into the effects of Al on the mechanical properties of the BC‐pectin composites as cell wall analogues. Our findings indicated that there were no significant effects of Al (4–160 μM) on the tensile stress, tensile strain or Young's modulus of the composites. This finding was consistent with cellulose, not pectin, being the major load bearing component in BC‐pectin composites, as is also the case in plant cell walls.     

The trafficking of the cellulose synthase complex in higher plants

Background

Cellulose is an important constituent of plant cell walls in a biological context, and is also a material commonly utilized by mankind in the pulp and paper, timber, textile and biofuel industries. The biosynthesis of cellulose in higher plants is a function of the cellulose synthase complex (CSC). The CSC, a large transmembrane complex containing multiple cellulose synthase proteins, is believed to be assembled in the Golgi apparatus, but is thought only to synthesize cellulose when it is localized at the plasma membrane, where CSCs synthesize and extrude cellulose directly into the plant cell wall. Therefore, the delivery and endocytosis of CSCs to and from the plasma membrane are important aspects for the regulation of cellulose biosynthesis.

Scope

Recent progress in the visualization of CSC dynamics in living plant cells has begun to reveal some of the routes and factors involved in CSC trafficking. This review highlights the most recent major findings related to CSC trafficking, provides novel perspectives on how CSC trafficking can influence the cell wall, and proposes potential avenues for future exploration.     

Chemistry‐based protein modification strategy for endocytic pathway analysis

Background information. The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large‐scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means. Results. Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor‐made chemical tools that are then applied to the step‐by‐step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B‐subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates. Conclusions. Our vectorial concept proposes a new chemical approach for traffic‐based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Trans-Golgi Network—An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.     

Rice BRITTLE CULM 3 (BC3) encodes a classical dynamin OsDRP2B essential for proper secondary cell wall synthesis

“Brittle culm” mutants found in Gramineae crops are suitable materials to study the mechanism of secondary cell wall formation. Through positional cloning, we have identified a gene responsible for the brittle culm phenotype in rice, brittle culm 3 (bc3). BC3 encodes a member of the classical dynamin protein family, a family known to function widely in membrane dynamics. The bc3 mutation resulted in reductions of 28–36% in cellulose contents in culms, leaves, and roots, while other cell wall components remained unaffected. Reductions of cell wall thickness and birefringence were observed in both fiber (sclerenchyma) and parenchymal cells, together with blurring of the wall’s layered structures. From promoter-GUS analyses, it was suggested that BC3 expression is directly correlated with active secondary cell wall synthesis. These results suggest that BC3 is tightly involved in the synthesis of cellulose and is essential for proper secondary cell wall construction.     

Rice cellulose synthase‐like D4 is essential for normal cell‐wall biosynthesis and plant growth

Cellulose synthase‐like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell‐wall polymers. The CSL D sub‐family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in‐depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map‐based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub‐family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C‐ or N‐terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype‐rescued transgenic plants expressing OsCSLD4–GUS under the control of its own promoter. Two phenotype‐altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell‐wall formation and plant growth.     

Intersection of endocytic and exocytic systems in Toxoplasma gondii

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome‐like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant‐like features suggest T. gondii endocytic trafficking involves transit through early and late endosome‐like compartments (ELCs) and potentially the trans‐Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin‐related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post‐invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host‐derived proteins, suggesting that DrpB is not required for parasite endocytosis.      

DKWSLLL,a Versatile DXXXLL‐Type Signal with Distinct Roles in the Cu+‐Regulated Trafficking of ATP7B

In the liver, the P‐type ATPase and membrane pump ATP7B plays a crucial role in Cu⁺ donation to cuproenzymes and in the elimination of excess Cu⁺. ATP7B is endowed with a COOH‐cytoplasmic (DE)XXXLL‐type traffic signal. We find that accessory (Lys ?3, Trp ?2, Ser ?1 and Leu +2) and canonical (D ?4, Leu 0 and Leu +1) residues confer the DKWSLLL signal with the versatility required for the Cu⁺‐regulated cycling of ATP7B between the trans‐Golgi network (TGN) and the plasma membrane (PM). The separate mutation of these residues caused a disruption of the signal, resulting in different ATP7B distribution phenotypes. These phenotypes indicate the key roles of specific residues at separate steps of ATP7B trafficking, including sorting at the TGN, transport from the TGN to the PM and its endocytosis, and recycling to the TGN and PM. The distinct roles of ATP7B in the TGN and PM and the variety of phenotypes caused by the mutation of the canonical and accessory residues of the DKWSLLL signal can explain the separate or joined presentation of Wilson's cuprotoxicosis and the dysfunction of the cuproenzymes that accept Cu⁺ at the TGN.      

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v‐SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)‐repressive conditions, although in wild‐type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post‐Golgi endosomes. The mislocalized Snc1 was co‐localized with an endocytic marker dye, FM4‐64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase‐activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP‐restricted form of Ypt32 GTPase. Furthermore, an endocytosis‐deficient mutant of Snc1 was localized to plasma membranes in PSS1‐repressed csg2Δ mutant cells as well as wild‐type cells. Thus, the PSS1‐repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post‐Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre‐vacuolar endosomes in the PSS1‐repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co‐ordinately involved in specific vesicular trafficking pathway.     

The Rab GTPase YPT‐1 associates with Golgi cisternae and Spitzenkörper microvesicles in Neurospora crassa

Vesicle traffic involves budding, transport, tethering and fusion of vesicles with acceptor membranes. GTP‐bound small Rab GTPases interact with the membrane of vesicles, promoting their association with other factors before their subsequent fusion. Filamentous fungi contain at their hyphal apex the Spitzenkörper (Spk), a multivesicular structure to which vesicles concentrate before being redirected to specific cell sites. The regulatory mechanisms ensuring the directionality of the vesicles that travel to the Spk are still unknown. Hence, we analyzed YPT‐1, the Neurospora crassa homologue of Saccharomyces cerevisiae Ypt1p (Rab1), which regulates different secretory pathway events. Laser scanning confocal microscopy revealed fluorescently tagged YPT‐1 at the Spk and putative Golgi cisternae. Co‐expression of YPT‐1 and predicted post‐Golgi Rab GTPases showed YPT‐1 confined to the Spk microvesicular core, while SEC‐4 (Rab8) and YPT‐31 (Rab11) occupied the Spk macrovesicular peripheral layer, suggesting that trafficking and organization of macro and microvesicles at the Spk are regulated by distinct Rabs. Partial colocalization of YPT‐1 with USO‐1 (p115) and SEC‐7 indicated the additional participation of YPT‐1 at early and late Golgi trafficking steps.     

Coxsackievirus B3 proteins directionally complement each other to downregulate surface major histocompatibility complex class I

Picornaviruses carry a small number of proteins with diverse functions that subvert and exploit the host cell. We have previously shown that three coxsackievirus B3 (CVB3) proteins (2B, 2BC, and 3A) target the Golgi complex and inhibit protein transit. Here we investigate these effects in more detail and evaluate the distribution of major histocompatibility complex (MHC) class I molecules, which are critical mediators of the CD8(+) T-cell response. We report that concomitant with viral protein synthesis, MHC class I surface expression is rapidly downregulated during infection. However, this phenomenon may not result solely from inhibition of anterograde trafficking; we propose a new mechanism whereby the CVB3 2B and 2BC proteins upregulate the internalization of MHC class I (and possibly other surface proteins), perhaps by focusing of endocytic vesicles at the Golgi complex. Thus, our findings indicate that CVB3 carries at least three nonstructural proteins that directionally complement one another; 3A disrupts the Golgi complex to inhibit anterograde transport, while 2B and/or 2BC upregulates endocytosis, rapidly removing proteins from the cell surface. Taken together, these effects may render CVB3-infected cells invisible to CD8(+) T cells and untouchable by many antiviral effector molecules. This has important implications for immune evasion by CVB3.