Promotes Vesicle Formation Research Articles

Transcriptome and ultrastructural analysis revealed the mechanism of Mercapto-palygorskite on reducing Cd content in wheat

Large areas of crop yields in northern China have faced with cadmium (Cd) contamination problems. Mercapto-modified palygorskite (MP), as a highly efficient immobilization material, could reduce Cd absorption in wheat and alleviate its biotoxicity. However, the molecular mechanism underlying MP-mediated Cd reduction and detoxification processes in wheat is not well understood. This aim of this study was to investigate the biochemical and molecular mechanisms underlying the reduction in Cd accumulation in wheat (Triticum aestivum L.). The results showed that MP application decreased the Cd concentration by 68.91–74.32% (root) and 70.68–77.2% (shoot), and significantly increased the glutathione (GSH) and phytochelatins (PCs) contents in root and shoot. In addition, with the application of MP, the percentage of Cd in the cell walls and organelles of wheat decreased, while that of Cd in soluble components was increased. The content of Cd in all components was significantly reduced. Ultrastructural analysis revealed that MP thickened the cell wall, promoted vesicle formation in the membrane and protected the integrity of intracellular organelles in wheat. Transcriptome analysis further confirmed the above results. MP upregulated the expression of several genes (CCR, CAD COMT and SUS) involved in cell wall component biosynthesis and promoted vesicle formation on cell membranes by upregulating the expression of PLC and IPMK genes. In addition, genes related to antioxidant synthesis (PGD, glnA and GSS) and photosynthesis (Lhca, Lhcb) were altered by MP to alleviate Cd toxicity in wheat. This present work will help to more thoroughly elucidate the molecular mechanism by which wheat defends against Cd contamination under MP application and provide and important research basis for the application of this material in the future.

The Spontaneous Vesicle-Micelle Transition in a Catanionic Surfactant System: A Chemical Trapping Study.

Typically, the formation of vesicles requires the addition of salts or other additives to surfactant micelles. However, in the case of catanionic surfactants, unilamellar vesicles can spontaneously form upon dilution of the micellar solutions. Our study explores the intriguing spontaneous vesicle-to-micelle transition in catanionic surfactant systems, specifically cetyltrimethyl ammonium bromide (CTAB) and sodium octylsulfonate (SOS). To gain insights into the changes occurring at the interface, we employ a chemical trapping method to characterize variations in the molarities of sulfonate headgroups, water, and bromide ions during the transition. Our findings reveal the formation of ion pairs between the cationic component of CTAB and the anionic component of SOS, leading to tight interfacial packing in CTAB/SOS solutions. This interfacial packing promotes vesicle formation at low surfactant concentrations. Due to the significant difference in critical micelle concentration (cmc) between CTAB and SOS, an increase in the stoichiometric surfactant concentration results in a substantial rise in the SOS-to-CTAB ratio within the interfacial region. This enrichment of SOS in the aggregates triggers the transition from vesicles to micelles. Overall, our study may shed new light on the design of morphologies in catanionic and other surfactant systems.

Hybrid organic–inorganic structures trigger the formation of primitive cell-like compartments

Alkaline hydrothermal vents have become a candidate setting for the origins of life on Earth and beyond. This is due to several key features including the presence of gradients of temperature, redox potential, pH, the availability of inorganic minerals, and the existence of a network of inorganic pore spaces that could have served as primitive compartments. Chemical gardens have long been used as experimental proxies for hydrothermal vents. This paper investigates-10pc]Please note that the spelling of the following author name in the manuscript differs from the spelling provided in the article metadata: Richard J. G. Löffler. The spelling provided in the manuscript has been retained; please confirm. a set of prebiotic interactions between such inorganic structures and fatty alcohols. The integration of a medium-chain fatty alcohol, decanol, within these inorganic minerals, produced a range of emergent 3 dimensions structures at both macroscopic and microscopic scales. Fatty alcohols can be considered plausible prebiotic amphiphiles that might have assisted the formation of protocellular structures such as vesicles. The experiments presented herein show that neither chemical gardens nor decanol alone promote vesicle formation, but chemical gardens grown in the presence of decanol, which is then integrated into inorganic mineral structures, support vesicle formation. These observations suggest that the interaction of fatty alcohols and inorganic mineral structures could have played an important role in the emergence of protocells, yielding support for the evolution of living cells.

Overexpression of synaptic vesicle protein Rab GTPase 3C promotes vesicular exocytosis and drug resistance in colorectal cancer cells.

Rab GTPase 3C (RAB3C) is a peripheral membrane protein that is involved in membrane trafficking (vesicle formation) and cell movement. Recently, researchers have noted the exocytosis of RAB proteins, and their dysregulation is correlated with drug resistance and the altered tumor microenvironment in tumorigenesis. However, the molecular mechanisms of exocytotic RABs in the carcinogenicity of colorectal cancer (CRC) remain unknown. Researchers have used various in silico datasets to evaluate the expression profiles of RAB family members. We confirmed that RAB3C plays a key role in CRC progression. Its overexpression promotes exocytosis and is related to the resistance to several chemotherapeutic drugs. We established a proteomic dataset based on RAB3C, and found that dystrophin is one of the proteins that is upregulated with the overexpression of RAB3C. According to our results, RAB3C-induced dystrophin expression promotes vesicle formation and packaging. A connectivity map predicted that the cannabinoid receptor 2 (CB2) agonists reverse RAB3C-associated drug resistance, and that these agonists have synergistic effects when combined with standard chemotherapy regimens. Moreover, we found high dystrophin expression levels in CRC patients with poor survival outcomes. A combination of the dystrophin and RAB3C expression profiles can serve as an independent prognostic factor in CRC and is associated with several clinicopathological parameters. In addition, the RAB3C-dystrophin axis is positively correlated with the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) genetic alterations in CRC patients. These findings can be used to provide novel combined therapeutic options for the treatment of CRC.

Spontaneous Self-Assembly of Thermoresponsive Vesicles Using a Zwitterionic and an Anionic Surfactant.

Spontaneous formation of vesicles from the self-assembly of two specific surfactants, one zwitterionic (oleyl amidopropyl betaine, OAPB) and the other anionic (Aerosol-OT, AOT), is explored in water using small-angle scattering techniques. Two factors were found to be critical in the formation of vesicles: surfactant ratio, as AOT concentrations less than equimolar with OAPB result in cylindrical micelles or mixtures of micellar structures, and salt concentration, whereby increasing the amount of NaCl promotes vesicle formation by reducing headgroup repulsions. Small-angle neutron scattering measurements reveal that the vesicles are approximately 30-40 nm in diameter, depending on sample composition. Small-angle X-ray scattering measurements suggest preferential partitioning of OAPB molecules on the vesicle inner layer to support vesicular packing. Heating the vesicles to physiological temperature (37 °C) causes them to collapse into smaller ellipsoidal micelles (2-3 nm), with higher salt concentrations (≥10 mM) inhibiting this transition. These aggregates could serve as responsive carriers for loading or unloading of aqueous cargoes such as drugs and pharmaceuticals, with temperature changes serving as a simple release/uptake mechanism.

Giant Lipid Vesicle Formation Using Vapor-Deposited Charged Porous Polymers.

In this study, we prepare giant lipid vesicles using vapor-deposited charged microporous poly(methacrylic acid- co-ethylene glycol diacrylate) polymer membranes with different morphologies and thicknesses. Our results suggest that vesicle formation is favored by thinner, more structured porous hydrogel substrates. Electrostatic interactions between the polymer and the lipid head groups affect vesicle yield and size distribution. Repulsive electrostatic interactions between the hydrogel and the lipid head groups promote vesicle formation; attractive electrostatic interactions suppress vesicle formation. Ionic strength and sugar concentration are also major parameters affecting the yield and size of giant vesicles. The presence of both ions and sugars in the hydration buffer results in increased vesicle yields. These results indicate that lipid-polymer interactions and osmotic effects in addition to the substrate morphology and surface charge are key factors affecting vesicle formation. Our data suggest that surface chemistry should be designed to tune electrostatic interactions with the lipid mixture of interest to promote vesicle formation. This vapor-deposited hydrogel fabrication technique offers tunability over the physicochemical properties of the hydrogel substrate for the production of giant vesicles with different sizes and compositions.

Influence of Specific Bacteriophage on the Level of Vesicle Formation and Morphology of Cells of Yersinia pseudotuberculosis.

Incubation of Yersinia pseudotuberculosis cells grown on a solid medium with pseudotuberculous diagnostic bacteriophage for 20 min at 37oC led to a significant decrease in the concentration of both components of the system. This effect was absent when the bacteria were grown in a fluid medium. At the same time, this incubation regimen promoted vesicle formation and typical morphological changes in bacteria grown in both surface and suspension cultures. Co-incubation of the bacteriophage with suspension of vesicles isolated from the suspension culture of Y. pseudotuberculosis grown at 10oC (but not 37oC) led to a decrease in plaque-forming activity of the bacteriophage.

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.

Self-assembly of phosphate amphiphiles in mixtures of prebiotically plausible surfactants.

The spontaneous formation of closed bilayer structures from prebiotically plausible amphiphiles is an essential requirement for the emergence of early cells on prebiotic Earth. The sources of amphiphiles could have been both endo- and exogenous (accretion of meteorite carbonaceous material or interstellar dust particles). Among all prebiotic possible amphiphile candidates, those containing phosphate are the least investigated species because their self-assembly occurs in a seemingly too narrow range of conditions. The self-assembly of simple phosphate amphiphiles should, however, be of great interest, as contemporary membranes predominantly contain phospholipids. In contrast to common expectations, we show that these amphiphiles can be easily synthesized under prebiotically plausible environmental conditions and can efficiently form bilayer structures in the presence of various co-surfactants across a large range of pH values. Vesiculation was even observed in crude reaction mixtures that contained 1-decanol as the amphiphile precursor. The two best co-surfactants promoted vesicle formation over the entire pH range in aqueous solutions. Expanding the pH range where bilayer membranes self-assemble and remain intact is a prerequisite for the emergence of early cell-like compartments and their preservation under fluctuating environmental conditions. These mixed bilayers also retained small charged solutes, such as dyes. These results demonstrate that alkyl phosphate amphiphiles might have played a significant role as early compartment building blocks.

Chain-Length-Dependent Autocatalytic Hydrolysis of Fatty Acid Anhydrides in Polyethylene Glycol

Autocatalytic hydrolysis of fatty acid anhydrides induced by the spontaneously formed vesicles has been studied for years. However, whether the reaction autocatalyzed by vesicles formed in diluted solutions applies also to macromolecular crowded conditions remains unknown. The aim of this study is to characterize hydrolysis behavior of fatty acid anhydrides and formation of vesicles in crowded media. Inert macromolecular crowding agents such as polyethylene glycol (PEG) and Dextran were used to probe the impact of external crowding on the autocatalytic hydrolysis of fatty acid anhydrides with varied hydrophobic chain length. Under stringent conditions of crowding, hydrolysis rates of octanoic anhydride, nonanoic anhydride, and decanoic anhydride were found to decrease, but the rates of lauric anhydride and oleic anhydride increased. These results suggest that the effect of the crowding agent on the hydrolysis of fatty acid anhydrides was chain-length-dependent. Characterization of the size and polydispersity of vesicles formed from hydrolyzed fatty acid anhydrides in crowding revealed that long-chain fatty acids formed monodisperse vesicles easier at lower concentrations of PEG. Measurement of the critical aggregation concentration of ionized fatty acid in the presence of PEG showed that crowding media promoted vesicle formation from long-chain fatty acids but inhibited those from fatty acids with fewer carbon atoms. Further investigation of the diffusion property of ionized fatty acids in crowding agents suggested that PEG might create more hydrophobic areas for long-chain fatty acids anhydrides, which subsequently promoted the unreacted anhydride in the aqueous phase to be solubilized in the formed vesicles. This research provides information for understanding the autocatalytic reaction accompanied by self-producing aggregates and the behavior of fatty acids in crowding media.

Molecular Assemblies of 4-(Hexadecyloxy)-N-(pyridinylmethylene)anilines at the Air−Water Interface and Cu(II)-Promoted Vesicle Formation via Metal Coordination

The molecular assemblies of 4-(hexadecyloxy)-N-(pyridinylmethylene)anilines (HPA) at the air-water interface on pure water and aqueous Cu(II) subphases have been investigated using in situ infrared reflection absorption spectroscopy (IRRAS). The Schiff base units were oriented with their long axes almost perpendicular to the water surface, and both imine and pyridinyl nitrogen atoms of the Schiff base units were coordinated to Cu(II) ions together with their geometrical conversions. The alkyl chains in the monolayers were quantitatively determined on the assumption that the HPA monolayers at the air-water interface were composed of sublayers of alkyl chains and Schiff base units, and the chain orientation angle on pure water was 30 +/- 2 degrees and increased to 37 +/- 2 degrees on the aqueous Cu(II) subphase. The HPA amphiphiles could not be dispersed in pure water but could self-organize into vesicles with metal-coordinated headgroups and interdigitated-packed alkyl chains in the presence of Cu(II) ions in aqueous solution. Transmission electron microscopy (TEM), differential scanning calorimetry (DSC), UV-vis spectroscopy, and small-angle X-ray diffraction (XRD) were used to investigate the aggregate structures and specific properties of the coordinated vesicles.

There goes the neighborhood. Focus on “Localized PtdIns 3,5-P2 synthesis to regulate early endosome dynamics and fusion”

the membranes of eukaryotic cells contain small amounts of phosphoinositides, which are phosphorylated forms of the membrane lipid phosphatidylinositol (PtdIns). Together, PtdIns and its phosphoinositide derivatives constitute ∼10% of the total cellular lipid in most cells. Although

Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network

The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin IIDeltaPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

Glyceraldehyde-3-phosphate Dehydrogenase Interacts with Rab2 and Plays an Essential Role in Endoplasmic Reticulum to Golgi Transport Exclusive of Its Glycolytic Activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.

Spontaneous vesicle formation from DSB/AOT mixed system

Spontaneous vesicles from the aqueous mixtures of dodecyl sulfonate betaine (DSB) and sodium bis (2-ethylhexyl) sulfosuccinate (AOT) at certain mixing ratios have been demonstrated by using calorimetry, freeze-fracture transmission electronic microscopy (TEM), negative-staining TEM and dynamic light scattering (DLS) methods. The addition of NaCl will promote vesicle formation and the heat effect of monodisperse vesicle system is greatest. Meanwhile the mechanism was analyzed from the viewpoint of packing parameterf, molecular geometry structure and interaction.

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.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Glyceraldehyde-3-phosphate Dehydrogenase Is Phosphorylated by Protein Kinase Cι/λ and Plays a Role in Microtubule Dynamics in the Early Secretory Pathway

The small GTPase Rab2 immunolocalizes to vesicular tubular clusters (VTCs) that function as transport complexes carrying cargo between the endoplasmic reticulum and the Golgi complex. Our previous studies showed that Rab2 promotes vesicle formation from VTCs and that the released vesicles are enriched in beta-coat protein, protein kinase C iota/lambda (PKCiota/lambda), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCiota/lambda kinase activity was necessary for vesicle formation, a search was initiated to identify the substrate(s) that potentiate Rab2 function within VTCs. In this study, we found that PKCiota/lambda phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCiota/lambda regulatory domain. Based on numerous observations that show (beta-COP) GAPDH associates with cytoskeletal elements, we examined the role of phospho-GAPDH in promoting microtubule (MT) binding to membrane. Using a quantitative microsomal binding assay, we found that membrane association of beta-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCiota/lambda kinase activity. Furthermore, normal rat kidney cells transfected with a constitutively activated form of Rab2 (Q65L) or with our anti-GAPDH polyclonal antibody displayed a dramatic change in MT organization. These combined results suggest that Rab2 stimulated PKCiota/lambda and GAPDH recruitment to VTCs, and the subsequent PKCiota/lambda phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.

Glyceraldehyde-3-phosphate Dehydrogenase Is Required for Vesicular Transport in the Early Secretory Pathway

Protein transport in the early secretory pathway requires Rab2 GTPase. This protein promotes the recruitment of soluble components that participate in protein sorting and recycling from pre-Golgi intermediates (vesicular tubular clusters (VTCs)). We previously reported that a constitutively activated form of Rab2 (Q65L) as well as Rab2 wild type promoted vesicle formation from VTCs. These vesicles contained Rab2, beta-COP, p53/gp58, and protein kinase Ciota/lambda but lacked anterograde-directed cargo. To identify other candidate Rab2 effectors, the polypeptide composition of the vesicles was further analyzed. We found that vesicles released in response to Rab2 also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To study the relationship of this enzyme to Rab2 function, we performed a quantitative binding assay to measure recruitment of GAPDH to membrane when incubated with Rab2. Rab2-treated microsomes showed a 5-10-fold increase in the level of membrane-associated GAPDH. We generated an affinity-purified anti-GAPDH polyclonal to study the biochemical role of GAPDH in the early secretory pathway. The antibody arrests transport of a reporter molecule in an assay that reconstitutes ER to Golgi traffic. Furthermore, the affinity-purified antibody blocked the ability of Rab2 to recruit GAPDH to membrane. However, the antibody did not interfere with Rab2 stimulated vesicle release. These data suggest that GAPDH is required for ER to Golgi transport. We propose that membranes incubated with anti-GAPDH and Rab2 form "dead end" vesicles that are unable to transport and fuse with the acceptor compartment.