Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants

Summary A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8mm CaCl₂ concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.     

Enzymatic characterization of subcellular samples from gypsy moth larval midgut following differential centrifugation and fractionation on Percoll-sucrose gradient

A method is described for isolation of an enriched fraction of plasma membranes from gypsy moth (Lymantria dispar) larval midgut tissue. Following differential centrifugation of tissue homogenate, a microsomal sample is obtained and fractionated on a Percoll®-sucrose gradient that yields 2 distinct regions of high protein concentration: one enriched in plasma membranes, the other in mitochondrial membranes. The procedure is relatively rapid, being completed within approximately 5 h. Protein yields and accompanying specific activities are reported for marker enzymes used to indicate the presence of plasma membranes (leucine aminopeptidase and alkaline phosphatase), endoplasmic reticulum (NADPH-cytochrome c reductase), and mitochondria (succinate dehydrogenase). The apparent differences between the plasma membrane enriched fraction vs. brush border membrane vesicles prepared from insect midguts are discussed, as is the suitability of the plasma membrane enriched fraction for ATP-dependent calcium ion transport studies. © 1992 Wiley-Liss, Inc.     

Alkaline phosphatase of basal lateral and brush border plasma membranes from intestinal epithelium

The alkaline phosphatases present on isolated brush border and basal lateral membranes of rat duodenal epitheilum were examined by means of a variety of biochemical assays and physical methods. The two alkaline phosphatases have similar pH optima of 9.6–9.8, similar substrate k_m's for p-nitrophenyl phosphate (PNPP) of 71 micromolar, similar responses to the inhibitors 2-mercaptoethanol, theophylline, phenylalanine, and ethylenediaminetetraacetic acid (EDTA), similar sensitivities to calcium, magnesium, zinc, sodium, and potassium, and similar insensitivities to digestion with trypsin or papain. The two enzymes also exhibit similar molecular weights on SDS-polyacrylamide gels in the range 124,000–150,000, and both enzymes show an R_f value of 0.092 on Triton X-100 polyacrylamide gels, indicating similar intrinsic charges. The V_max of the brush border enzyme is ten times greater than that of the basal lateral enzyme, 140 μmoles/mg-h as opposed to 14 μmoles/mg-h. The differences in V_max are a reflection of the known distribution of alkaline phosphatase in rat duodenum, there being more alkaline phosphatase activity present on the brush border than on the basal lateral surface. One other major difference was observed between the two enzymes, the stimulation of the basal lateral and not the brush border alkaline phosphatase by SDS, Triton X-100, or cholate. We conclude that the enzymes are very similar to one another and probably perform similar membrane functions.     

Stimulation of intestinal Na+/d-glucose cotransport by monoclonal antibodies

Summary The small intestinal brush border membrane is endowed with a number of transport systems. Monoclonal antibodies were produced against integral membrane proteins and tested for their ability to bind to such membranes. For this purpose papain-digested, deoxycholate-extracted BBMVs from rabbit small intestine were used to immunize mice. Of the 765 hybridoma supernatants tested, 119 gave a significantly higher extent of binding to the crude antigen preparation as compared with the background. The monoclonal antibodies were also tested for their ability to influence the sodium-dependent uptake of solutes into intact BBMVs. Two monoclonal antibodies clearly showed stimulation of secondary actived-glucose transport, whereas sodium-dependent uptake ofl-alanine andl-proline was not affected. Hydrophobically labeled, i.e. intrinsic, membrane proteins of 175, 78 and 65 kilodaltons could be immunoprecipitated by both monoclonal antibodies, the 78 kDa band corresponding in all likelihood to the Na⁺/glucose cotransporter.     

GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens

Cytoplasmic aggregation, the rapid translocation of cytoplasm and subcellular components to the site of pathogen penetration, is one of the earliest reactions of plant cells against attack by microorganisms. We have investigated cytoplasmic aggregation during Arabidopsis-oomycete interactions. Infection by non-pathogenic Phytophthora sojae was prevented in the plant epidermal cell layer, whereas Peronospora parasitica isolates Cala2 (avirulent) and Noks1 (virulent) could both penetrate into the mesophyll cell layer. Epidermal cell responses to penetration by these oomycetes were examined cytologically with a range of transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-tagged cell components. These included plants containing GFP-TUA6 for visualizing microtubules, GFP-hTalin for actin microfilaments, GFP-tm-KKXX for endoplasmic reticulum (ER), and STtmd-GFP for the Golgi apparatus. In all interactions, actin microfilaments were actively re-arranged and formed large bundles in cytoplasmic strands focused on the penetration site. Aggregation of ER membrane and accumulation of Golgi bodies at the infection site were observed, suggesting that production and secretion of plant materials were activated around the penetration site. Microtubules did not become focused on the penetration site. No difference was evident between the responses of epidermal cells in the non-host, incompatible and compatible interactions. This result indicates that the induction of cytoplasmic aggregation in Arabidopsis epidermal cells was neither suppressed by the virulent strain of Peronospora, nor effective in stopping infection.     

Docosahexaenoic acid impairs the maturation of very low density lipoproteins in rat hepatic cells

One mechanism of the lipid-lowering effects of the fish oil n-3 fatty acids [e.g., docosahexaenoic acid (DHA)] in cell and animal models is induced hepatic apolipoprotein B100 (apoB) presecretory degradation. This degradation occurs post-endoplasmic reticulum, but whether DHA induces it before or after intracellular VLDL formation remains unanswered. We found in McA-RH7777 rat hepatic cells that DHA and oleic acid (OA) treatments allowed formation of pre-VLDL particles and their transport to the Golgi, but, in contrast to OA, with DHA pre-VLDL particles failed to quantitatively assemble into fully lipidated (mature) VLDL. This failure required lipid peroxidation and was accompanied by the formation of apoB aggregates (known to be degraded by autophagy). Preventing the exit of proteins from the Golgi blocked the aggregation of apoB but did not restore VLDL maturation, indicating that failure to fully lipidate apoB preceded its aggregation. ApoB autophagic degradation did not appear to require an intermediate step of cytosolic aggresome formation. Taken with other examples in the literature, the results of this study suggest that pre-VLDL particles that are competent to escape endoplasmic reticulum quality control mechanisms but fail to mature in the Golgi remain subject to quality control surveillance late in the secretory pathway.     

Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cells

Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES.     

Studies on the submicrosomal fractions of bovine oligodendroglia: Lipid composition and glycolipid biosynthesis

Oligodendroglia were isolated from bovine brain, and a crude, microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.     

Effects of inositol 1,4,5-trisphosphate on calcium release from the endoplasmic reticulum and Golgi apparatus in mouse mammary epithelial cells: a comparison during pregnancy and lactation

It has been established that inositol 1,4,5-trisphosphate(IP3) is responsible for the mobilization of calcium(Ca2+) from intracellular locations in a wide variety of tissues, and that this response triggers the stimulation of several hormones and neurotransmitters. However, these phenomena have yet to be examined in the mammary epithelium. Ca2+ uptake from the medium into the endoplasmic reticulum(ER) and Golgi apparatus in vitro in both pregnant and lactating mouse mammary epithelial cells was studied and a strong Ca2+ release from these organelles into the medium with the use of IP3 was shown. The Ca2+ uptake and its release due to IP3 was also usually greater during pregnancy than lactation.     

Targeting of tail‐anchored membrane proteins to subcellular organelles in Toxoplasma gondii

Proper protein localization is essential for critical cellular processes, including vesicle‐mediated transport and protein translocation. Tail‐anchored (TA) proteins are integrated into organellar membranes via the C‐terminus, orienting the N‐terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C‐terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early‐branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow‐up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C‐terminal sequence.      

A Novel Membrane Protein Complex on the Endoplasmic Reticulum and Early Golgi Compartments in the Yeast Saccharomyces cerevisiae

A novel membrane protein, Yml067c in the systematic ORF name, was discovered as a component of immunoisolated vesicles of the early Golgi compartment of the yeast Saccharomyces cerevisiae (Cho et al., FEBS Lett. 469, 151-154 (2000)). Conserved sequences having sequence similarity to Yml067c were widely distributed in the eukaryotes and one of them, Yal042w, was found in the Saccharomyces genome database. In the yeast cell, Yml067c and Yal042w were found to form a heterooligomeric complex by immunoprecipitation of their tagged derivatives from the detergent-solubilized membrane. Cell fractionation and indirect immunofluorescent staining indicated that the majority of these proteins were localized on the ER membrane. Therfore, the Yml067c-Yal042w complex should shuttle between the ER and the early Golgi compartment as well as the p24-family proteins.     

Cell fractionation analysis of human CD8 glycoprotein transport between endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells

We have set up an analytical cell fractionation procedure to dissect, by a non-morphological method, the anterograde transport of proteins from endoplasmic reticulum, intermediate compartment and Golgi complex in tissue cultured cells. Using this procedure after pulse-chase labelling of cells expressing human CD8 glycoprotein, we obtained results that: (1) support the view that the intermediate compartment is a distinct station in the export from the endoplasmic reticulum to the Golgi complex; and (2) strongly suggests that the O -glycosylation process starts after the intermediate compartment, presumably in the cis -Golgi complex.     

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.     

The migration of labeled phosphatidylcholine from the nuclear-associated endoplasmic reticulum to plasma membranes in L-929 cells

Summary The nuclear-associated endoplasmic reticulum of L-929 cells was found to contain the highest amount of labeled phosphatidylcholine after a 60 min incubation with¹⁴C-choline. Radioactivity was otherwise distributed relatively evenly among other membrane-containing organelles (nuclei, mitochondria, plasma membranes and endoplasmic reticulum membranes). During a 120 min chase following removal of isotope and addition of cold choline chloride, there was a considerable reduction in labeled phosphatidylcholine in the NER and nuclei. The decrease in radioactivity in these fractions was matched by an almost identical increase in the fraction containing mitochondria and plasma membranes. Separation of mitochondria and plasma membranes by centrifugation on discontinuous gradients showed that¹⁴C-choline labeled phosphatidylcholine appeared most rapidly in the plasma membranes. The results indicate that phospholipid molecules migrate within a short period of time from their site of synthesis in the NER to plasma membranes.     

Co-localisation of membranes and actin in growing infected cells of Glycine max root nodules

The root nodule of Glycine max (L.) Merr. is almost spherical at maturity, and its central tissue consists of infected cells filled with numerous symbiosomes containing bacteroids, interspersed with uninfected cells. During the growth of the nodule, the volume of each infected cell and the number of bacteroids per cell increases, and thus abundant membranes are required for the proliferation of symbiosomes. In expanding infected cells, there are areas adjacent to the nucleus that are devoid of bacteroids, but these areas are filled with numerous membranes and actin filaments, surrounded by endoplasmic reticulum membranes, indicating a perinuclear reservoir of newly formed membranes and a role for actin in delivering membranes to proliferating symbiosomes.     

Physical membrane displacement: Reconstitution in a cell-free system and relationship to cell growth+

Summary Physical membrane displacement is a process common to all forms of vesicle budding as well as cell enlargement and pleomorphic shape changes. Cell-free reconstitution of membrane budding has been achieved with transitional endoplasmic reticulum fractions from both plants and animals where 50 to 70 nm transition vesicles have been observed to bud from the part-rough, part-smooth membrane elements that define transitional endoplasmic reticulum. This budding phenomenon requires ATP, is facilitated by cytosol and guanine nucleotides, and is both time- and temperature-dependent. The transitional endoplasmic reticulum buds that form when concentrated by preparative free-flow electrophoresis will attach specifically to cis Golgi apparatus membranes immobilized on nitrocellulose as an acceptor compartment. Golgi apparatus membranes derived from the trans compartment do not serve as an efficient acceptor compartment. Transfer of the vesicles once formed is rapid, nearly complete and no longer dependent upon added ATP. Transfer shows a strict temperature dependency corresponding to that of the intact cell where at temperatures of 16°C or below, vesicles form but do not attach to cis Golgi whereas at temperatures of greater than 16°C, vesicles both form and fuse. The principle ATPase of transitional endoplasmic reticulum which may be involved in the budding process has been identified, characterized and isolated. A 38 kDa cis Golgi apparatus associated protein also has been identified as a potential candidate as a docking protein. Transfer between trans Golgi apparatus and the plasma membrane also has been studied by cell-free analysis. Here, transfer has been found to be stimulated by NADH or NADH plus ascorbate. The role of NADH is unknown but the ability of plant and Golgi apparatus to oxidize NADH is inhibited by brefeldin A, a compound known to block membrane trafficking even at the level of the trans Golgi network. NADH oxidase activity of plasma membranes also has been described and is inhibited as well by brefeldin. Recent observations suggest that brefeldin A may block both the formation of vesicles at the trans Golgi apparatus as well as auxin hormone-stimulated cell elongation in plants. This once again raises the possibility of whether or not plant cell elongation is obligatorily mediated by membrane input from the Golgi apparatus. The latter seems unlikely based on two additional lines of evidence. The first is that auxin-induced cell elongation in plants shows no sharp temperature transition over the range of 4 to 24°C, whereas production of secretory vesicles from the trans Golgi apparatus appears to be largely prevented at temperatures of 18°C or less. Secondly, the sodium selective ionophore, monensin, which effectively blocks the formation of functional secretory vesicles at the trans Golgi apparatus, is also largely without effect on auxin-induced cell elongation for periods of 4 h or longer. Taken together the findings suggest that the action of brefeldin A on vesicle budding at the Golgi apparatus and cell enlargement, are not directly correlated but may represent a common action of the drug on some constituent essential to membrane displacement mechanisms.Abbreviations BFA brefeldin A - IAA indole-3-acetic acid; 2, 4-D 2, 4-dichlorophenoxyacetic acid - NSF N-ethylmaleimide-sensitive factor Much of the information summarized in this report was presented as a plenary lecture at the XV International Botanical Congress Tokyo, Yokohama, Japan, August 28–September 3, 1993.     

Ca++-transport across basal-lateral plasma membranes from rat small intestinal epithelial cells

Summary Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5mm MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 g/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na+K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 g/ml oligomicin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal-lateral membranes possess two distinct mechanisms for Ca-transport:a) ATP-driven Ca-transport andb) Na/Ca-exchange.     

Profound architectural and functional readjustments of the secretory pathway in decidualization of endometrial stromal cells

Certain cell types must expand their exocytic pathway to guarantee efficiency and fidelity of protein secretion. A spectacular case is offered by decidualizing human endometrial stromal cells (EnSCs). In the midluteal phase of the menstrual cycle, progesterone stimulation induces proliferating EnSCs to differentiate into professional secretors releasing proteins essential for efficient blastocyst implantation. Here, we describe the architectural rearrangements of the secretory pathway of a human EnSC line (TERT-immortalized human endometrial stromal cells (T-HESC)). As in primary cells, decidualization entails proliferation arrest and the coordinated expansion of the entire secretory pathway without detectable activation of unfolded protein response (UPR) pathways. Decidualization proceeds also in the absence of ascorbic acid, an essential cofactor for collagen biogenesis, despite also the secretion of some proteins whose folding does not depend on vitamin C is impaired. However, even in these conditions, no overt UPR induction can be detected. Morphometric analyses reveal that the exocytic pathway does not increase relatively to the volume of the cell. Thus, differently from other cell types, abundant production is guaranteed by a coordinated increase of the cell size following arrest of proliferation.