Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

The Arabidopsis thaliana RER1 gene family: its potential role in the endoplasmic reticulum localization of membrane proteins

Many endoplasmic reticulum (ER) proteins are known to be localized to the ER by a mechanism called retrieval, which returns the molecules that are exported from the ER to the Golgi apparatus back to the ER. Signals are required to be recognized by this retrieval system. In the work on yeast Saccharomyces cerevisiae, we have demonstrated that transmembrane domains of a subset of ER membrane proteins including Sec12p, Sec71p and Sec63p contain novel ER retrieval signals. For the retrieval of these proteins, a Golgi membrane protein, Rer1p, is essential (Sato et al., Mol. Biol. Cell 6 (1995) 1459–1477; Proc. Natl. Acad. Sci. USA 94 (1997) 9693–9698). To address the role of Rer1p in higher eukaryotes, we searched for homologues of yeast RER1 from Arabidopsis thaliana. We identified three cDNAs encoding Arabidopsis counterparts of Rer1p with an amino acid sequence identity of 39–46% to yeast Rer1p and named AtRER1A, AtRER1B, and AtRER1C1. AtRer1Ap and AtRer1Bp are homologous to each other (85% identity), whereas AtRer1C1p is less similar to AtRer1Ap and AtRer1Bp (about 50%). Genomic DNA gel blot analysis indicates that there are several other AtRER1-related genes, implying that Arabidopsis RER1 constitutes a large gene family. The expression of these three AtRER1 genes is ubiquitous in various tissues but is significantly higher in roots, floral buds and a suspension culture in which secretory activity is probably high. All the three AtRER1 cDNAs complement the yeast rer1 mutant and remedy the defect of Sec12p mislocalization. However, the degree of complementation differs among the three with that of AtRER1C1 being the lowest, again suggesting a divergent role of AtRer1C1p.     

The role of NADH oxidase in growth and physical membrane displacement

Summary An NADH oxidase activity of the plasma membrane that was growth factor- and hormone-stimulated has been reported and characterized extensively. The activity is constitutively activated in cancer. Thiol groups are important to its activity and its activity as a thiol oxidoreductase or thiol interchange activity may be important to its functioning in growth and membrane trafficking. The activity is inhibited in the presence of GDP and brefeldin A and appears to be found not only at the plasma membrane but also with the Golgi apparatus. The phenotype of an inhibited NADH oxidase is a failure of cells to enlarge. The hypothesis under investigation is that the hormone- and growth factor-stimulated NADH (quinone)/thiol oxidase functions as an essential component of physical membrane displacements associated with cell elongation, vesicle budding and pleomorphic membrane changes in both animal and plant cells.     

ATP stimulates the uptake of S-dinitrophenylglutathione by rat liver plasma membrane vesicles

Incubation of inverted plasma membrane vesicles from rat liver with micromolar concentrations of S-dinitrophenylglutathione (DNP-SG) in the presence of ATP resulted in the uptake of DNP-SG into the vesicles. ATP-dependent DNP-SG accumulation was half-maximal with 9 μM DNP-SG, while the K_m for ATP was 320 μM. Glutathione disulfide (GSSG), but not reduced glutathione, inhibited the ATP-dependent accumulation of DNP-SG by the vesicles, suggesting that the same, ATP-dependent transport system is responsible for the extrusion of glutathione conjugates and GSSG from liver cells.     

Fractionation of suspension cultures of wild carrot and kinetics of membrane labeling

Summary Wild carrot (Daucus carota L.) cells, grown in suspension culture, were labeled with radioactive precursors and fractionated into constituent membranes to be analyzed for specific radioactivity. Results show rapid incorporation of [³H] leucine into endoplasmic reticulum (ER)-, Golgi apparatus-, and plasma membrane/tonoplast-enriched fractions. The time lag between incorporation into ER and its appearance in Golgi apparatus or plasma membrane/tonoplast were less than 5 minutes. With an average time of 3–4 minutes for cisternal formation estimated from studies with monensin, and an average of 5 cisternae per dictyosome (total transit time of 15–20 minutes), it was not possible to account for early incorporation of radioactivity into plasma membranes by passage of proteins from ER to plasma membrane via the Golgi apparatus. To account for the findings, it would appear that at least some proteins were delivered to the plasma membrane via the first membranes that exited (i.e., mature face vesicles) from the Golgi apparatus post-pulse and that some of these proteins had been translated and inserted into membranes at or near the mature face of the Golgi apparatus.     

Amino acid-dependent sodium transport in plasma membrane vesicles from rat liver

Summary Thel-alanine-dependent transport of sodium ions across the plasma membrane of rat-liver parenchymal cells was studied using isolated plasma membrane vesicles. Sodium uptake is stimulated specifically by thel-isomer of alanine and other amino acids, whose transport is sodium-dependent in rat-liver plasma membrane vesicles. Thel-alanine-dependent sodium flux across the membrane is inhibited by an excess of Li⁺ ions, but not by K⁺ or choline ions. Sodium transport is sensitive to-SH reagents and ionophores, and is an electrogenic process: a membrane potential (negative inside) can enhancel-alanine-dependent sodium accumulation. The data presented provide further evidence for a sodium-alanine cotransport mechanism.     

Membrane flow in plants: preparation and kinetics of labelling of plasma membranes from growing pollen tubes of tobacco

Summary Growing pollen tubes of tobacco germinated in suspension culture, were labelled with [³H]leucine and after varying times of chase with unlabelled leucine at 23, 16, or 4°C, were separated into plasma membrane-enriched and plasma membrane-depleted fractions by aqueous two-phase partition. At 23°C, the specific radioactivity of the plasma membrane increased with time to a maximum at 60 min. At 16°C and 4°C, labelling of the plasma membrane was respectively 40% and 10% that at 23°C. However, if labelling was at 23°C and subsequent transfer was at 4°C, plasma membrane labelling was much less affected and labelling of the plasma membrane was 60% that at 23°C. Additionally, quantitation of various morphological parameters revealed no accumulations of 50–70 nm transition vesicles in the space between endoplasmic reticulum and cis Golgi apparatus that might suggest formation of a low temperature compartment similar to those described for mammalian cells and tissues. Similarly, growth of pollen tubes was reduced but not blocked even at temperatures of 12°C. The results suggest that tube elongation is accompanied by a steady state flow of membranes to the cell surface that is relatively insensitive to interruption by low temperatures. Whereas leucine incorporation is reduced by low temperature even at 16°C, the flow pathway to the cell surface, including the endoplasmic reticulum to Golgi apparatus transfer step, as well as elongation growth does not exhibit a pronounced low temperature block in this tip growing system.     

Allosteric activation of plasma membrane receptors--physiological implications and structural origins

Allosteric modulation of receptors has physiological not just pharmacological significance. Thus, the chemical context in which an agonist signal is received can have a major impact on the nature of the physiological response by modifying receptor sensitivity and/or maximal activity-even the nature of the signalling response. In addition, recognising that an endogenous activator is the allosteric modulator of a known receptor, rather than the agonist of a novel receptor, has the potential to solve, in dramatic fashion, key physiological questions. What is an allosteric modulator and why are allosteric effects on receptors so diverse and frequently complex? What is the scope of allosteric effects? Can the existence of endogenous modulators be predicted from a receptor's amino acid sequence? How should screening for endogenous allosteric modulators be undertaken? These questions form the framework of this mini-review on physiological and structural aspects of receptor allostery.     

Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes

Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs. Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein. A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by [(14)C]N-ethylmaleimide. The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide. Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs. The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids.     

Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells

Background information. The GA (Golgi apparatus) has an essential role in membrane trafficking, determining the assembly and delivery of UPs (uroplakins) to the APM (apical plasma membrane) of superficial UCs (uroepithelial cells) of urinary bladder. UPs are synchronously and uniformly delivered from the GA to the APM by DFVs (discoidal‐ or fusiform‐shaped vesicles); however, the mechanism of UP delivery is not known. We have used the culture model of UCs with the capacity to undergo terminal differentiation to study the process of uniform delivery of DFVs to the APM and to elucidate the mechanisms involved. Results. By three‐dimensional localization using confocal microscopy of immunofluorescence‐labelled GA‐related markers [GM130 (cis‐Golgi matrix protein of 130 kDa), GS15 (Golgi Snare 15 kDa), GS28 and giantin], uroepithelial differentiation‐related markers (UPs), MTs (microtubules; α‐tubulin) and intermediate filaments [CK7 (cytokeratin 7) and CK20], we found that in non‐differentiated, UP‐negative UCs the GA is mostly organized as a single ribbon‐like structure close to the nucleus, whereas in differentiated, UP‐positive UCs the GA is fragmented and spread almost through the entire cell. The FRAP (fluorescence recovery after photobleaching) experiments on the UCs transfected with GalT (trans‐Golgi/TGN enzyme β1,4‐galactosyltransferase) fused to fluorescent protein showed that Golgi‐resident enzyme cycles freely within ribbon‐like GA but not within fragmented GA. By CLEM (correlative light—electron microscopy), we examined the GA fragments in cells expressing UPs. We found that GA fragments are fully functional and similar to the GA fragments that are formed after nocodazole treatment. Furthermore, we demonstrated that the reorganization of GA into a fragmented form is associated with the impairment of the MT organization in the basal, central and subapical cytoplasm and the accumulation of intermediate filaments in the apical cytoplasm that could affect the kinetics of MT star leading to the peripheral fragmentation of the GA in the differentiated UCs. Conclusions. The fragmentation of the GA and the subsequent spreading of GA to the cell periphery represent one of the key events that promote the uniform delivery of UPs over the entire APM of differentiating UCs and thus are of major importance in the final proper formation and maintenance of the blood—urine barrier.     

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca²⁺ export mechanisms in rat brain synaptic membranes. Ca²⁺ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca²⁺ pump and by a low affinity/high capacity ATP-independent Na⁺-Ca²⁺ exchanger. Both Ca²⁺ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP>NAD>NADPH>NADH with IC₅₀ values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca²⁺ pump and with IC₅₀ values between 8 and 10 mM for the Na⁺-Ca²⁺ exchanger. Oxidizing agents such as DCIP and ferricyanide inhibited the ATP-driven Ca²⁺ efflux mechanism but not the Na⁺-Ca²⁺ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca²⁺ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na⁺-Ca²⁺ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by eithercAMP-dependent protein kinase, Ca²⁺ calmodulin-dependent protein kinases, or protein kinase C.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - EGTA ethylenglycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid - DCIP dichlorophenol-indophenol     

Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells.

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.     

Golgi-localized KIAA0725p regulates membrane trafficking from the Golgi apparatus to the plasma membrane in mammalian cells

Mammals have three members of the intracellular phospholipase A₁ protein family (phosphatidic acid preferring-phospholipase A₁, p125, and KIAA0725p). In this study, we showed that KIAA0725p is localized in the Golgi, and is rapidly cycled between the Golgi and cytosol. Catalytic activity is important for targeting of KIAA0725p to Golgi membranes. RNA interference experiments suggested that KIAA0725p contributes to efficient membrane trafficking from the Golgi apparatus to the plasma membrane, but is not involved in brefeldin A-induced Golgi-to-endoplasmic reticulum retrograde transport.

Structured summary

MINT-8019765: KIAA0725 (uniprotkb:O94830) and Beta-COP (uniprotkb:P53618) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8019775: KIAA0725 (uniprotkb:O94830) and GM130 (uniprotkb:Q5PXD5) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Involvement of endoplasmic reticulum in the origins of dictyosome-like structures of guinea pig spermatocytes

Summary Dictyosome-like structures (DLS) are formed in early spermatocytes first as single saccules. These saccules occur in association with forms of endoplasmic reticulum (ER) characterized by a paucity of ribosomes and luminal content, by a constriction of the lumina, and by a tendency to fragment or form myelin figures during fixation. Nascent DLS and the unusual ER cisternae share many characteristics in common including a pattern of staining with fixatives containing tannic acid where the membranes appear thin due to the inner membrane leaflet being unstained or poorly stained. DLS also appear to form in the region conventional Golgi apparatus but always in association with ER forms that frequently occupy portions of the Golgi apparatus zone.An ability to stain with phosphotungstic acid at low pH exhibited by DLS is given also by the specialized ER forms. One possibility for DLS formation suggested by the present study is that DLS cisternae differentiate from ER membranes after which they ultimately associate into the stacked configurations that characterize mature DLS.     

p97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly

p97ATPase‐mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47‐mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47‐binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47‐mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD‐p97/p47‐FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD‐p97/p47‐FTCD complex in p97/p47‐mediated Golgi membrane fusion.     

The NADH oxidizing system of the plasma membrane and metabolic signal control

Summary The development of ideas concerning plasma membrane redox reactions in normal and transformed animal cells is described, with emphasis on transferrin and ceruloplasmin. Control by hormones and growth factors, as well as the NAD⁺/NADH ratio in the cell are important in distinguishing the two types of cells.     

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.     

Interaction of the plasma membrane with the cytoskeleton: An overview

The intent of this review was to point out the diversity of cellular functions thought to be mediated by PM—cytoskeleton interactions. Based upon possible molecular mechanism, the functions were categorized into those involving PM proteins which are dispersed and those involving clustered proteins. Functions associated with dispersed proteins are thought to mediate the stabilization and shape of the PM. Clustering of PM proteins provides the driving force inducing their interaction with the cytoskeleton. Clustering by external ligands, pH or ionic exchanges, etc., is also a means of transmembrane signalling. Various methods used to explore cytoskeletal—PM mediated functions were evaluated. The methods were considered separately under biophysical, morphological and biochemical headings. This made it easier to point out current and potential values of the methods as well as their limitations. Each method taken separately is insufficient to elucidate molecular mechanisms regulating cytoskeletal—PM reactions, but combined they hold great promise of future solutions.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.