Subcellular localization of mannosyl transferase and glycoprotein biosynthesis in castor bean endosperm

Differential and sucrose density gradient centrifugation have shown that the mannosyl transferase present in germinating castor bean endosperm cells which catalyses the synthesis of mannosyl-phosphoryl-polyisoprenol is exclusively located in the endoplasmic reticulum membrane. This intracellular location was confirmed using both ribosome-denuded microsomes isolated in the presence of EDTA and rough-surfaced microsomes isolated in the presence of excess Mg²⁺ added to maintain ribosome-membrane attachment. Separation of organelles following the incubation of crude particulate fractions with GDP[¹⁴C]mannose demonstrated that most of the mannolipid thus formed remained associated with the microsomal fraction. When organelles were isolated from intact tissue which had previously been incubated with GDP[¹⁴C]mannose, [¹⁴C]glycoprotein was found to be associated with other cellular fractions in addition to the microsomes, in particular the glyoxysomes. The kinetics of radioactive labelling of these organelles suggest that [¹⁴C]glycoprotein appears initially in the microsomal fraction and subsequently accumulates in the glyoxysomes. Subfractionation of isolated, [¹⁴C]glycoprotein-labelled glyoxysomes established that over 80% of the total radioactivity was present in the membrane, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis of solubilized glyoxysomal membranes showed that the [¹⁴C]sugar moiety was associated with several, but not all, constituent polypeptides.Abbreviations ER endoplasmic reticulum - TCA trichloroacetic acid - SDS sodium dodecylsulphate - GDP guanosine diphosphate     

Serological and developmental relationships between endoplasmic reticulum and glyoxysomal proteins of castor bean endosperm

Glyoxysomes isolated from the endosperm of castor bean (Ricinus communis L.) by sucrose density gradient centrifugation were fractionated into their matrix protein and membrane components. Antisera were raised in rabbits against both the matrix proteins and sodium dodecyl sulphate (SDS)-solubilized membrane proteins. SDS-polyacrylamide gel electrophoresis (PAGE) analysis established that such antisera precipitate all major polypeptide components present in their respective glyoxysomal mixedantigen preparations. Furthermore, when soluble constituents recovered from the microsomal vesicles or solubilized microsomal membranes were challenged with the appropriate glyoxysomal antiserum, serological determinants were again found to be present. Intact endosperm tissue was incubated with [³⁵S]methionine and the kinetics of ³⁵S-incorporation into protein recovered in immunoprecipitates when the glyoxysomal matrix fraction or the soluble fraction released from the microsomes were incubated with anti-glyoxysomal matrix serum were followed. [³⁵S]antigens rapidly appeared in the microsomal fraction whereas a lag period preceded their appearance in glyoxysomes. Interupting such kinetic experiments by the addition of an excess of unlabelled methionine resulted in a rapid decrease in the microsomal content of [³⁵S]antigens and a concomitant increase in glyoxysomal content.Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ER endoplasmic reticulum     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

Incorporation of D-[14C]galactose into organelle glycoprotein in castor bean endosperm

Excised casto bean (Ricinus communis L.) endosperm tissue supplied with [¹⁴C]galactose incorporates radioactivity into particulate cell components. Fractionation of homogenates established that ¹⁴C-labeled trichloroacetic acid-insoluble material was located primarily in the microsomal and glyoxysomal fractions. The capacity of the tissue to incorporate [¹⁴C]galactose into organelle glycoprotein varied during seedling development, increasing during the first 3 days of germination and subsequently declining. The kinetics of incorporation into the major organelle fractions of 2-day old endosperm tissue showed that the endoplasmic reticulum was immediately labeled whereas a lag period preceded the labeling of glyoxysomes. Sub-fractionation of the isolated organelles established that the greatest proportion of the [¹⁴C]-galactose labeled glycoprotein was located in the membrane, although a significant incorporation into the matrix protein was also observed.The results indicate that the addition of the carbohydrate moiety to the polypeptide cores occurs in the endoplasmic reticulum during or immediately after their synthesis on membrane-bound ribosomes.Abbreviations ER endoplasmic reticulum - SDS sodium dodecyl sulphate - TCA trichloroacetic acid     

Studies of sporogenesis in the rhodophyta

Summary Post-meiotic tetraspore mother cells of Corallina officinalis L. have been studied by light and electron microscopy. During the course of post-meiotic cellular reorganisation each nucleus becomes surrounded by a complex of precisely oriented endoplasmic reticulum, termed nuclear endoplasmic reticulum. A distinctive feature of this relationship is an electron dense substance in contact with the nuclear surface and extending as groundplasm between the ER cisternae as far as the outer limits of the complex, where it gives place to the ribosome-containing matrix of the general cytoplasm. There is circumstantial evidence to indicate that the extracisternal electron dense material is a product of nucleo-cytoplasmic interaction, and that it is involved in the assembly of ribosomes.The nuclear endoplasmic reticulum appears to be active in the production of smaller swollen cisternal elements, which form frequently anastomosing reticular tracts in the regions between adjacent nuclei. There is structural evidence of vesicular transport of material from the swollen cisternae to the proximal (forming) face of the Golgi apparatus.These events are thought to be of fundamental importance in achieving the cellular reorganisation and transformation which occurs after the second meiotic division.The authors thank Mr. D. C. Williams for technical assistance. One of us (MCP) gratefully acknowledges the financial support provided by a Natural Environment Research Council Studentship.     

Studies on the structure and function of 16S ribosomal RNA using structure-specific chemical probes

Recent technological developments permit us to examine the accessibility of specific atoms on any nucleotide in any large RNA molecule to certain chemical probes. This can provide detailed information about the higher order structure of large RNA molecules, including secondary and tertiary structure, protein-RNA contacts, binding sites for functional ligands and possible biologically significant conformational changes. Here, we summarize recent studies on (i) the conformation of naked 16S rRNA under a variety of ionic conditions, and (ii) the behaviour of 16S rRNA in active and inactive 30S subunits, as defined by Zamir, Elson and their colleagues. The latter study reveals a reciprocal conformational change in the vicinity of the decoding region of 16S rRNA in 30S ribosomal subunits. This conformational change appears to be a rearrangement of tertiary and/or quaternary structure involving several universally conserved nucleotides. No reproducible effects are seen elsewhere in the molecule, suggesting that the active-inactive transition is a result of the observed conformational change.     

Degradation of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in the vacuole

To understand how plant cells exert quality control over the proteins that pass through the secretory system we examined the transport and accumulation of the bean (Phaseolus vulgaris L.) vacuolar storage protein phaseolin, structurally modified to contain a helix-breaking epitope and carboxyterminal HDEL, an endoplasmic reticulum (ER)-retention signal. The constructs were expressed in tobacco (Nicotiana tabacum L.) with a seedspecific promoter. The results show that phaseolin-HDEL accumulates in the protein-storage vacuoles, indicating that HEDL does not contain sufficient information for retention in the ER. However, the ER of seeds expressing the phaseolin-HDEL construct contain relatively more phaseolin-HDEL compared to phaseolin in the ER of seeds expressing the phaseolin construct. This result indicates that the flow out of the ER is retarded but not arrested by the presence of HDEL. Introduction into phaseolin of the epitope himet (Hoffman et al., 1988, Plant Mol. Biol. 11, 717–729) greatly reduces the accumulation of HiMet phaseolin compared to normal phaseolin. However, the increased abundance within the ER is similar for both phaseolin-HDEL and HiMet phaseolin-HDEL. Using immunocytochemistry with specific antibodies, HiMet phaseolin was found in the ER, the Golgi stack, and in transport vesicles indicating that it was transport competent. It was also present at an early stage of seed development in the protein-storage vacuoles, but was not found there at later stages of seed development. Together these results support the conclusion that the HiMet epitope did not alter the structure of the protein sufficiently to make it transport incompetent. However, the protein was sufficiently destabilized to be degraded by vacuolar proteases.Abbreviations ER endoplasmic reticulum - BiP binding protein - IgG immunoglobulin G - M_r relative molecular mass The mention of vendor or product does not imply that they are endorsed or recommended by the US Department of Agriculture over vendors of similar products not mentionedThis work was supported by a grant from the National Science Foundation (Cell Biology) to M.J. Chrispeels and a fellowship from the Ministry of Education and Science, Spain-Fullbright Program to J.J. Pueyo. We thank H. Pelham for a gift of the constructs containing c-myc-SEKDEL and cmyc-FEHDEL and for a gift of anti-HDEL monoclonal antibodies. The original HiMet phaseolin construct was made by L. Hoffman and the phaseolin-HDEL or KDEL and HiMet-HDEL or KDEL constructs were made by D. Hunt as part of his doctoral research.     

Beta2 integrins target Rap GTPases to the plasma membrane by means of degranulation

We report the novel observation that engagement of β2 integrins on human neutrophils is accompanied by increased levels of the small GTPases Rap1 and Rap2 in a membrane-enriched fraction and a concomitant decrease of these proteins in a granule-enriched fraction. In parallel, we observed a similar time-dependent decrease of gelatinase B (a marker of specific and gelatinase B-containing granules) but not myeloperoxidase (a marker of azurophil granules) in the granule fraction, and release of lactoferrin (a marker of specific granules) in the extracellular medium. Furthermore, inhibition of Src tyrosine kinases, or phosphoinositide 3-kinase with PP1 or LY294002, respectively, blocked β2 integrin-induced degranulation and the redistribution of Rap1 and Rap2 to a membrane-enriched fraction. Consequently, the β2 integrin-dependent exocytosis of specific and gelatinase B-containing granules occurs via a Src tyrosine kinase/phosphoinositide 3-kinase signaling pathway and is responsible for the translocation of Rap1 and Rap2 to the plasma membrane in human neutrophils.     

Differential proteomic analysis of the endoplasmic reticulum from developing and germinating seeds of castor (Ricinus communis) identifies seed protein precursors as significant components of the endoplasmic reticulum

The endoplasmic reticulum is a major compartment of storage protein and lipid biosynthesis. Maximal synthesis of these storage compounds occurs during seed development with breakdown occurring during germination. In this study, we have isolated four independent preparations of ER from both developing and germinating seeds of castor bean (Ricinus communis) and used 2-D DIGE, and a combination of PMF and MS/MS sequencing, to quantify and identify differences in protein complement at both stages. Ninety protein spots in the developing seeds are up-regulated and 19 individual proteins were identified, the majority of these are intermediates of seed storage synthesis and protein folding. The detection of these transitory storage proteins in the ER is discussed in terms of protein trafficking and processing. In germinating seed ER 15 spots are elevated, 5 of which were identified, amongst them was malate synthetase which is a component of the glyoxysome which is believed to originate from the ER. Notably no proteins involved in complex lipid biosynthesis were identified in the urea soluble ER fraction indicating that they are probably all integral membrane proteins.     

A novel adaptive fluorescent probe for cell labelling

In this study we describe the synthesis and characterisation of a new hydrazone-based fluorescent compound that is able to selectively label the endoplasmic reticulum (ER) in yeast and mammalian living cells. The fluorescence properties of the compound depended on the DMSO/water ratio and on the pH. NMR experiments allowed determination of the conformation adopted in various environments. Apart from the convenient synthetic procedure, our compound displays low cell toxicity and blue emission compatible with filters routinely used in fluorescence microscopy.     

Specializations of the outer mitochondrial membrane during oocyte development

Summary A coating of electron dense material is present on the cytoplasmic surface of outer mitochondrial membranes in medium-sized hamster oocytes. The coating is not present at earlier or later stages of oocyte development. Its possible relationship to the synthesis and transport of mitochondrial protein is discussed. Associations between endoplasmic reticulum, ribosomes, glycogen and the outer mitochondrial membrane are also described and discussed.     

Activation of galanin receptor 2 stimulates large conductance Ca-dependent K (BK) channels through the IP3 pathway in human embryonic kidney (HEK293) cells

The large conductance Ca²⁺-activated K⁺ (BK) channels are widely distributed in the brain, and act as intracellular calcium sensors in neurons. They play an important feedback role in controlling Ca²⁺ flux and Ca²⁺-dependent processes, including neurotransmitter release and cellular excitability. In this study, the effects of the neuropeptide galanin on BK channels were examined by determining the whole-cell currents and single-channel activities in human embryonic kidney (HEK293) cells co-expressing GalR2 and the BK alpha subunit. Galanin enhanced the currents of BK channels, in a concentration-dependent and PTX-independent manner, with an ED50 value of 71.8 ± 16.9 nM. This activation was mediated by GalR2, since its agonist AR-M1896 mimicked the effect of galanin, and since galanin did not facilitate BK currents in cells co-expressing cDNAs of BK and GalR1 or GalR3. The galanin-induced BK current persisted after replacement with Ca²⁺-free solution, suggesting that extracellular Ca²⁺ is not essential. Chelating intracellular Ca²⁺ by either the slow Ca²⁺ buffer EGTA or the fast Ca²⁺ buffer BAPTA abolished galanin-mediated activation of BK channels, indicating the important role of intracellular Ca²⁺. The role of Ca²⁺ efflux from the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) was confirmed by application of thapsigargin, an irreversible inhibitor that depletes Ca²⁺ from SR/ER. Moreover, the inositol-1,4,5-triphosphate receptor (IP3R) was identified as the mediator responsible for increased intracellular Ca²⁺ activating BK channels. Taken together, activation of GalR2 leads to elevation of intracellular Ca²⁺ is due to Ca²⁺ efflux from ER through IP3R sequentially opening BK channels.     

Gsdma3 is required for hair follicle differentiation in mice

Apoptosis can result from activation of three major pathways: the extrinsic, the intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. While the two former pathways are known to be operational in human polymorphonuclear neutrophils (PMNs), the existence of the ER stress-mediated pathway, generally involving caspase-4, has never been reported in these cells. Recently, we have documented that arsenic trioxide (ATO) induced apoptosis in human PMNs by a mechanism that needs to be further investigated. In this study, using immunofluorescence and electron microscopy, we present evidence of ER alterations in PMNs activated by the ER stress inducer arsenic trioxide (ATO). Several key players of the unfolded protein response, including GRP78, GADD153, ATF6, XBP1 and eIF2α are expressed and activated in PMNs treated with ATO or other ER stress inducers. Although caspase-4 is expressed and activated in neutrophils, treatment with a caspase-4 inhibitor did not attenuate the pro-apoptotic effect of ATO at a concentration that reverses caspase-4 processing and activation. Our results demonstrate for the first time that the ER stress-mediated apoptotic pathway operates in human neutrophils.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Folding of viral envelope glycoproteins in the endoplasmic reticulum

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.     

Mice carrying a conditional Serca2 allele for the generation of Ca handling-deficient mouse models

Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca²⁺ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2^null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2^flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2^flox mice with MLC-2v^wt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2^wt/floxMLC-2v^wt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2^flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2^flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca²⁺ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.     

Characterization of residues in the cytoplasmic domain of the LDL receptor required for exit from the endoplasmic reticulum

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE₈₁₄ and/or ExD₈₃₇ had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.     

Lipid rafts,microdomain heterogeneity and inter‐organelle contacts: Impacts on membrane preparation for proteomic studies

In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra‐membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter‐membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.