Direct association of messenger RNA labeled in the presence of fluoroorotate with membranes of the endoplasmic reticulum in rat liver

Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.     

甜菊愈伤组织分生区细胞中膜内含物的超微结构研究

生长在分化培养基上的甜菊（Ｓｔｅｖｉａ ｒｅｂａｕｄｉａｎａ）愈伤组织分生区细胞中存在双膜和多膜内含物。电镜观察表明,这些膜内含物是由一圈或多圈呈同民贺或卷绕状排列的内质网包围部分细胞质而形成的。双膜内含物内外层膜的靠细胞质表面有核糖体附着,而多膜内含物仅在其最外层潴泡的外膜上偶有和量核糖体附着。附着细胞液泡化程度的提高,多膜内含物通过液泡膜内陷而转移到液泡中或通过消化其中被包围的细胞质及内膜而转     

Localization of calmodulin in rat cerebellum by immunoelectron microscopy

Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

苜蓿组织培养中球形胚发生时特异蛋白质和同工酶分析

试验在苜蓿组织培养中,对球形胚形成过程中特异蛋白质表达的模式、过氧化物酶及酯酶同工酶酶谱变化进行研究,结果表明：苜蓿组织培养中从胚性愈伤组织到球形胚发育的进程中,顺序消失和出现了11种中小分子量多肽;过氧化物酶同工酶酶谱发生了显著的变化;酯酶同工酶酶谱变化不大,但其总活力对于维持体细胞胚胎发生是必须的。     

荔枝雄花性别决定过程中细胞超微结构的变化

荔枝雄花雌蕊原基在大孢子母细胞减数分裂后开始衰退.内质网历经增生扩展,穿壁相连,同心缠绕,多条平行弯曲,不规则堆叠.内质网和高尔基体产生许多囊泡,囊泡在细胞内含物的降解和运输过程中起着重要的作用.线粒体在雌蕊原基细胞衰败的前、中期数量增加,后期分批降解.过氧化物酶体在雌蕊原基细胞衰败的中期紧挨核短暂出现.细胞核的染色质凝集断裂;核周腔扩大,形成胀泡;染色质趋边,外泄.细胞原生质表现出有序的、在膜包裹下的降解,首先是核糖体,而后依次是:过氧化物酶体、内质网、高尔基体、线粒体、核.雌蕊原基的衰败历程可能是一种程序性细胞死亡的过程.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

ATTACHMENT OF RIBOSOMES TO MEMBRANES DURING POLYSOME FORMATION IN MOUSE SARCOMA 180 CELLS

Addition of nutrients to starved mouse S-180 cells leads to rapid conversion of ribosomal monomers to polysomes. During this process, a portion of the ribosomes originally found in the 17,000 g (10 min centrifugation) supernatant of cell lysates becomes firmly attached to structures sedimenting at 500 g (5 min centrifugation). Electron microscopy of sections of the intact cells showed the change from randomly distributed ribosomal particles to clusters. Association with membranes also became evident. The material sedimenting at 500 g comprised nuclei enclosed in an extensive endoplasmic reticulum (ER) network. This fraction prepared from recovering cells showed numerous ribosome clusters associated with the ER network. The appearance of many of these clusters indicated that the ribosomal particles were not directly bound to the membranes. RNase treatment released about 40% of the attached ribosomes as monomers, and ethylenediaminetetraacetic acid released 60% as subunits. It is suggested that during polysome formation a portion of the ribosomes becomes attached to the membranes through the intermediary of messenger RNA.     

The effect of neuraminidase treatment of ribosome-free membranes on their ribosomal reattachment ability

Ribosome-free membranes prepared by means of 2M lithium chloride lose 90% of their ability to reattach ribosomes after digestion with neuraminidase for 1 hour. It can be shown that this is not due to proteolytic degradation of the membranes by contaminating proteases presant in neuraminidase preparations. It was found that neuraminidase troatment of rat liver RER results in the release of approximately 50% of the bound ribosomes. Ribosomal reattachment ability can be restored to the neuraminidase-treated membranes by incubation with N-acetylneuraminic acid and nucleotide triphosphates.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

Laminated cisternae of the rough endoplasmic reticulum induced by coronavirus MHV-A59 infection

The infection of murine fibroblasts of the sac- line with a coronavirus, mouse hepatitis virus strain A59 (MHV-A59), results in a novel modification to some cisternae of the rough endoplasmic reticulum (RER). From 8 hours post infection (h.p.i.) we see in thin sections pairs of cisternae closely, stably and uniformly aligned. Serial sectioning shows that the regions of pairing or lamination extend for many thousands of nm in two dimensions, with the spacing between the juxtaposed membranes remaining very uniform at about 18 nm. These structures appear coincident with the onset of accumulation of the viral glycoprotein E1 in the RER membrane but 2 hours after the viral glycoprotein E2 can first be detected there. Ribosomes are excluded from the paired cisternal surfaces, while budding of progeny virions has never been seen at the cisternal membranes facing the cytosol, although ribosomes bind there. The lumina of paired cixternae are usually devoid of virions which, however, accumulate in areas where the paired cisternae diverge. Electron immunocytochemistry shows that both E1 and E2 glycoproteins are abundant in the paired cisternae. Following labelling for the E1 glycoprotein we see a periodic fine structure, rows of "beads" with a centre to centre spacing of about 7.5 nm, in the region between the paired membranes. In oblique sections of this region in cells fixed as if for the immunoperoxidase labelling, but omitting all its steps we see parallel rows of "beads" separated by about 7 nm. We suggest that the membrane spanning viral glycoprotein E1 together with viral nucleocapsids may be involved in laminating cisternae of the RER.     

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity

We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.     

Taxol induces microtubule-rough endoplasmic reticulum complexes and microtubule-bundles in cultured chondroblasts

Taxol induces a vast increase in the number of microtubules (MTs) in functional chondroblasts. The drug also induces a marked change in MT distribution. In control cultures, anti-tubulin stains long, fine, sinuous filaments radiating from a perinuclear center. In taxol-treated cells, anti-tubulin stains stubby, straight, chevron-like structures that assume a striking antipodal distribution. Such MT-bundles are relatively stable: they persist for over 48 h after removal of taxol, and even for 16-24 h in Colcemid. Many of these supernumerary MTs bind to, and align on, the cytoplasmic face of the rough endoplasmic reticulum (RER). In binding, the MTs displace the numerous ribosomes that normally stud the surface of the cisternae of the RER. The bound MTs form a remarkably uniform layer with center-to-center spacings of 40 nm. The attached parallel arrays of MTs achieve lengths of over 10 microns. These bound MTs not only dislodge ribosomes from the RER surface, but they also zip together adjacent ER complexes, forming tiers of two to eight cisternae. Numerous cytoplasmic bundles of hexagonally-ordered MTs are also induced. When closely aligned, the MTs assume a crystalline configuration with a six-fold symmetry, a central MT being surrounded by six equidistant MTs. A single cell can have over 100 MT-bundles and the number of MTs per bundles varies from 2-30. The forces aggregating cytoplasmic MT-bundles probably differ from those that bind MTs to the RER. Taxol also fragments the prominent Golgi complex that characterizes actively secreting chondroblasts. No obvious morphologic relationship has yet been detected between these induced MTs and other organelles such as intermediate-sized filaments, microfilaments, mitochondria, Golgi cisternae, or secretory vesicles.     

Taxol Induces Microtubule-Rough Endoplasmic Reticulum Complexes and Microtubule-Bundles in Cultured Chondroblasts

Abstract. Taxol induces a vast increase in the number of microtubules (MTs) in functional chondroblasts. The drug also induces a marked change in MT distribution. In control cultures, anti-tubulin stains long, fine, sinuous filaments radiating from a perinuclear center. In taxol-treated cells, anti-tubulin stains stubby, straight, chevron-like structures that assume a striking antipodal distribution. Such MT-bundles are relatively stable: they persist for over 48 h after removal of taxol, and even for 16–24 h in Colcemid. Many of these supernumerary MTs bind to, and align on, the cytoplasmic face of the rough endoplasmic reticulum (RER). In binding, the MTs displace the numerous ribosomes that normally stud the surface of the cisternae of the RER. The bound MTs form a remarkably uniform layer with center-to-center spacings of 40 nm. The attached parallel arrays of MTs achieve lengths of over 10 μm. These bound MTs not only dislodge ribosomes from the RER surface, but they also zip together adjacent ER complexes, forming tiers of two to eight cisternae. Numerous cytoplasmic bundles of hexagonally-ordered MTs are also induced. When closely aligned, the MTs assume a crystalline configuration with a six-fold symmetry, a central MT being surrounded by six equidistant MTs. A single cell can have over 100 MT-bundles and the number of MTs per bundles varies from 2–30. The forces aggregating cytoplasmic MT-bundles probably differ from those that bind MTs to the RER. Taxol also fragments the prominent Golgi complex that characterizes actively secreting chondroblasts. No obvious morphologic relationship has yet been detected between these induced MTs and other organelles such as intermediate-sized filaments, microfilaments, mitochondria, Golgi cisternae, or secretory vesicles.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.     

Protein Translocation across the Rough Endoplasmic Reticulum

The rough endoplasmic reticulum is a major site of protein biosynthesis in all eukaryotic cells, serving as the entry point for the secretory pathway and as the initial integration site for the majority of cellular integral membrane proteins. The core components of the protein translocation machinery have been identified, and high-resolution structures of the targeting components and the transport channel have been obtained. Research in this area is now focused on obtaining a better understanding of the molecular mechanism of protein translocation and membrane protein integration.Protein translocation across the rough endoplasmic reticulum (RER) is an ancient and evolutionarily conserved process that is analogous to protein export across the cytoplasmic membranes of eubacterial and archaebacterial cells both with respect to the mechanism and core components. The RER membrane of eukaryotic cells is contiguous with the nuclear envelope and is morphologically composed of interconnected cisternae and tubules. Electron microscope images of mammalian cells and tissues revealed that the cisternal regions of the cytoplasmic surface of the endoplasmic reticulum are densely studded by membrane-bound ribosomes (Palade 1955a,b), giving rise to the term “rough ER.” The RER-bound ribosomes in en face images are often arranged in spirals or hairpins (Palade 1955a; Christensen and Bourne 1999), indicative of polyribosomes that are actively engaged in protein translation.Consistent with this high density of membrane-bound ribosomes, the RER is a major site of protein biosynthesis in eukaryotic cells. The nuclear envelope, the Golgi, lysosome, peroxisome, plasma membrane, and endosomes are biosynthetically derived from the rough ER. The three major groups of proteins that are synthesized by RER-bound ribosomes include secretory proteins, integral membrane proteins destined for ER-derived membranes, and the lumenal-resident proteins of the ER, Golgi, nuclear envelope, and lysosome. For those membranes that are not physically linked to the ER (e.g., the lysosome), integral membrane and lumenal proteins are delivered to their destination by vesicular transport pathways. Bioinformatics analysis of fully sequenced eukaryotic genomes indicates that roughly 30% of open reading frames encode integral membrane proteins (Wallin and von Heijne 1998); hence, a major role of the RER is the biosynthesis of membrane proteins. An important class of membrane proteins that are integrated into the RER has single carboxy-terminal TM spans and are known as tail-anchored (TA) membrane proteins. The posttranslational integration pathway for TA proteins has been a subject of several recent reviews (Borgese and Fasana 2011; Shao and Hegde 2011), thus we will not address the TA pathway in this article.     

Mechanism of RNA redistribution of the 17,000g postmitochondrial supernatant after incubation at 37 degrees C and its impact on other related biochemical investigations

Evidence for stripping of ribosomes from RER in the 17,000g PMS of the liver of the cancer patient was obtained in the 1.35 M region which is the region between the SER and RER. RNA/protein ratios for the SER, 1.35 M region, and RER of 0.001, 0.083, and 0.235, respectively, for this liver are consistent with the degranulation of RER compared with RNA/protein ratios for SER and RER from normal livers of 0.025 +/- 0.003, and 0.35 + 0.030, respectively. A RNA/protein ratio of 0.235 was obtained for the RER region of the cancer patient. The EM revealed that the region of the RER in the cancer liver was ribosomal and not at all RER. This ribosomal material is reminiscent of the ribosomes banding in the 1.35- to 2 M domain of the RER isolated from the reconstitution experiments (SER + polyribosomes + supernatant). It was suggested that the ribosomal material isolated from the cancer liver could therefore be indicative of polyribosomal lesion or degradation. The 0.25-1.35 M interface (SER), 1.35 M region, and 1.35-2 M interface (RER) characteristics are therefore exploitable for diagnostic potential and further understanding of the molecular basis of cancer.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.