Linear Sucrose Gradients Research Articles

Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells

We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts.

A double-stranded RNA mycovirus in Botrytis cinerea.

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M1 and M2 with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Extracellular Trafficking of Myocilin in Human Trabecular Meshwork Cells

Myocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Isolation of Clathrin‐Coated Vesicles by Differential and Density Gradient Centrifugation

Clathrin-coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans-Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D(2)O-sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described. Furthermore, we describe a protocol in which differential centrifugation and a series of discontinuous sucrose gradients are used to isolate CCVs from rat liver. An approach to analyzing CCV purity by electron microscopy is also described.

The Cytosolic Domains of Ca2+-sensitive Adenylyl Cyclases Dictate Their Targeting to Plasma Membrane Lipid Rafts

Lipid rafts are specialized, cholesterol-rich domains of the plasma membrane that are enriched in certain signaling proteins, including Ca(2+)-sensitive adenylyl cyclases. This restrictive localization plays a key role in the regulation of the Ca(2+)-stimulable AC8 and the Ca(2+)-inhibitable AC6 by capacitative calcium entry. Interestingly, AC7, a Ca(2+)-insensitive AC, is found in the plasma membrane but is excluded from lipid rafts (Smith, K. E., Gu, C., Fagan, K. A., Hu, B., and Cooper, D. M. F. (2002) J. Biol. Chem. 277, 6025-6031). The mechanisms governing the specific membrane targeting of adenylyl cyclase isoforms remain unknown. To address this issue, a series of chimeras were produced between the raft-targeted AC5 and the non-raft-targeted AC7, involving switching of their major domains. The AC5-AC7 chimeras were expressed in HEK 293 cells and lipid rafts were isolated from the bulk plasma membrane by either detergent-based or non-detergent-based fractionation methods. Additionally, confocal imaging was used to investigate the precise cellular targeting of the chimeras. Surprisingly, the two tandem six-transmembrane domains of AC5 were not required for localization to lipid rafts. Rather, AC5 localization depended on the complete cytoplasmic loops (C1 and C2); constructs with mixed domains were either retained in the endoplasmic reticulum or degraded. Similar conclusions are drawn for the lipid raft localization of the Ca(2+)/calmodulin-stimulable AC8; again, the C1 and C2 domains are critical. Thus, protein-protein interactions may be more important than protein-lipid interactions in targeting these calcium-sensitive enzymes to lipid rafts.

Annexin A2 is a novel RNA-binding protein.

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.

Putative virulence factors are released in association with membrane vesicles from Burkholderia cepacia.

Like many other Gram-negative bacteria, Burkholderia cepacia naturally releases membrane vesicles (n-MVs) during normal growth. Through filtration and differential centrifugation, n-MVs from clinical isolates of the IIIa and V genomovars were isolated and their characteristics compared. Electron microscopy revealed that they were spherical, 30-220 nm in diameter, and bilayered. Virulence factors thought to play a role in pathogenicity (e.g., lipase, phospholipase-N, and protease, including a metalloprotease) were found associated with n-MVs, while peptidoglycan zymogram analysis also revealed 26, 28, 36, and 66 kDa peptidoglycan-degrading enzymes. n-MVs were often contaminated with flagella and pili when isolated by traditional methods, and a new strategy using a linear isopycnic sucrose gradient was utilized. For better characterization, this was applied to a representative genomovar IIIa strain (C5424) and showed that n-MVs consisted of a subset of specific outer membrane and periplasmic proteins as well as lipopoly saccharide possessing only a putative minor O-side chain polymer. This finding suggests that certain components are selected by B. cepacia during n-MV formation, and since some are putative virulence factors, this property could help deliver the factors to tissue, thereby aiding infection.

Synaptotagmin IX, a possible linker between the perinuclear endocytic recycling compartment and the microtubules.

The pericentriolar endocytic recycling compartment (ERC) is involved in receptor and lipid recycling as well as in the delivery of internalized cargo from early endosomes to the trans Golgi network (TGN). We show that synaptotagmin (Syt) IX, a member of the Syt family of proteins, localizes to the ERC and is required for export from the ERC to the cell surface. We demonstrate that rat basophilic leukemia (RBL-2H3) mast cells endogenously express Syt IX mRNA and protein. Localization studies employing fractionation on linear sucrose gradients combined with confocal microscopy by indirect immunofluorescence or stable expression of a Syt IX-green fluorescent fusion protein demonstrate that Syt IX colocalizes with internalized transferrin (Tfn) and with Rab 11 at the perinuclear ERC. Syt IX also colocalizes with tubulin at the microtubules organizing center (MTOC) and remains associated with tubulin clusters formed in taxol-treated cells. Moreover, Syt IX coimmunoprecipitates with tubulin from intact RBL cells, and chimeric fusion proteins comprising either the C2A or the C2B domain of Syt IX are able to pull down tubulin from RBL cell lysates. To study the functional role of Syt IX, we have stably transfected RBL cells with Syt IX sense or antisense cDNA and monitored the routes of Tfn internalization and recycling in cells that overexpress (RBL-Syt IX+) or display substantially reduced (<90%) levels of Syt IX (RBL-Syt IX-). In these cells, Tfn binding and internalization into early endosomes and the ERC are unaltered. However, recycling from the ERC to the cell surface is significantly slowed down in the RBL-Syt IX- cells. These results therefore indicate that Syt IX is involved in regulating transport from the ERC to the cell surface, and suggest that it may play a role in linking vesicles that exit the ERC with the microtubules network.

Structural analyses of purified human immunodeficiency virus type 1 intracellular reverse transcription complexes.

Retroviruses copy their RNA genome into a DNA molecule, but little is known of the structure of the complex mediating reverse transcription in vivo. We used confocal and electron microscopy to study the structure of human immunodeficiency virus type 1 (HIV-1) intracellular reverse transcription complexes (RTCs). Cytoplasmic extracts were prepared 3, 4, and 16 h after acute infection by Dounce homogenization in hypotonic buffer. RTCs were purified by velocity sedimentation, followed by density fractionation in linear sucrose gradients and dialysis in a large pore cellulose membrane. RTCs had a sedimentation velocity of approximately 350 S and a density of 1.34 g/ml and were active in an endogenous reverse transcription assay. Double labeling of nucleic acids and viral proteins allowed specific visualization of RTCs by confocal microscopy. Electron microscopy revealed that RTCs are large nucleoprotein structures of variable shape consisting of packed filaments ca. 6 nm thick. Integrase and Vpr are associated with discrete regions of the 6-nm filaments. The nucleic acids within the RTC are coated by small proteins distinct from nucleocapsid and are partially protected from nuclease digestion.

A Novel Intracellular K+/H+ Antiporter Related to Na+/H+ Antiporters Is Important for K+ Ion Homeostasis in Plants

In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.

Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa

The profile of sedimentation on a 4–20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA. Their activity was maximal at pH 8.0 and increased in the presence of Ca 2+ ions. Both enzymes were inhibited by the chelating agent 1,10-phenanthroline but their thermal inactivation kinetics were different. A second protease peak was observed at 6.8 S and corresponded to a metallo-endoprotease that hydrolyzed with a maximal activity at pH 8.0 only the amide bonds of peptide substrates having a threonine residue at the P1' position. A last protease peak identified at 9.0 S contained a protease that preferentially acted on tripeptides, such as Val-Pro-Leu (diprotin B) and Thr-Val-Leu, releasing the C-terminal residue. Unlike the proteases identified in the two other peaks, its activity was maximal at acid pH (5.0) and was inhibited by the serine protease inhibitors. Together these results show the potential of A. ventricosa as a source of specific proteases.

Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas: EVIDENCE FOR CASPASE-8-GANGLIOSIDE INTERACTION IN T CELLS

In this investigation we show that the death-inducing signaling complex (DISC) associates with glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement. We primarily analyzed the ganglioside pattern and composition of GEM after triggering through CD95/Fas and observed that GM3 is the main ganglioside constituent of GEM. Stimulation with anti-CD95/Fas did not cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon CD95/Fas engagement, as revealed by the analysis of linear sucrose gradient fractions. It indicates that the DISC associates with GEM; no changes were observed in the distribution of caspase-9. The disruption of GEM by methyl-beta-cyclodextrin prevented DNA fragmentation, as well as CD95/Fas clustering on the cell surface, demonstrating a role for GEM in initiating of Fas signaling. These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway.

Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid.

We have examined the specificity of oleate as an activator of phospholipase D2 (PLD2) and whether it can be used to study PLD2 localization and its involvement in cell function. Oleate stimulates PLD activity in intact RBL-2H3 mast cells. Comparing PLD1- with PLD2-overexpressing cells, oleate enhanced PLD activity only in PLD2-overexpressing cells. Membranes were also sensitive to oleate and when membranes prepared from PLD1- and PLD2-overexpressing cells were examined, oleate further increased PLD activity only in membranes from PLD2-overexpressing cells. Overexpressed green fluorescent protein (GFP)-PLD2 fusion protein was localized at the plasma membrane and GFP-PLD1 was found in an intracellular vesicular compartment. Oleate was used to examine whether overexpressed PLD2 co-localized with endogenous PLD2. RBL-2H3 mast cell homogenates were fractionated on a linear sucrose gradient and analysed for both oleate-stimulated activity and ADP ribosylation factor 1-stimulated PLD1 activity. The oleate-stimulated activity co-localized with markers of the plasma membrane including the beta-subunit of the FcepsilonRI and linker for activation of T cells. Fractionation of homogenates from PLD2-overexpressing cells demonstrated that the overexpressed PLD2 fractionated in an identical location to the endogenous oleate-stimulated activity and this activity was greatly enhanced in comparison with control membranes. Examination of membranes prepared from COS-7, Jurkat and HL60 cells indicated a relationship between oleate-stimulated PLD2 activity and PLD2 immunoreactivity. We examined whether oleate could be used to activate secretion and membrane ruffling in adherent RBL-2H3 mast cells. Oleate did not stimulate secretion but did stimulate membrane ruffling, which was short-lived. We conclude that oleic acid is a selective activator of PLD2 and can be used for localization studies, but its use as an activator of PLD2 in intact cells to study function is limited due to toxicity.

The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi.

Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.

The Role of N-Glycosylation in Transport to the Plasma Membrane and Sorting of the Neuronal Glycine Transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.

Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.

The translation of mammalian selenoprotein mRNAs requires the 3' untranslated region that contains a selenocysteine insertion sequence (SECIS) element necessary for decoding an in-frame UGA codon as selenocysteine (Sec). Selenoprotein biosynthesis is inefficient, which may be due to competition between Sec insertion and termination at the UGA/Sec codon. We analyzed the polysome distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, a member of the glutathione peroxidase family of selenoproteins, in rat hepatoma cell and mouse liver extracts. In linear sucrose gradients, the sedimentation velocity of PHGPx mRNA was impeded compared to CuZn superoxide dismutase (SOD) mRNA, which has a coding region of similar size. Selenium supplementation increased the loading of ribosomes onto PHGPx mRNA, but not CuZn SOD mRNA. To determine whether the slow sedimentation velocity of PHGPx mRNA is due to a block in elongation, we analyzed the polysome distribution of wild-type and mutant mRNAs translated in vitro. Mutation of the UGA/Sec codon to UGU/cysteine increased ribosome loading and protein synthesis. When UGA/Sec was replaced with UAA or when the SECIS element core was deleted, the distribution of the mutant mRNAs was similar to the wild-type mRNA. Addition of SECIS-binding protein SBP2, which is essential for Sec insertion, increased ribosome loading and translation of wild-type PHGPx mRNA, but had no effect on the mutant mRNAs. These results suggest that elongation is impeded at UGA/Sec, and that selenium and SBP2 alleviate this block by promoting Sec incorporation instead of termination.

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Autoregulation of the androgen receptor at the translational level:: Testosterone induces accumulation of androgen receptor mrna in the rat ventral prostate polyribosomes

Several studies have documented that androgens have the ability to autoregulate their own receptor levels; however, the mechanism of such autoregulation remains poorly understood. Along these lines, our laboratory has shown that testosterone increased androgen receptor (AR) protein levels and binding in the castrated rat ventral prostate within 1 h. Ongoing protein synthesis was required for the testosterone effect, as the protein synthesis inhibitor cycloheximide blocked this effect. Testosterone and/or actinomycin D, an mRNA synthesis inhibitor, did not affect the steady-state AR mRNA levels. Therefore, we suggest that the early events induced by testosterone are posttranscriptional and that protein synthesis is required for the maintenance of AR protein and AR mRNA levels. In addition, we hypothesize that the testosterone posttranscriptional effect is primarily through the sequestering of AR mRNA in the prostate polyribosomes. To test this hypothesis, total RNA was isolated from prostate polyribosomes of controls and testosterone-treated rats and AR mRNA levels were quantitated by competitive reverse transcription–polymerase chain reaction. Polyribosomes profiles on linear sucrose gradients showed no difference in the sedimentation characteristics of ribosomal particles from the vehicle-treated control or testosterone-treated animals. Furthermore, because both polyribosomal preparations can direct protein synthesis to the same extent in a cell-free system, testosterone does not increase the efficiency of translation. However, competitive reverse transcription–polymerase chain reaction revealed that testosterone increases AR mRNA associated with polyribosomes by threefold after 1 h of treatment compared with control. These data suggest a rapid testosterone-mediated posttranscriptional mechanism, in which testosterone regulates the stability of the AR mRNA by sequestering it in polyribosomes, and consequently increasing its translation.