Phosphorylation-Related Crosstalk Between Distant Regions of the Core Region of the Coat Protein Contributes to Virion Assembly of Plum Pox Virus

Eukaryotic proteins are often targets of posttranslational modifications (PTMs). Capsid protein (CP) of plum pox virus (PPV), a member of genus Potyvirus, has been reported to be prone to phosphorylation in four serines at the N-terminal region. CP phosphorylation has been proposed to influence PPV infection by regulating CP accumulation in coordination with a second PTM, O-GlcNAcylation. In this study, a further proteomic characterization of PPV CP phosphorylation revealed additional phospho-targets, thus evidencing even greater complexity of the network of PTMs affecting this protein. In particular, two new phosphorylation targets, T254 and T313, at protein distal core, appear to be highly relevant for infection. Although abolishing phosphorylation at these positions does not have a severe effect on infectivity or viral accumulation, phospho-mimicking at either of these targets disrupts cell-to-cell movement. Strand-specific reverse transcription-quantitative PCR analysis and fractionation by centrifugation in a continuous sucrose gradient enabled us to conclude that such a deleterious effect is not related to failures in replication but is a consequence of inaccurate virion assembly. The analysis of spontaneous compensatory mutations at the CP core identified in a multiple phospho-mimicking mutant disclosed a functional dialogue between distant phospho-targets, which was further supported by an in silico PPV virion model, built on the watermelon mosaic virus atomic structure. Therefore, whereas joint and opposite action of O-GlcNAcylation and phosphorylation at the N-terminal disordered protrusion of CP appears to regulate protein stability, we propose that phosphorylations at the core region control assembly and disassembly of viral particles.

Abstract 390: VLDL Exits From The Endoplasmic Reticulum In A Specialized VLDL-transporting-vesicle (VTV) In Rat Primary Hepatocytes.

Transport of very low-density lipoprotein (VLDL) from its site of synthesis, the endoplasmic reticulum (ER), to the Golgi is required for its eventual secretion from hepatocytes. This step represents a potential therapeutic target in controlling VLDL export and thus control of its metabolic derivative, LDL, the major carrier of cholesterol and determinant of atherosclerosis. The present study was designed to understand how VLDL exits from the ER at the molecular level. We developed an in vitro ER-budding assay in which rat liver ER (500 ug) was pre-loaded with 14 C-triacylglycerol (TAG) to mark VLDL and 3 H-proteins to mark newly synthesized proteins. The ER was incubated with rat liver cytosol (1 mg), GTP, and ATP at 37 o C for 30 min. The reaction mix was fractionated on a continuous sucrose gradient and the distribution of 14 C-TAG and 3 H-protein across the gradient was determined. 14 C-TAG was found in the light density region of the gradient, the expected place for TAG-rich VLDL carrying vesicles whereas 3 H-proteins appeared in the mid portion, the expected place in the gradient for protein vesicles. We examined the distribution of apolipoprotein B100 (apoB100), a marker for VLDL and albumin (a typical liver secretory protein) across the same gradient by Western blotting. As expected, apoB100 was distributed in light fractions whereas albumin was mainly in the mid portion of the gradient. These data show that VLDL and albumin are transported in vesicles of differing density. We hypothesize that a specialized vesicle is utilized for VLDL transport, which we name the VLDL-transporting-vesicle (VTV). Our results show that the release of VTV from rat liver ER requires cytosol, GTP, Sar1 (a GTPase), ATP, and incubation at 37 o C. VTV was sealed as judged by apoB100 signal post proteinase K treatment. VTVs concentrate ApoB100, Sar1, and exclude ER-resident protein calnexin. VTV fuses with liver cis -Golgi and delivers its cargo, VLDL, to the Golgi lumen. 2D-gels and electron microscopy data reveal that VTVs are different in their protein composition and are larger in size when compared to albumin carrying vesicles. In conclusion, we have identified and characterized a new ER-derived vesicle, VTV, which transports nascent VLDL from the ER to the Golgi in primary hepatocytes.

Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells

Functional thyrotropin-releasing hormone (TRH) receptors have been expressed in Xenopus laevis oocytes following the microinjection of total and poly(A)+ RNA from GH3 rat anterior pituitary tumour cells. Under voltage-clamp conditions, application of the peptide induced a biphasic Ca(2+)-dependent chloride current. The amplitude of the initial, fast, component of the response was dependent on the concentration of the hormone and on the amount of mRNA injected. Size fractionation of poly(A)+ RNA on a continuous sucrose gradient and Northern blot analysis indicated that the receptor was encoded by an mRNA of approx. 3.5 kb. A 3.28 kbp cDNA encoding the TRH receptor has been cloned and sequenced. Full functionality of the predicted 412-amino-acid receptor protein was demonstrated by functional expression of cell surface receptors in Xenopus oocytes after both cytoplasmic injection of sense RNA transcribed in vitro from this cDNA and nuclear injection of the cDNA under the control of the Herpes simplex virus thymidine kinase promoter. The predicted protein contains seven putative membrane-spanning domains and shows significant sequence identify with some G-protein-coupled receptors. RNA blot analysis indicates that the mRNA for the TRH receptor is exclusively expressed in the pituitary gland. Expression studies performed with clones in which the 3′ region of the mRNA has been successively shortened indicate that the 3′ terminal region is not an important determinant for efficient functional expression in oocytes.

Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

Two enzymes involved in the synthesis of O-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells.

Microsomal membranes from mouse lymphoma BW5147 cells were fractionated on a continuous sucrose gradient and assayed for two enzymes involved in the synthesis of O-linked oligosaccharides. Both enzymes were recovered in membranes that were less dense than the membranes containing the endoplasmic reticulum marker enzymes, glucosidase I and II. UDP-Gal:N-acetylgalactosamine-beta 1, 3-galactosyltransferase had a distribution that coincided with that of the galactosyltransferase that acts on asparagine-linked oligosaccharides. This latter enzyme has been immunolocalized to the trans Golgi elements. The UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase was recovered in a membrane fraction of intermediate density, between the endoplasmic reticulum and trans Golgi markers. These findings are consistent with the assembly of O-linked oligosaccharides occurring in at least two different Golgi compartments.

A 45 000 Molecular Weight Human Renin Precursor is Synthesized in a Cell-Free Translation System

1. Human kidney mRNA species were isolated and fractionated through a continuous sucrose gradient ultracentrifugation. 2. mRNA fractions were translated by using a rabbit reticulocyte lysate and [35S]metbionine as tracer. Double immunoprecipitation was carried out with highly specific anti-human renin and anti-rabbit γ-globulin antisera. 3. A 15S mRNA has been found to direct synthesis of a 45 000 molecular weight protein immunoprecipitable with anti-human renin. This protein is considered to be the ultimate precursor of renin (preprorenin).

Virosome preparation: differences between influenza and rubella hemagglutinin adsorption

Rubella and influenza virosomes were prepared from preformed liposomes or dried lipid films with or without nonionic detergent (β-D-octylglucoside. The preformed liposomes and lipid films were prepared from lecithin and dicetyl phosphate (3.5:1). Viral hemagglutinin rosettes were prepared from purified viruses after solubilization with Triton X-100 (1%), centrifugation through a continuous sucrose gradient containing 30 mM octylglucoside, and dialysis. Analysis of virosomes by sucrose density gradient centrifugation, hemagglutination assay, and electron microscopy revealed that rubella hemagglutinin did not require the presence of detergent to form virosomes, whereas influenza hemagglutinin could absorb efficiently to liposomes only in the presence of detergent.

High content of tyrosine and arginine in peptides and proteins from the growth hormone producing cells of the adenohypophysis.

Fluorescence histochemistry has demonstrated an abundance of tyrosine and arginine residues in the growth hormone (GH) producing cells of the pituitary of several mammals. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. One granular fraction, rich in GH, was found to contain proteins or peptides rich in tyrosine and arginine. Gel chromatography of the acid-soluble components from sedimented pituitary granules revealed that the arginine- and tyrosine-rich material was heterogeneous. The arginine-containing peptides and proteins could be separated into several peaks with molecular weights from 5000-10,000 and higher. The tyrosine-containing material comprised one peptide with a molecular weight of 5000-10,000 and another much smaller peptide. Since GH itself is not excessively rich in either tyrosine or arginine, the tyrosine- and arginine-containing proteins or peptides probably constitute as yet unidentified granular components, distinct from GH itself.