A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein.

Members of the Bunyaviridae family mature by a budding process in the Golgi complex. The site of maturation is thought to be largely determined by the accumulation of the two spike glycoproteins, G1 and G2, in this organelle. Here we show that the signal for localizing the Uukuniemi virus (a phlebovirus) spike protein complex to the Golgi complex resides in the cytoplasmic tail of G1. We constructed chimeric proteins in which the ectodomain, transmembrane domain (TMD), and cytoplasmic tail (CT) of Uukuniemi virus G1 were exchanged with the corresponding domains of either vesicular stomatitis virus G protein (VSV G), chicken lysozyme, or CD4, all proteins readily transported to the plasma membrane. The chimeras were expressed in HeLa or BHK-21 cells by using either the T7 RNA polymerase-driven vaccinia virus system or the Semliki Forest virus system. The fate of the chimeric proteins was monitored by indirect immunofluorescence, and their localizations were compared by double labeling with markers specific for the Golgi complex. The results showed that the ectodomain and TMD (including the 10 flanking residues on either side of the membrane) of G1 played no apparent role in targeting chimeric proteins to the Golgi complex. Instead, all chimeras containing the CT of G1 were efficiently targeted to the Golgi complex and colocalized with mannosidase II, a Golgi-specific enzyme. Conversely, replacing the CT of G1 with that from VSV G resulted in the efficient transport of the chimeric protein to the cell surface. Progressive deletions of the G1 tail suggested that the Golgi retention signal maps to a region encompassing approximately residues 10 to 50, counting from the proposed border between the TMD and the tail. Both G1 and G2 were found to be acylated, as shown by incorporation of [3H]palmitate into the viral proteins. By mutational analyses of CD4-G1 chimeras, the sites for palmitylation were mapped to two closely spaced cysteine residues in the G1 tail. Changing either or both of these cysteines to alanine had no effect on the targeting of the chimeric protein to the Golgi complex.     

Identification of Ca2+-stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light-grown shoots than from dark-grown ones, whereas no differences in activity between plasma membranes from light- and dark-grown roots were seen. Maximum activity of the membrane-bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.     

The Fluorane Derivatives: Methods for the Standardization of Biological Stains: Part II

In this paper the methods are given which are used in determining whether to approve the sale of certain dyes of the fluorane group as certified biological stains. The methods have been worked out by the Commission on Standardization of Biological Stains in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Dept. of Agriculture. The dyes for which the methods are given in the present paper are: Fluorescein, eosin yellowish, ethyl eosin, eosin bluish, erythrosin, phloxine B, and rose bengal. For each of these dyes methods are given under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

The Production of Basic Fuchsin Suitable for the Feulgen Technic

Much difficulty has been experienced in obtaining basic fuchsins satisfactory for the Feulgen technic. A method of purification employing sulfur dioxide is described which has been found to improve many unsatisfactory fuchsins. It is also shown that if the best grade of commercial pararosanilin base is used and proper precautions observed in converting it to the chloride or the acetate, a product can be obtained which gives excellent results in the Feulgen technic. The methods by which the base can be converted to the chloride or acetate are described in detail. Precautions are given for avoiding, in the conversion of the base to the dye salts, contamination of the product with the impurities which interfere with the staining. The use of the acetate is recommended because of its greater solubility.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Lipoprotein synthesis and secretion by cultured rat hepatocytes. Parallel inhibition of secretion of VLDL, HDL and albumin by monensin

The biosynthesis and secretion of very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) by cultured normal rat hepatocytes was investigated with particular emphasis on its modification by monensin. This acidic ionophore coordinately inhibited the rates of secretion of the several VLDL apolipoproteins and the VLDL lipids, suggesting an effect late in the process of biosynthesis and secretion, probably at the stage of exiting from the Golgi apparatus. The secretion of immunoreactive albumin into the medium was comparably inhibited, implying that the pathway and mechanisms involved in albumin secretion may be closely similar to those for VLDL synthesis and secretion. Secretion of phospholipids and of apolipoproteins E and A-I in the HDL fraction increased progressively with time over 18 h in control incubations but was strongly inhibited by monensin. During extended incubation with monensin at high concentrations (10 microM), there was a net release to the medium of a number of hepatocyte proteins, including some that comigrated with apolipoprotein A-I and apolipoprotein C, making it appear that monensin increased the secretion of these apolipoproteins. However, using labeled amino acids, it was shown by autoradiography and by immunoprecipitation that secretion of newly-synthesized, radioactive apolipoprotein A-I and apolipoprotein C was actually inhibited by monensin. These results are compatible with the conclusion that HDL synthesis and secretion may occur by mechanisms closely related to those for synthesis and secretion of albumin and VLDL.     

Synthesis of peptides by fragment condensation on a solid support. II. A scheme for preparation of 4,8-disubstituted vasopressins evaluated on 8-arginine-vasopressin.

A convenient scheme for the synthesis of 4,8-disubstituted vasopressins has been designed and its usefulness evaluated in the preparation of one of the parent hormones, 8-arginine-vasopressin. The main feature of the scheme involves preparation of two protected tripeptide fragments, Z-Cys(Bzl)-Tyr(Bzl)-Phe and Boc-Asn(Mbh)-Cys(Bzl)-Pro which are incorporated in a synthesis on a solid support. Both tripeptides were prepared conventionally with the carboxyl groups protected as benzyl esters. The benzyl-ester groups were removed by transesterification with 2-dimethylaminoethanol and subsequent hydrolysis. To avoid racemization in the coupling step with the fragment containing a C-terminal phenylalanine, N-hydroxysuccinimide was added. After removal of the peptide from the resin, deprotection, oxidation and desalting, final purification was effected by ion-exchange chromatography. Apart from the main product, which exhibited full pressor activity, only small amounts of impurities could be isolated.