Mutations Affecting Synthesis of β-Galactosidase Activity in the Yeast KLUYVEROMYCES LACTIS

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac^-) were isolated and characterized. All of the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal^-). Since the Gal^- and Lac^- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.——To further characterize the nature of the Lac^- phenotype, the basal and inducible level of β-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of β-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of β-galactosidase activity.     

Polyribosomes in isolated liver cells. Preparative procedures, effects of incubation and correlation with protein synthesis.

Methods have been derived which permit the isolation of undergraded polyribosomes from isolated rat liver cells. Under the conditions used the polyribosome profile of hepatocytes immediately after isolation was essentially identical with that from intact liver. However, during incubation of cells in complex physiological media there was a progressive dissociation of polyribosomes. The addition of a variety of factors that produce reaggregation of polyribosomes in rat liver in vivo did not prevent dissociation during cell incubations. Although large polyribosomes were lost most rapidly, the albumin-synthesizing capacity of isolated cells was not selectively lost when compared with total protein synthesis. The significance of these results for the use of isolated hepatocytes in the study of liver protein synthesis is discussed.     

Identification of a precursor protein to the major glycoproteins of mouse mammary tumor virus.

Mouse mammary tumor virus-producing cultures of mouse mammary tumor cells synthesize a viral-related polypeptide of molecular weight of 73,000 (gp 73) which is rapidly labeled during a short pulse but disappears during the chase concomitantly with the appearance of label in the virion glycoproteins gp 49 and gp 37.5/33.5. The addition of the protein synthesis-inhibitor cycloheximide to the chase medium has little effect on this conversion. Treatment of the proposed precursor with alpha-chymotrypsin leads to the formation of a polypeptide of molecular weight 49,000, similar to the major virion glycoprotein. A comparison of tryptic digest maps of the glycoproteins involved supports the hypothesis that both the viral glycoproteins gp 49 and gp 37.5/33.5 are derived from gp 73.     

Utilization of diversity indices in evaluating the effect of a paper mill effluent on bottom fauna

Hydrobiologia - Bottom fauna surveys of the Lower Sabine River in the vicinity of Orange, Texas were performed from 1967 to 1969. During this time samples were taken before and after effluent from...     

Discovery of potent and selective Spleen Tyrosine Kinase inhibitors for the topical treatment of inflammatory skin disease

The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.     

MARCH6 and TRC8 facilitate the quality control of cytosolic and tail‐anchored proteins

Misfolded or damaged proteins are typically targeted for destruction by proteasome‐mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome‐mediated degradation of the soluble misfolded reporter, mCherry‐CL1, involves two ER‐resident E3 ligases, MARCH6 and TRC8. mCherry‐CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail‐anchored protein heme oxygenase‐1 (HO‐1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO‐1 following intramembrane proteolysis. Our results highlight how ER‐resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail‐anchored proteins.     

Prelysosomal divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis

The routes followed by epidermal growth factor and transferrin during their endocytosis by human epithelial cells were compared in double-label studies by using density gradient centrifugation of cell homogenates and fluorescence microscopy with intact cells. Gradient centrifugation studies of cells incubated with radioactively labeled epidermal growth factor and transferrin indicated that both ligands initially were associated with a class of vesicles having a density of 1.037 g/mL and then were rapidly transferred to a membrane compartment having a slightly higher density (1.039 g/mL). Subsequently, the two ligands diverged. Epidermal growth factor ultimately was transferred to a membranous compartment containing lysosomal enzymes (density (1.08 g/mL) where it was degraded. Transferrin was released intact from the cells; very little was transferred to lysosomes. Using fluorescently labeled ligands, it was observed that after cells were warmed to 37 degrees C for 5 min, transferrin and epidermal growth factor gave coincident, punctate fluorescent patterns, strongly suggesting they were localized within the same endocytic vesicles. Subsequently, the epidermal growth factor signal was observed in lysosomes whereas the transferrin signal became weaker and diffuse and did not coincide with the punctate epidermal growth factor fluorescence. The time course of the divergence of the radioactive and fluorescent ligands coupled with the previous morphologic studies on the pathway of epidermal growth factor internalization [Willingham, M. C., & Pastan, I. (1982) J. Cell Biol. 94, 207-212] suggests that the sorting process is prelysosomal and possibly Golgi associated.     

Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores

Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.     

Microinjection of anticlathrin antibodies into fibroblasts does not interfere with the receptor-mediated endocytosis of alpha2-macroglobulin

Affinity-purified antibodies prepared against the major coat protein of brain coated vesicles, clathrin, were microinjected into cultured fibroblasts, and their intracellular distribution was followed by immunofluorescence microscopy and ultrastructural immunocytochemistry. Microinjected anticlathrin antibodies were concentrated on coated regions of the plasma membrane and the GERL apparatus. When an excess of anticlathrin antibodies was injected into the cytosol, coated pits on the plasma membrane were covered by anticlathrin antibody but still functioned to cluster an internalize alpha2-macroglobulin. These results are discussed in terms of the role of clathrin in the pathway of receptor-mediated endocytosis. Our data indicate that in cultured fibroblasts coated pits are stable elements permanently attached to the plasma membrane.     

M?ssbauer spectroscopic studies of the terminal dioxygenase protein of benzene dioxygenase from Pseudomonas putida.

Mössbauer spectra obtained from the terminal dioxygenase protein of the benzene dioxygenase system from Pseudomonas putida show that it contains [2Fe--2S] centres similar to those of the two-iron plant-type ferredoxins. In the oxidized form the two iron atoms within the centre are high-spin ferric but with considerable inequivalence. In the reduced form the centre contains one extra electron, and this is localized on one of the iron atoms, which becomes high-spin ferrous.