title: Geldanamycin-associated inhibition of intracellular trafficking is attributed to a co-purified activity.
abstract: Geldanamycin, an ansamycin antibiotic that specifically inhibits heat-shock protein-90 (HSP90) and its endoplasmic reticulum homologue, glucose-regulated protein-94 (GRP94), accelerates the degradation of selected cellular proteins. We showed previously that geldanamycin inhibits maturation and transport of the epidermal growth factor receptor in addition to accelerating its degradation (Supino-Rosin, L., Yoshimura, A., Yarden, Y., Elazar, Z., and Neumann, D. (2000) J. Biol. Chem. 275, 21850-21855). Here we demonstrate that the additional activities of geldanamycin on intracellular transport and protein maturation are related to its supply source. By combining chemical separation of Streptomyces hygroscopicus var. geldanus extracts and biological screens, we show that the geldanamycin-associated effects on intracellular transport and protein maturation are not mediated by geldanamycin itself but are due to the presence of an additional component(s). Chromatography of S. hygroscopicus var. geldanus extracts on a silica-gel column allowed separation between the inhibition of intracellular trafficking and geldanamycin-mediated degradation. One fraction that was devoid of geldanamycin blocked secretion of a soluble form of the erythropoietin receptor, retarded maturation of the epidermal growth factor receptor without enhancing its degradation, and blocked anterograde transport of a temperature-sensitive mutant of the vesicular stomatitis virus G protein (VSVGtsO45) from the early Golgi cisternae. This fraction was enriched (>95%) in 17-demethylgeldanamycin. However, as synthetically derived 17-demethylgeldanamycin did not inhibit intracellular trafficking, we concluded that 17-demethylgeldanamycin is not the active component. We thus propose that a compound(s) that co-purifies with benzoquinone ansamycins inhibits intracellular transport. Taken together, our data demonstrate that the inhibitory effects on protein maturation and intracellular trafficking, previously attributed to geldanamycin, are mediated by another distinct moiety.
authors.affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
authors.forename: Eran
authors.initials: E
authors.lastname: Barzilay
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ben-Califa
authors.forename: Lia
authors.initials: L
authors.lastname: Supino-Rosin
authors.forename: Yoel
authors.initials: Y
authors.lastname: Kashman
authors.forename: Koret
authors.initials: K
authors.lastname: Hirschberg
authors.forename: Zvulun
authors.initials: Z
authors.lastname: Elazar
authors.forename: Drorit
authors.initials: D
authors.lastname: Neumann
mesh_terms: D000818:Animals; D016227:Benzoquinones; D001692:Biological Transport; D019556:COS Cells; D002845:Chromatography; D002850:Chromatography, Gel; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006596:Hexosaminidases / Q000378:metabolism; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D021381:Protein Transport; D011809:Quinones / Q000494:pharmacology*; D017467:Receptors, Erythropoietin / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014759:Viral Envelope Proteins / Q000737:chemistry
chemical_list: D016227:Benzoquinones; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; C028391:G protein, vesicular stomatitis virus; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011809:Quinones; D017467:Receptors, Erythropoietin; D014759:Viral Envelope Proteins; C018551:glucose-regulated proteins; D066246:ErbB Receptors; D006596:Hexosaminidases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C001277:geldanamycin