title: Hyaluronan synthase mediates dye translocation across liposomal membranes.
abstract: BACKGROUND
  Hyaluronan (HA) is made at the plasma membrane and secreted into the extracellular medium or matrix by phospolipid-dependent hyaluronan synthase (HAS), which is active as a monomer. Since the mechanism by which HA is translocated across membranes is still unresolved, we assessed the presence of an intraprotein pore within HAS by adding purified Streptococcus equisimilis HAS (SeHAS) to liposomes preloaded with the fluorophore Cascade Blue (CB).
  
  
  RESULTS
  CB translocation (efflux) was not observed with mock-purified material from empty vector control E. coli membranes, but was induced by SeHAS, purified from membranes, in a time- and dose-dependent manner. CB efflux was eliminated or greatly reduced when purified SeHAS was first treated under conditions that inhibit enzyme activity: heating, oxidization or cysteine modification with N-ethylmaleimide. Reduced CB efflux also occurred with SeHAS K48E or K48F mutants, in which alteration of K48 within membrane domain 2 causes decreased activity and HA product size. The above results used liposomes containing bovine cardiolipin (BCL). An earlier study testing many synthetic lipids found that the best activating lipid for SeHAS is tetraoleoyl cardiolipin (TO-CL) and that, in contrast, tetramyristoyl cardiolipin (TM-CL) is an inactivating lipid (Weigel et al, J. Biol. Chem. 281, 36542, 2006). Consistent with the effects of these CL species on SeHAS activity, CB efflux was more than 2-fold greater in liposomes made with TO-CL compared to TM-CL.
  
  
  CONCLUSIONS
  The results indicate the presence of an intraprotein pore in HAS and support a model in which HA is translocated to the exterior by HAS itself.
authors.affiliation: Department of Biochemistry & Molecular Biology, The University of Oklahoma Health Sciences Center (940 S, L, Young Blvd), Oklahoma City, OK 73104, USA.
authors.forename: Andria P
authors.initials: AP
authors.lastname: Medina
authors.forename: Jialing
authors.initials: J
authors.lastname: Lin
authors.forename: Paul H
authors.initials: PH
authors.lastname: Weigel
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry*; D002308:Cardiolipins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics; D000076002:Hyaluronan Synthases; D008081:Liposomes / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010539:Permeability; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013291:Streptococcus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D002308:Cardiolipins; D005456:Fluorescent Dyes; D008081:Liposomes; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C066850:Cascade Blue; D014453:Glucuronosyltransferase; D000076002:Hyaluronan Synthases