title: Modification of the cytoplasmic domain affects the subcellular localization of Golgi glycosyl-transferases.
abstract: Our goal was to engineer a Golgi glycosyltransferase epitope-tagged on its cytoplasmically exposed, short, N-terminal domain that gave normal subcellular localization. Partial replacement of the cytoplasmic tail of human alpha-2,6-sialyltransferase (SialylT) with the negatively charged myc or FLAG epitope resulted in almost complete mislocalization of the chimera expressed in Vero cells. A granular cytoplasmic staining pattern was seen by immunofluorescence. Spacing the negatively charged residues progressively outward from the negative N-terminus resulted in increasingly more normal localization of myc or FLAG-tagged protein to a juxtanuclear Golgi-like distribution. Substitution of a neutrally charged VSV-G sequence for these tags resulted in normal localization of the chimera to the juxtanuclear Golgi region. Insertion of the myc epitope within the N-terminal domain of the short form of bovine beta-1,4-galactosyltransferase (GalT) gave a chimeric protein that mislocalized in BHK cells. No signal was detected with a monoclonal anti-epitope antibody indicating that the myc epitope was masked. Placement of myc or FLAG epitopes at the NH2-terminus of human N-acetylglucosaminyltransferase I (GlcNAc-T) resulted in chimeric proteins that in Vero cells displayed little Golgi localization. We conclude that positioning of negative charge, in particular, close to the membrane, typically produces a failure of type II Golgi glycosyltransferases to exit the ER/CGN, presumably due to quality control mechanisms. These proteins may be successfully epitope-tagged on their N-terminal domain either using a neutral or positively charged sequence or spacing any negatively charged sequence out from the membrane.
authors.affiliation: Department of Biochemistry and Anacrobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: R
authors.initials: R
authors.lastname: Pepperkok
authors.forename: P
authors.initials: P
authors.lastname: Bender
authors.forename: T E
authors.initials: TE
authors.lastname: Kreis
authors.forename: B
authors.initials: B
authors.lastname: Storrie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002451:Cell Compartmentation; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012799:Sialyltransferases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013329:Structure-Activity Relationship; D014709:Vero Cells; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
chemical_list: D000939:Epitopes; D011993:Recombinant Fusion Proteins; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase