title: Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins.
abstract: We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yang
authors.forename: B
authors.initials: B
authors.lastname: Storrie
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D016259:Genes, myc / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics; D009237:N-Acetyllactosamine Synthase / Q000235:genetics / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000096:biosynthesis / Q000235:genetics; D014709:Vero Cells
chemical_list: C053330:Golgi membrane glycoproteins; C084004:LMAN1 protein, human; C501458:Lman1 protein, rat; D037241:Mannose-Binding Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D009237:N-Acetyllactosamine Synthase; D012799:Sialyltransferases; D015739:Nocodazole