Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells.
We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.
Stults
N L
NL
Department of Biochemistry, University of Georgia, Athens 30602.
Fechheimer
M
M
Cummings
R D
RD
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D003703:Demecolcine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D005690:Galactose / Q000378:metabolism; D006020:Glycopeptides / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008358:Mannose / Q000378:metabolism; D010053:Ovary; D011499:Protein Processing, Post-Translational; D012794:Sialic Acids / Q000032:analysis; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D014316:Tritium
D006020:Glycopeptides; D006023:Glycoproteins; D012794:Sialic Acids; D013463:Sulfuric Acid Esters; D014316:Tritium; C007482:dimethyl sulfate; D008358:Mannose; D005690:Galactose; D003703:Demecolcine