Abstract
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.
Highlights
Glycoproteins to the cell surface was not affected by colcemid
Colcemid treatment had no the medium was removed and thecells were washed 3 to 4 times with detectable effect on the transport of newly synthesized glycoproteins to the cell surface. These results suggest that microtubules are not required for inter- or intra-Golgi trafficking during glycoprotein biosynthesisand do not facilitate
While the endoplasmic reticulum in control cells had an extended reticular network (Fig. le), in the presence of colcemid it was retracted from the cell periphery, and itslattice-like reticular morphology wasaltered (Fig. If).These results indicate, under the conditions of the colcemid treatment, that disassembly of microtubules in Chinese hamster ovary (CHO) cells is accompaniedby fragmentation of the Golgi apparatus and disruption of the ER
Summary
To determine the incorporation of radiolabeled sugar into total cell glycoproteins, aliquots of the solubilized phosphotungstic acidftrichloroacetic acid precipitates were assayed for protein content (Lowry et al, 1951) using BSA as the standard and for radioactivity using a Beckman LS 1801 liquid scintillation counter. Lectin Affinity Chromatography of ~ ~ ~ o l ~GelyclopeepdtidesLectin affinity chromatography of radiolabeled glycopeptides on columns (1-2-ml bed volume) of Con A-Sepharose, RCA I-agarose, and tomato lectin-Sepharose was carried out at room temperatureas described previously (Merkle and Cummings, 1987a).Fractions (1-2 ml) were collected and monitored for radioactivity by liquid scintillation counting. The resulting Pronase digests were desialylated and passed over RCA I-agarose to assess trypsin-released cell surface and cell-associated glycopeptides having terminal pl,4-Iinked galactose residues
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
