Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells

Ivan De Curtis, Kathryn E. Howell, Kai Simons

Research output: Contribution to journalArticle

Abstract

Incubation of cultured cells at 20 °C blocks the transport of newly synthesized plasma membrane proteins, and the proteins accumulate intracellularly in a terminally glycosylated form. When baby hamster kidney cells are infected with the ts O45 mutant of vesicular stomatitis virus, and incubated at 20 °C, the terminally glycosylated spike glycoprotein G of the virus accumulates in the membranes of a tubular network localized on the trans side of the Golgi cisternae, the trans-Golgi network (TGN). We have used the G protein of ts O45 as a marker for the TGN and isolated a TGN fraction using a combination of conventional cell fractionation techniques and immunoisolation. The TGN was separated from the bulk of the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and endosomes, while the activity of trans-Golgi marker galactosyltransferase copurified with the G protein. Using G protein as the TGN marker we have determined that the TGN was enriched 25-fold in the final fraction relative to the total homogenate. Several polypeptides (Mr 75,000, 87,000, 92,000, and 120,000) copurified with the G protein in the isolated TGN fraction and most likely represent resident markers of the compartment.

Original languageEnglish
Pages (from-to)248-265
Number of pages18
JournalExperimental Cell Research
Volume175
Issue number2
DOIs
Publication statusPublished - 1988

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trans-Golgi Network
Cricetinae
Kidney
GTP-Binding Proteins
Cell Membrane
Viruses
Galactosyltransferases
Cell Fractionation
Vesicular Stomatitis
Endosomes
Lysosomes
Endoplasmic Reticulum
Blood Proteins
Cultured Cells
Glycoproteins
Mitochondria
Membrane Proteins
Peptides
Membranes

ASJC Scopus subject areas

  • Cell Biology

Cite this

Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells. / De Curtis, Ivan; Howell, Kathryn E.; Simons, Kai.

In: Experimental Cell Research, Vol. 175, No. 2, 1988, p. 248-265.

Research output: Contribution to journalArticle

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