A single mutation in Chinese hamster ovary cells impairs both Golgi and endosomal functions.

Abstract

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071). DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification. Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 X DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae. That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

• Authors: A R Robbins, C Oliver, J L Bateman, S S Krag, C J Galloway, I Mellman
• Journal: The Journal of cell biology (J Cell Biol) Vol. 99 Issue 4 Pt 1 Pg. 1296-308 (Oct 1984) ISSN: 0021-9525 [Print] United States
• PMID: 6480694 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Sialic Acids
• Viral Proteins
• Tritium
• N-Acetylneuraminic Acid

Topics

• Animals
• Cell Fusion
• Cell Line
• Cell Transformation, Viral
• Cricetinae
• Cricetulus
• Endocytosis
• Female
• Golgi Apparatus (ultrastructure)
• Microscopy, Electron
• Mutation
• N-Acetylneuraminic Acid
• Organoids (ultrastructure)
• Ovary
• Sialic Acids (analysis)
• Sindbis Virus (genetics)
• Tritium
• Viral Proteins (genetics, isolation & purification)