Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum

C. V. Dang, D. C H Yang, T. D. Pollard

Research output: Contribution to journalArticle

Abstract

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, surcrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as 'cytoskeletal' preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

Original languageEnglish (US)
Pages (from-to)1138-1147
Number of pages10
JournalJournal of Cell Biology
Volume96
Issue number4
StatePublished - 1983

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Methionine-tRNA Ligase
Rough Endoplasmic Reticulum
Detergents
Ribosomes
Octoxynol
Endoplasmic Reticulum
Microtubules
Membranes
Buffers
Staining and Labeling
Antigens
Amino Acyl-tRNA Synthetases
Acridine Orange
Egtazic Acid
Normal Distribution
Keratins
Fluorescent Dyes
Fluorescence Microscopy
Actins
Electron Microscopy

ASJC Scopus subject areas

  • Cell Biology

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Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum. / Dang, C. V.; Yang, D. C H; Pollard, T. D.

In: Journal of Cell Biology, Vol. 96, No. 4, 1983, p. 1138-1147.

Research output: Contribution to journalArticle

Dang, C. V. ; Yang, D. C H ; Pollard, T. D. / Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum. In: Journal of Cell Biology. 1983 ; Vol. 96, No. 4. pp. 1138-1147.
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