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

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 detergentinsoluble 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, sucrose, and CaCI2 (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 detergentinsoluble 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.

The Phosphoinositide-dependent Kinase, PDK-1, Phosphorylates Conventional Protein Kinase C Isozymes by a Mechanism That Is Independent of Phosphoinositide 3-Kinase

“…One possibility is the endoplasmic reticulum. However, endoplasmic reticulum-associated proteins retain significant detergent insolubility (27), suggesting that this is not the membrane fraction where protein kinase C becomes processed by phosphorylation. Whether the membrane fraction represents the plasma membrane remains to be elucidated.…”

Interaction between mRNA, ribosomes and the cytoskeleton

“…1). In addition, immunohistochemical studies using antibodies to ER components have shown that membrane material is retained in the cell matrix (Dang et al, 1983;Mirande et al, 1985) and the presence of ER residues has been confirmed by electron microscopy (Dang et al, 1983;Ramaekers et al, 1983). The presence of labelled phospholipids in the deoxycholate-solubilized cell matrix (Lenk et al, 1977;Vedeler et al, 1991) again demonstrates the presence of membrane material in the so-called 'cytoskeletal fraction'.…”

Aminoacyl-tRNA synthetase complexes: beyond translation

“…In particular, association of translational components such as tRNA, ARSs and elongation factors with the cytoskeletal framework (Dang et al, 1983;Mirande et al, 1985a;Sanders et al, 1996) and colocalization of these components have been described (Barbarese et al, 1995). ARSs can be classified into two groups based on their structural features (Eriani et al, 1990;Burbaum and Schimmel, 1991;Cusack et al, 1991).…”