Subcellular topology of rat liver methionyl-, leucyl-, and arginyl-tRNA synthetases.
We have investigated the distribution of methionyl-, leucyl-, and arginyl- tRNA synthetases in primary liver fractions obtained by differential centrifugation of homogenates in isotonic sucrose: 78-93% of synthetase activities are recovered in the cytosolic fraction. Microsomes contain only 4.8%, 19.4%, and 6.4% of the methionyl-, leucyl-, and arginyl-tRNA synthetases activities, respectively. This proportion increases up to 11.3%, 26.1%, and 20.7%, respectively, when the homogenization medium is supplemented with 5 mM Mg2+ and 25 mM K+. The presence of protease inhibitors in the homogenization medium does not increase the proportion of synthetases recovered in microsomes. After subfractionation of microsomes by isopycnic centrifugation, the distributions of the 3 synthetases display a second peak overlapping that of at a density of 1.12. In addition, methionyl- and leucyl- tRNA synthetases display a second peak overlapping that of RNA. This suggests that a small proportion of these synthetases (0.7% and 5.71% of total activities, respectively) bind to the d domain of the ER. The Golgi complex, the plasma membranes, and the peroxisomes lack aminoacyl-tRNA synthetase activity. The 3 synthetases are readily detached from membranes when intact microsomes are washed with 250 mM sucrose alone or containing 5 mM PPi, or 320 mM KCl. The binding of methionyl-tRNA synthetases to microsomes was measured in vitro, at 4 degrees C, with a sample of the cytosolic fraction as a source of synthetase. Microsomes stripped of their bound polysomes display a binding capacity that is not significantly different from that of unstripped microsomes. Even in the presence of cations, the amount of synthetase bound to the membranes remained low by comparison with the cytosolic content.
Amar-Costesec
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A
International Institute of Cellular and Molecular Pathology, Université de Louvain, Brussels, Belgium.
Turu
C
C
D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis*; D000818:Animals; D001126:Arginine-tRNA Ligase / Q000032:analysis*; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007935:Leucine-tRNA Ligase / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008718:Methionine-tRNA Ligase / Q000032:analysis*; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology
D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase; D001126:Arginine-tRNA Ligase; D007935:Leucine-tRNA Ligase