Canavanine inhibits vimentin assembly but not its synthesis in chicken embryo erythroid cells.
In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine-vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo.
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002172:Canavanine / Q000494:pharmacology*; D002642:Chick Embryo; D003599:Cytoskeleton / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D007381:Intermediate Filament Proteins / Q000097:blood*; D007700:Kinetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D014746:Vimentin
D007381:Intermediate Filament Proteins; D014746:Vimentin; D002172:Canavanine; D001120:Arginine