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NH2-terminal acetylation of Dictyostelium discoideum actin in a cell-free protein-synthesizing system
Journal article   Open access   Peer reviewed

NH2-terminal acetylation of Dictyostelium discoideum actin in a cell-free protein-synthesizing system

Peter Rubenstein, Patricia Smith, Jon Deuchler and Kent Redman
The Journal of biological chemistry, Vol.256(15), pp.8149-8155
08/10/1981
DOI: 10.1016/S0021-9258(18)43400-X
PMID: 6894920
url
https://doi.org/10.1016/S0021-9258(18)43400-XView
Published (Version of record) Open Access

Abstract

We present here a new method for inhibiting protein acetylation in a rabbit reticulocyte cell-free protein-synthesizing system. This procedure utilizes S-acetonyl coenzyme A, a nonreactive acetyl-CoA analogue, as an inhibitor of the NH2-terminal protein acetyltransferase in this lysate. With this procedure, we can make, in vitro, Dictyostelium discoideum actin which is 85% nonacetylated but fully translated. With the fully translated but nonacetylated actin as a substrate, the actin can be almost completely acetylated post-translationally in an acetyl-CoA-dependent system after the actin has left the ribosome. Using formylated and nonformylated [35S]Met-tRNAfMet as a source of label and in conjunction with detailed peptide mapping experiments with trypsin and thermolysin, the in vitro acetylation is shown to occur at the NH2 terminus of the newly synthesized actin. Furthermore, the initiator methionine residue, contrary to expectation, is not cleaved off but remains stable for at lest 50 min. thus, in the acetylating reticulocyte lysate system, the primary complete translation product in actin synthesis is Ac-Met-Asp and not Ac-Asp.
 Acetylation Acetyltransferases - metabolism Actins - metabolism Amino Acid Sequence Animals Dictyostelium - metabolism Kinetics Peptide Fragments - analysis Protein Biosynthesis Rabbits Reticulocytes - metabolism Thermolysin

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