Post‐translational processing of the amino terminus affects actin function

Abstract

We have studied the importance of N‐terminal processing for normal actin function using the Drosophila Act88F actin gene transcribed and translated in vitro. Despite having different charges as determined by two‐dimensional (2D) gel electrophoresis, Act88F expressed in vivo and in vitro in rabbit reticulocyte lysate bind to DNase I with equal affinity and are able to copolymerise with bulk rabbit actin equally well. Using peptide mapping and thin‐layer electrophoresis we have shown that bestatin ([3‐amino‐2‐hydroxy‐4‐phenyl‐butanoyl]‐l‐leucine), an inhibitor of aminopeptidases, can inhibit actin N‐terminal processing in rabbit reticulocyte lysate. Although processed and unprocessed actins translated in vitro are able to bind to DNase I equally well, unprocessed actins are less able to copolymerise with bulk actins. This effect is more pronounced when bulk rabbit actin is used but is still seen with bulk Lethocerus actin. Also, the unprocessed actins reduce the polymerisation of the processed actin translated in vitro with the bulk rabbit actin. This suggests that individual actins do interact, even in non‐polymerising conditions. The reduced ability of unprocessed actin to polymerise shows that correct post‐translational modification of the N terminus is required for normal actin function.

Actin Residue Glu93 Is Identified as an Amino Acid Affecting Myosin Binding

“…The three main actin spots are labeled I-III in order of increasing basicity (41) and might be expected to correspond to the three charge groups above. When the Act88F gene is transcribed and translated in vitro under conditions allowing complete N-terminal processing, including acetylation, ACT88F runs, as predicted, at spot II (42). However, the most basic actin, labeled III, on two-dimensional gels of the whole fly actin extract is the mature ACT88F isoform (43), which we refer to as ACT88F-III.…”

Mammary Derived Growth Inhibitor Is Not a Distinct Protein but a Mix of Heart-type and Adipocyte-type Fatty Acid-binding Protein

“…38). Two additional variants were found (presumed N terminus of variant 1, Ac-Asp-Ala-; and of variant 2, Asp-Ala-), which were probably due to N-terminal processing as observed for class II actin (37,39). Formation of fully processed class II actin (AcAsp-) in the rabbit reticulocyte lysate could be decreased by inclusion of acetylation inhibitors (39) as also observed by us Here we clearly demonstrate for the first time that A-FABP is expressed in cells of the lactating bovine mammary gland as well as H-FABP.…”

Cell biology of embryonic migration

“…N-Terminal aminopeptidation and acetylation is involved in the early maturation of the actin monomers and is shown to be essential for normal actin function (Hennessey et al, 1991;Redman and Rubenstein, 1984;Rubenstein and Martin, 1983). An emerging role in cell motility belongs to post-translational arginylation of actin and actin-associated proteins (Wong et al, 2007).…”