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Nature of Actin Amino-Terminal Acetylation COMMENTARY

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Nature of Actin Amino-Terminal Acetylation COMMENTARY COMMENTARY NATure of actin amino-terminal acetylation COMMENTARY Peter A. Rubensteina,1 and Kuo-kuang Wena Actins constitute a highly structurally conserved family discovery of this processing pathway, however, neither of proteins found in virtually all eukaryotic cells, in the identity of the enzyme that carries out the final which they participate in processes such as production acetylation step of the pathway nor the functional sig- of contractile force, structural stabilization of the cell, nificance of this processing has been established. In cell motility, endocytosis, and exocytosis (1). The actin PNAS, two papers from the same group [Drazic et al. monomer, or G-actin, has a nucleotide-binding cleft (7) and Goris et al. (8)] make significant contributions separating two large domains. Each of these is sepa- toward solving these two questions. rated into two subdomains, with the N terminus A number of protein N-acetyltransferases had pre- appearing as an arm which originates from subdomain viously been characterized, but no enzyme had been 1 (Fig. 1). In the context of the actin filament (F-actin), a found that would acetylate the N terminus of actin two-stranded helix, subdomain 1 is located on the fila- following removal of its initiator methionine residue. ment exterior. This position allows the N-terminal re- Here, Drazic et al. (7) show that a previously poorly gion to be a site of interaction for myosin and a number described enzyme called NAA80/NATH or NAT6/ of other actin-binding regulatory proteins (2, 3). Actins Fus2 (9) had the requisite activity for such a task. Based are characterized by an acidic N-terminal region con- on a series of in vitro studies, they first demonstrated sisting of two to four acidic amino acids in which the N- that the enzyme had its highest activity with a peptide terminal amino acid is N-acetylated. However, in ma- beginning ME but also showed high activity on pep- ture actins, the initiator methionine is missing. In the tides beginning DDDI and EEEI, the N termini of beta early 1980s, a series of papers reported the discovery and gamma cytoplasmic actins. Subsequently, they of a unique processing pathway leading to the produc- demonstrated that in NAA80 knockout tissue culture tion of the pure actin (4–6). For class I actins, in which cells only the acetylation of the two actins was essen- the initiator methionine directly precedes the eventual tially affected, and reintroduction of NAA80 into the N-terminal acid residue, the methionine is acetylated. cell restored actin acetylation. Then the acetyl-methionine is removed proteolytically, Goris et al. (8) extend these studies to the kinetic exposing the N-terminal acidic amino acid, which is and structural levels. Initially the enzyme was thought then acetylated to produce the mature form of the pro- to work via a ping-pong mechanism (10). Goris et al. tein. Examples of these actins are the beta and gamma (8) demonstrate that the enzyme forms a ternary com- cytoplasmic actins found in mammalian cells. For class plex containing actin and the acetyl-CoA after which II actins, which include the striated and smooth muscle acetylation occurs. Recognition of this mechanism led actins in mammalian cells, the N-terminal processing is to the isolation of an inhibitor which approximated the more complex. These proteins are produced from ternary complex when bound to the enzyme. The au- genes which encode a Met-Cys-acidic residue N termi- thors cocrystallized the inhibitor with the Drosophila nus. For these actins, following removal of the initiator form of NAT6, with the same specificity as the human acetyl-methionine, the new N-terminal cysteine is N- enzyme, and determined the structure of the complex acetylated. The acetyl-cysteine is then removed pro- via X-ray crystallography. They demonstrated that the teolytically to expose the eventual N-terminal acidic specificity of the enzyme resided in a more open ac- residue, which is then acetylated to produce the mature tive site cleft and a much more cationic surface in this actin N terminus. The conservation of this unique pro- cleft to accommodate the N-terminal negatively cessing pathway coupled with the demonstrated in- charged residues than in other N-acetyltransferases. volvement of the N terminus in actin filament function The difference in specificity of the enzyme in vitro suggests that proper N-terminal processing of actin and in vivo was surprising. In vitro, the highest activity may have significant functional interactions. Since the was in acetylating an N-terminal Met when the next aDepartment of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242 Author contributions: P.A.R. and K.-k.W. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion articles 10.1073/pnas.1718336115 and 10.1073/pnas.1719251115. 1To whom correspondence should be addressed. Email: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1803804115 PNAS Latest Articles | 1of3 Downloaded by guest on September 29, 2021 Pointed ends A B M6 Subdomain 4 Subdomain 2 M5 M4 M3 Subdomain 3 Subdomain 1 M2 M1 Barbed ends Fig. 1. The actin monomer (A, Protein Data Bank ID code 2BTF) and the filament (B, ref. 15). The N-terminal peptide (red) and the ADP bound in the nucleotide cleft (cyan) are shown. The four subdomains are denoted on the monomer, and M denotes the individual monomers in the filament. amino acid was acidic. However, elimination of NAA80 showed nucleation or Arp2/3 and formin-dependent nucleation. However, the accumulation of actin in which the Met had been removed and it caused a twofold slower filament elongation rate from pre- the N-terminal amino group was exposed. Subsequent work formed actin seeds compared with acetylated actin; and with showed that in the cell NATB was responsible for the initial Met mDia1, but not mDia2, formin the unacetylated actin polymerized acetylation, and NAA80 then carried out the final acetylation fol- only about 40% as fast as did the acetylated actin. Of the two lowing removal of the acetyl-Met. This result underscores the im- formins, only the mDia 1 shows a strong elongation activity as portance of establishing the activity of a protein in vivo, where the well as nucleation. The effect of altered acetylation on filament biochemical context may be very different from what occurs elongation is unexpected, because the N terminus is far removed in vitro, to gain insight into its true function. The studies carried from the monomer–monomer interaction sites that result in fila- out by this group did not specifically demonstrate that this en- ment formation. Thus, the acetylation effects can only occur through zyme carried out the ultimate acetylation of the class II actin acidic propagated conformational changes from the amino terminus to amino-terminal residue. However, the demonstrated specificity of the interfaces that are important in longitudinal strand and in the enzyme would make it the likely candidate for class II process- cross-strand contacts within the actin filament. In the latter case, ing. Furthermore, Drosophila melanogaster actins are class II ac- the possibility of the N terminus’ affecting strand–strand interactions tins, and the NAA80 used for the crystallization studies was the in the middle of the actin filament has been demonstrated (11). Drosophila homolog. This enzyme was also shown to have the The identification of actin N-terminal acetylating enzyme, its same substrate specificity as the human form, further supporting structure determination, the ability to eliminate it from cells, and this hypothesis. the demonstration that its absence affects actin function at the cell Discovery of a posttranslational protein modification and the and molecular level opens the door to many avenues of in- manner in which it is carried out is often much easier to establish vestigation that should provide new insight into the regulation of than the functional significance of that modification. Drazic et al. actin function. Elimination of the N-acetyl group from actin would (7) made a series of observations that shed light on the functional introduce a positive charge, thereby decreasing net negative significance of N-terminal actin acetylation. They showed NAA80 charge density of the amino-terminal fragment. Earlier studies distributed diffusely throughout the cytoplasm instead of associ- based on genetically altered yeast actin demonstrated that the ating with ribosomes as do most NATs. They demonstrated that degree of negative charge density affects actin-activated myosin NAA80 knockout cells displayed faster motility and faster gap ATPase activity (2). Decreased negative charge density also in- closure in a wound-healing assay. They also showed the acetyla- creases the propensity of the actin to form spontaneous bundles. tion was important for controlling cell morphology. Elimination of Changes in myosin activation could easily be tied to alterations in the enzyme resulted in a decreased G/F actin ratio and an in- cell motility associated with NAA80 elimination, while increased crease in filopodia- and lamellipodia-containing cells, all consis- bundling could very well lead to the decreased G/F actin ratio and tent with the increased cell motility they observed. To gain insight increased filament stability that would facilitate lamellipodia and into these differences in cell behavior, the authors purified actin filopodia formation as observed in the knockout cells. The ability from control and knockout cells and compared their polymeriza- to isolate active nonacetylated actin allows testing of these hy- tion properties. Absence of acetylation did not affect overall actin potheses specifically in the context of actin posttranslational pro- polymerization rates. It also did not affect spontaneous filament cessing, which was not possible earlier. Further investigation of 2of3 | www.pnas.org/cgi/doi/10.1073/pnas.1803804115 Rubenstein and Wen Downloaded by guest on September 29, 2021 these effects could provide significant new insight into allosteric The actin amino-terminal fragment has been a source of regulation of actin function.
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