Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin–actin interactions in budding yeast

Abstract

The evolutionarily conserved Mdm20 protein (Mdm20p) plays an important role in tropomyosin-F-actin interactions that generate actin filaments and cables in budding yeast. However, Mdm20p is not a structural component of actin filaments or cables, and its exact function in cable stability has remained a mystery. Here, we show that cells lacking Mdm20p fail to N-terminally acetylate Tpm1p, an abundant form of tropomyosin that binds and stabilizes actin filaments and cables. The F-actin-binding activity of unacetylated Tpm1p is reduced severely relative to the acetylated form. These results are complemented by the recent report that Mdm20p copurifies with one of three acetyltransferases in yeast, the NatB complex. We present genetic evidence that Mdm20p functions cooperatively with Nat3p, the catalytic subunit of the NatB acetyltransferase. These combined results strongly suggest that Mdm20p-dependent, N-terminal acetylation of Tpm1p by the NatB complex is required for Tpm1p association with, and stabilization of, actin filaments and cables.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

“…Previous studies have observed that an increment in tropomyosin expression partially restores functional defects in actin stress fibers (31,37). In contrast, Tpm1 overexpression in wildtype HeLa cells significantly reduced the area and number of focal adhesions, in agreement with other expressed tropomyosin isoforms (38) and suggestive of a compromised Tpm1 function, most probably by its reduced Nt-acetylation.…”

“…Interestingly, NatB mediated Ntacetylation of Nt-unspliced tropomyosin and iMet-retaining forms of actin is conserved in yeast and mammals. NatB-deficient yeast strain exhibit defects in actin cable formation similar to observed in the strains with actin and tropomyosin mutations (10,31). These findings suggest that such defects are caused by the lack of acetylated actin and tropomyosins.…”

“…Destabilization of yeast actin cables caused by the absence of Nt-acetylation of tropomyosin and actin was observed in the yeast naa20-Δ strain (10,31). To determine whether hNatB activity is required in human cells for the maintenance of the integrity of actin microfibrils, we inhibited the expression of either hNAA20 or hNAA25 in HeLa cells and examined the F-actin network by labeling it with fluorescent phalloidin.…”

“…It was previously shown that NatB-mediated Nt-acetylation of Tpm1 is important for the formation of stable yeast F-actin structures (10,31). In the case of Tpm1, Nt-acetylation can be mimicked by the expression of an amino-terminally extended form of Tpm1 in vitro (37).…”

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…Mutations in these complexes lead to a number of pleiotropic phenotypes, but in recent years, a growing number of these defects have been specifically linked to single protein substrates. For example, telomeric silencing, membrane trafficking, L-A viral assembly, and tropomyosin-actin interactions are mediated by the specific N-terminal acetylation of Orc1, Arl3, gag, and Tpm1, respectively (Tercero and Wickner, 1992;Singer and Shaw, 2003;Behnia et al, 2004;Geissenhoner et al, 2004;Setty et al, 2004). In many of these cases, the loss of acetylation reduces the affinity of these substrates for a partner protein, providing a detailed mechanistic explanation for the observed phenotype.…”