N-terminal acetylation of cellular proteins creates specific degradation signals

Abstract

The retained N-terminal methionine (Met) residue of a nascent protein is often N-terminally acetylated (Nt-acetylated). Removal of N-terminal Met by Met-aminopeptidases frequently leads to Nt-acetylation of the resulting N-terminal alanine (Ala), valine (Val), serine (Ser), threonine (Thr), and cysteine (Cys) residues. Although a majority of eukaryotic proteins (for example, more than 80% of human proteins) are cotranslationally Nt-acetylated, the function of this extensively studied modification is largely unknown. Using the yeast Saccharomyces cerevisiae, we found that the Nt-acetylated Met residue could act as a degradation signal (degron), targeted by the Doa10 ubiquitin ligase. Moreover, Doa10 also recognized the Nt-acetylated Ala, Val, Ser, Thr, and Cys residues. Several examined proteins of diverse functions contained these N-terminal degrons, termed AcN-degrons, which are a prevalent class of degradation signals in cellular proteins.

Fig. 1

Destabilizing N-terminal residues. (A) CHX chases, for 0, 1, and 2 hours in wild-typeS. cerevisiaeexpressing CK-e^K-Ura3 (lanes 1 to 3) or CL-e^K-Ura3 (lanes 4 to 6). Cell extracts were fractionated by SDS–polyacrylamide gel electrophoresis, followed by immunoblotting with anti-Ha and anti-tubulin, the latter a loading control. (B) As in (A) but chases for 0 and 2 hours with XZ-e^K-Ura3 (X = Cys, Met, or Ser; Z = Trp, Val, or Leu) in wild-type versus ubr1Δ cells. (C) As in (A) but with MZ-e^K-Ura3 (Z = Leu or Lys) in wild-type cells. (D) As in (A) but chases for 0, 0.5, and 1.5 hours with XL-e^K-Ura3 (X = Gly, Val, Ala, or Thr) in wild-type cells. (E) As in (A) but with CL-e^K-Ura3 in wild-type cells (lanes 1 to 3) versus doa10Δ cells (lanes 4 to 6). (F) As in (E) but chases for 0, 0.5, 1, and 2 hours with ML-e^K-Ura3. (G) Lanes 1 and 2, short-lived ML-e^K-Ura in the MG132-sensitive pdr5Δ S. cerevisiae, in the absence and presence of MG132, respectively. Lanes 3 and 4, same as lanes 1 and 2 but with long-lived MK-e^K-Ura3.

Fig. 2

Doa10 as an N-recognin. (A) Extracts from wild-type, doa10Δ, and nat3Δ S. cerevisiae that expressed XZ-α2^3-67-e^K-Ura3 (XZα2) (X = Met, Arg, or Gly; Z = Asn or Lys) were immunoblotted with anti-^AcNtMATα2 (which selectively recognized Nt-acetylated MNα2) or (separately) with anti-Ha, which recognized both Nt-acetylated and unacetylated MNα2, or with anti-tubulin. XZα2 (“α2”), Nt-acetylated XZα2 (“Ac-α2”), and tubulin are indicated. Asterisks denote a protein cross-reacting with anti-^AcNtMATα2. (B) As in (A) but CHX chases for 0, 0.5, 1, and 2 hours with MNα2, in wild-type, doa10Δ, and nat3Δ cells. (C) Indicated amounts of the Ntacetylated Ac-MNKIPIKDLLNC peptide versus its unacetylated counterpart were spotted onto membrane and assayed for their binding to anti-^AcNtMATα2. (D) X-peptide pulldown with peptides XNKIPIKDLLNC (X = Met, ^AcMet, or Gly) (lanes 2 to 4) or XIFSTDTGPGGC (X = Gly, Met, Arg, or Phe) (lanes 5 to 8) and extract of S. cerevisiae that expressed Doa10_myc13. Lane 1, input extract (5%). (E) SPOT assay with purified, flag-tagged Doa10_f and spot-arrayed synthetic peptides XZ-e^K(3-11) (X = Gly, Ala, Val, Pro, Ser, Thr, or Cys; Z = Leu or Lys) and their Nt-acetylated XZ-e^K(3-11)counterparts. XZ residues are indicated at the top of the membrane. (F) Quantitation, using a PhosphorImager, of ³⁵S-pulse chases with MATα2_f and its mutant derivatives (fig. S6, B and C). Solid circles, ^MNMATα2_f; open circles,^MKMATα2_f; upright triangles, ^GNMATα2_f (initially ^MGNMATα2_f) in ubc4Δ cells; inverted triangles, ^MNMATα2_f in ubc4Δ doa10Δ cells.

Fig. 3

^AcN-degrons in yeast proteins. (A) Lanes 1 to 3, CHX chase for 0, 1, and 2 hours in wild-type S. cerevisiaeexpressing Tbf1_ha. Lanes 4 to 6, 7 to 9, and 10 to 12, analogous patterns but in doa10Δ cells with^MKTbf1_ha (Lys at position 2), ^GTbf1_ha(initially ^MGTbf1_ha), and wild-type Tbf1_ha, respectively. (B) As in (A) but with wild-type Slk19_ha and its mutant derivatives in wild-type versus doa10Δ cells. (C) As in (A) but with wild-type Ymr090w_ha and its mutant derivatives in wild-type versus doa10Δ cells. (D) As in (C) but with wild-type Ymr090w_ha in wild-type versus doa10Δ and ard1Δ cells. (E) As in (A) but with wild-type His3_ha in wild-type versus doa10Δ cells. (F) As in (E), with wild-type His3_ha in wild-type versus doa10Δ and ard1Δ cells. (G) As in (A) but CHX chases for 0, 0.5, 1, and 2 hours with wild-type Aro8_hain wild-type versus doa10Δ and ard1Δ cells. (H) As in (A) but with wild-type Hsp104_ha and its mutant derivatives in wild-type versus doa10Δ cells.

Fig. 4

N^α-terminal acetylases, Met-aminopeptidases, and the Doa10 branch of the N-end–rule pathway. (A) The Doa10-mediated branch of the S. cerevisiaeN-end–rule pathway (see fig. S1C for the Ubr1-mediated branch of this pathway). The red arrow on the left indicates the MetAP-mediated removal of N-terminal Met. This Met is retained if a residue at position 2 is nonpermissive (too large) for MetAPs. If the retained N-terminal Met or N-terminal Ala, Val, Ser, Thr, and Cys are followed by acetylation-permissive residues, the above N-terminal residues are usually Ntacetylated (–7). The resulting N-degrons are termed ^AcN-degrons. The term “secondary” refers to the necessity of modification (Nt-acetylation) of a destabilizing N-terminal residue before a protein can be recognized by a cognate Ub ligase (fig. S1C). Proteins containing ^AcN-degrons are targeted for ubiquitylation (and proteasome-mediated degradation) by the Doa10 E3 Ub ligase. Although Gly or Pro can be made N-terminal by MetAPs, and although Doa10 can recognize Ntacetylated Gly and Pro (Fig. 2E), few proteins with N-terminal Gly or Pro are Nt-acetylated (– 7). (B) The Ubr1 and Doa10 branches of the N-end–rule pathway. Both branches target, through different mechanisms, the N-terminal Cys residue (yellow rectangles), with oxidized Cys marked by an asterisk.