Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway

doi:10.1016/j.molcel.2013.03.018

. 2013 May 23;50(4):540-51.

doi: 10.1016/j.molcel.2013.03.018. Epub 2013 Apr 18.

Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway

Anna Shemorry¹, Cheol-Sang Hwang, Alexander Varshavsky

Affiliations

PMID: 23603116
PMCID: PMC3665649
DOI: 10.1016/j.molcel.2013.03.018

Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway

Anna Shemorry et al. Mol Cell. 2013.

. 2013 May 23;50(4):540-51.

doi: 10.1016/j.molcel.2013.03.018. Epub 2013 Apr 18.

Authors

Anna Shemorry¹, Cheol-Sang Hwang, Alexander Varshavsky

Affiliation

¹ Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.

PMID: 23603116
PMCID: PMC3665649
DOI: 10.1016/j.molcel.2013.03.018

Abstract

N(α)-terminal acetylation of cellular proteins was recently discovered to create specific degradation signals termed Ac/N-degrons and targeted by the Ac/N-end rule pathway. We show that Hcn1, a subunit of the APC/C ubiquitin ligase, contains an Ac/N-degron that is repressed by Cut9, another APC/C subunit and the ligand of Hcn1. Cog1, a subunit of the Golgi-associated COG complex, is also shown to contain an Ac/N-degron. Cog2 and Cog3, direct ligands of Cog1, can repress this degron. The subunit decoy technique was used to show that the long-lived endogenous Cog1 is destabilized and destroyed via its activated (unshielded) Ac/N-degron if the total level of Cog1 increased in a cell. Hcn1 and Cog1 are the first examples of protein regulation through the physiologically relevant transitions that shield and unshield natural Ac/N-degrons. This mechanistically straightforward circuit can employ the demonstrated conditionality of Ac/N-degrons to regulate subunit stoichiometries and other aspects of protein quality control.

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Figures

**Figure 1. The Ac/N-degron of Cog1**
(A) Cycloheximide (CHX)-chases were performed at 30°C with wt or *doa10Δ S. cerevisiae* expressing either wt Cog1, termed MD-Cog1^wt, or its MK-Cog1 derivative in which Asp2 was replaced with Lys2. Both proteins were C-terminally ha-tagged. At the indicated times of chase, proteins in cell extracts were fractionated by SDS-PAGE and assayed by immunoblotting with anti-ha antibody. (B) ³⁵S-pulse-chase with MD-Cog1^wt or P-Cog1 in wt, *doa10Δ*, or *naa10Δ* (*ard1Δ*) *S. cerevisiae*, the latter strain lacking the catalytic subunit of the non-cognate (for MD-Cog1^wt) NatA Nt-acetylase (Figure S2). Cog1 proteins were C-terminally tagged with 3 flag epitopes modified to contain a Met residue in each epitope, to increase ³⁵S-Met in Cog1. (C) Same as in B but another ³⁵S-pulse-chase. It included *naa20Δ* (*nat3Δ*) *S. cerevisiae* lacking the catalytic subunit of the cognate (for MD-Cog1^wt) NatB Nt-acetylase (Figure S2). (D) Quantification of data in C.◆, MD-Cog1^wt in wt cells. ▲, MD-Cog1^wt in *naa20Δ* cells. ■, P-Cog1 in wt cells. (E) Anti-Cog1^AcNt antibody specific for Nt-acetylated MD-Cog1^wt (see Figure S4A–C) was used for immunoblotting in CHX-chase assays with MD-Cog1^wt and P-Cog1 (C-terminally tagged with 3 flag epitopes) in either wt or *naa20Δ* (*nat3Δ*) *S. cerevisiae*. (F) Same as in E, except that membrane was reprobed with anti-flag antibody. (G) Quantification of anti-Cog1^AcNt-specific immunoblotting patterns in E using a linear scale, with the level of MD-Cog1^wt at time zero in wt cells taken as 100%. ◆, MD-Cog1^wt in wt cells. ▲, MD-Cog1^wt in *naa20Δ* cells. ■, P-Cog1 in wt cells. (H) Same as in G but a semi-log plot of the flag-specific Cog1 immunoblotting patterns in F. Same designations as in G. See also Figures S1, S2, S5, and Tables S1 and S2.

**Figure 2. Stabilization of Cog1 in *S. cerevisiae* Lacking the Not4 E3 Ubiquitin Ligase**
(A) CHX-chases with yeast expressing MD-Cog1^wt C-terminally tagged with three flag epitopes. Lane M and red stars, M_r markers of 37, 50, and 100 kDa, respectively. MD-Cog1^wt in wt yeast (lanes 1–3), and in *not4Δ* (lanes 4–6), *not4Δ ubr1Δ* (lanes 7–9), and *not4Δ doa10Δ* mutants (lanes 10–12). (B) Quantification of immunoblots in H, with the level of MD-Cog1^wt in *not4Δ* cells at the beginning of chase taken as 100%. ■, MD-Cog1^wt C in *not4Δ* cells (black curve). ◆, MD-Cog1^wt in wt cells (red curve).▲, MD-Cog1^wt in *not4Δ ubr1Δ* cells (green curve). X,MD-Cog1^wt in *not4Δ doa10Δ* cells (blue curve). (C) Same as in A but an independent CHX-chase, and immunoblotting with anti-Cog1^AcNt antibody specific for Nt-acetylated MD-Cog1^wt. Lane M and red star, an M_r marker of 50 kDa. MD-Cog1^wt in wt yeast (lanes 1–3), and in *naat20Δ* (*nat3Δ*) (lanes 4–6), and *not4Δ* mutants lanes 7–9). See also Figure S5.

**Figure 3. Stabilization of Overexpressed, Short-Lived Cog1 by Coexpressed Cog2-Cog4**
(A) CHX-chases with endogenous $MD - Cog 1_{3 ha}^{wt}$ (C-terminally tagged with 3 ha epitopes) expressed from the chromosomal *COG1* locus and the native P_COG1 promoter in wt, *doa10Δ*, and *naa20Δ* (*nat3Δ*) cells. (B) Same as in A but an independent CHX-chase. *S. cerevisiae* (in 2% glucose) expressing endogenous $MD - Cog 1_{3 ha}^{wt}$ and carrying a plasmid that could express the $MD - Cog 1_{3 f}^{wt}$ decoy but only in the presence of galactose. Lanes 4–6, same as lanes 1–3 but in *naa20Δ* cells. (C) Stabilization of overexpressed MD-Cog1^wt (C-terminally tagged with 3 flag epitopes) by coexpressed Cog2 and Cog3. Lane M and red stars, M_r markers of 37, 50, and 100 kDa, respectively. Lanes 1–3, wt *S. cerevisiae* in 2% glucose, expressing MD-Cog1^wt from the P_CUP1 promoter on a low copy plasmid and carrying a high copy plasmid that expressed, only in galactose, both Cog2 and Cog3 (C-terminally tagged with ha) from the bidirectional P_GAL1/10 promoter. Lanes 4–6, same as lanes 1–3 but with cells in 2% galactose. Asterisk on the right indicates a protein crossreacting with anti-ha. (D) Same as in C but cells also carried a second high copy plasmid expressing Cog4 (C-terminally tagged with ha) from the P_GAL1/10 promoter. (E) Quantification of data in C for MD-Cog1^wt. ◆, MD-Cog1^wt in cells that did not coexpress other COG subunits. ■, MD-Cog1^wt in cells that coexpressed (in galactose) Cog2 and Cog3. (F) Quantification of data in D for MD-Cog1^wt. ◆, MD-Cog1^wt in cells that did not coexpress other COG subunits. ■, MD-Cog1^wt in cells that coexpressed (in galactose) Cog2-Cog4. See also Figure S1.

**Figure 4. Subunit Decoy Technique and the Cause of Stability of Endogenous Cog1**
(A) The subunit decoy technique. “CogX” denotes a Cog1-interacting COG subunit (Cog2 or Cog3) that can shield the Ac/N-degron of Cog1. “Normally expressed” refers to levels of expression from endogenous promoters and chromosomal loci. The normally expressed Cog1-tag1 bears a C-terminal tag denoted as “tag1”, whereas the otherwise identical but overexpressed Cog1-tag2 decoy bears a different C-terminal tag (“tag2”). In the absence of decoy, the bulk of (normally expressed) Cog1-tag1 would occur as a CogX-Cog1-tag1 complex in which the Ac/N-degron of Cog1 is largely sequestered. By contrast, in the presence of overexpressed Cog1-tag2 decoy, the bulk of both Cog1-tag1 and Cog1-tag2 would not be in the complex with CogX (i.e., their Ac/N-degron would be active), given relatively low levels of a (normally expressed) CogX “shielding” protein. (B) Lane M and red stars, M_r markers of 37, 50, and 100 kDa, respectively. Lanes 1–4, stability of endogenous $MD - Cog 1_{13 myc}^{wt}$ (C-terminally tagged with 13 myc epitopes) in the absence of the $MD - Cog 1_{3 f}^{wt}$ decoy (C-terminally tagged with 3 flag epitopes). CHX-chase with $MD - Cog 1_{13 myc}^{wt}$ expressed from the chromosomal *COG1* locus and the native P_COG1 promoter in wt cells in the presence of vector alone. Lanes 5–8, same as lanes 1–4 but cells carried a plasmid that expressed the $MD - Cog 1_{3 f}^{wt}$ decoy from the P_ADH1 promoter. An asterisk denotes a protein crossreacting with anti-flag. (C) Quantification of data in B. ◆, endogenous $MD - Cog 1_{13 myc}^{wt}$ in wt cells that did not express the $MD - Cog 1_{3 f}^{wt}$ decoy. ■, endogenous $MD - Cog 1_{13 myc}^{wt}$ in wt cells that expressed $MD - Cog 1_{3 f}^{wt}$ . (D) Same as in C but with wt and *naa20Δ* cells expressing the endogenous $MD - Cog 1_{3 ha}^{wt}$ in 2% galactose, i.e., in the presence of the coexpressed $MD - Cog 1_{3 f}^{wt}$ decoy. Immunoblotting with anti-ha, specific for $MD - Cog 1_{3 ha}^{wt}$ . (E) Same as in D but also probed (in a parallel immunoblot) with anti-flag, specific for $MD - Cog 1_{3 f}^{wt}$ . (F) Quantification of data in D, E. ◆, endogenous $MD - Cog 1_{3 ha}^{wt}$ in wt cells grown in 2% glucose. ■, endogenous $MD - Cog 1_{3 ha}^{wt}$ in wt cells grown in 2% galactose, i.e., in the presence of the $MD - Cog 1_{3 f}^{wt}$ decoy. ▲, $MD - Cog 1_{3 f}^{wt}$ decoy. See also Figures S4 and S5.

**Figure 5. Hcn1 and Repression of Its Ac/N-degron by Cut9**
(A) CHX-chases in wt or *naa30Δ* (*mak3Δ*) *S. cerevisiae* expressing the wt *S. pombe* Hcn1, termed ML-Hcn1^wt, C-terminally tagged with 3 flag epitopes. *naa30Δ* cells lacked the catalytic subunit of the cognate NatC Nt-acetylase (Figure S2). Lane 1 and red stars, M_r markers of 10, 15, 20, 37, and 50 kDa, respectively. (B) ³⁵S-pulse-chase with ML-Hcn1^wt in wt and *naa30Δ* (*mak3Δ*) *S. cerevisiae*. Lane 7, vector alone. (C) Quantification of data in B. ◆, ML-Hcn1^wt in wt cells. ■, ML-Hcn1^wt in *naa30Δ* cells. (D) Lanes 1–4, CHX-chase with wt cells in 2% galactose (and without methionine) that expressed ML-Hcn1^wt from the methionine-repressible P_MET25 promoter on a low copy plasmid and carried a vector alone (no Cut9 expression). Note the metabolic instability of ML-Hcn1^wt (lower panel). Lanes 5–8, same as lanes 1–4 but with a low copy plasmid (instead of control vector) expressing Cut9 from the galactose-inducible P_GAL1 promoter, with both ML-Hcn1^wt and Cut9 C-terminally tagged with 3 flag epitopes. Note the metabolic stabilization of ML-Hcn1^wt(lower panel), including a strong increase of its level at the beginning of the chase. (E) Quantification of data in D. ◆, ML-Hcn1^wt in the absence of co-expressed Cut9. ■, ML-Hcn1^wt in the presence of co-expressed Cut9. See also Figure S1.

**Figure 6. Conditionality of Ac/N-degrons**
This diagram summarizes the functional understanding of the dynamics of Nt-acetylated proteins vis-à-vis the Ac/N-end rule pathway attained in the present study, in conjunction with results that initially revealed the Ac/N-end rule pathway (Hwang et al., 2010b). See also Figure S1.

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References

1. Arnesen T, Van Damme P, Polevoda B, Helsens K, Evjenth R, Colaert N, Varhaug JE, Vandekerckhove J, Lillehaug JR, Sherman F, et al. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast to humans. Proc Natl Acad Sci USA. 2009;106:8157–8162. - PMC - PubMed
1. Barford D. Structure, function and mechanism of the anaphase promoting complex (APC/C) Q Rev Biophys. 2011;44:153–190. - PubMed
1. Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li GW, Zhou S, King D, Shen PS, Weibezahn J, et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell. 2012;151:1042–1054. - PMC - PubMed
1. Chen J, Archer T. Regulting SWI/SNF subunit levels via protein-protein interactions and proteasomal degradation: BAF155 and BAF170 limit expression of BAF57. Mol Cell Biol. 2005;25:9016–9027. - PMC - PubMed
1. Collart M, Panasenko O, Nikolaev S. The Not3/5 subunit of the Ccr4-Not complex: A central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells. Cell Signal. 2012;25:743–751. - PubMed

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[1] Arnesen T, Van Damme P, Polevoda B, Helsens K, Evjenth R, Colaert N, Varhaug JE, Vandekerckhove J, Lillehaug JR, Sherman F, et al. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast to humans. Proc Natl Acad Sci USA. 2009;106:8157–8162. - PMC - PubMed

[2] Arnesen T, Van Damme P, Polevoda B, Helsens K, Evjenth R, Colaert N, Varhaug JE, Vandekerckhove J, Lillehaug JR, Sherman F, et al. Proteomics analyses reveal the evolutionary conservation and divergence of N-terminal acetyltransferases from yeast to humans. Proc Natl Acad Sci USA. 2009;106:8157–8162. - PMC - PubMed

[3] Barford D. Structure, function and mechanism of the anaphase promoting complex (APC/C) Q Rev Biophys. 2011;44:153–190. - PubMed

[4] Barford D. Structure, function and mechanism of the anaphase promoting complex (APC/C) Q Rev Biophys. 2011;44:153–190. - PubMed

[5] Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li GW, Zhou S, King D, Shen PS, Weibezahn J, et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell. 2012;151:1042–1054. - PMC - PubMed

[6] Brandman O, Stewart-Ornstein J, Wong D, Larson A, Williams CC, Li GW, Zhou S, King D, Shen PS, Weibezahn J, et al. A ribosome-bound quality control complex triggers degradation of nascent peptides and signals translation stress. Cell. 2012;151:1042–1054. - PMC - PubMed

[7] Chen J, Archer T. Regulting SWI/SNF subunit levels via protein-protein interactions and proteasomal degradation: BAF155 and BAF170 limit expression of BAF57. Mol Cell Biol. 2005;25:9016–9027. - PMC - PubMed

[8] Chen J, Archer T. Regulting SWI/SNF subunit levels via protein-protein interactions and proteasomal degradation: BAF155 and BAF170 limit expression of BAF57. Mol Cell Biol. 2005;25:9016–9027. - PMC - PubMed

[9] Collart M, Panasenko O, Nikolaev S. The Not3/5 subunit of the Ccr4-Not complex: A central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells. Cell Signal. 2012;25:743–751. - PubMed

[10] Collart M, Panasenko O, Nikolaev S. The Not3/5 subunit of the Ccr4-Not complex: A central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells. Cell Signal. 2012;25:743–751. - PubMed

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Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway

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Control of protein quality and stoichiometries by N-terminal acetylation and the N-end rule pathway

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