The N-terminal rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) targeted by the N-end rule pathway include a set called N-degrons. The N-end rule pathway consists of two main branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway.
I also report the discovery of the other branch of the N-‐end regulation pathway, the Ac/N-‐end regulation pathway, which recognizes N-‐terminally acetylated residues as N-‐. The Ac/N-‐End regulatory pathway, the Arg/N-‐end regulatory pathway, and the steric sequestration of Nα-‐terminal.
CHAPTER 1
The 1986 discovery of the N-end regulatory pathway identified the first specific pathway of the Ub system (17-20). The physiological functions of the N-‐end regulatory pathway are remarkably broad and still being discovered. In eukaryotes, the N-end regulatory pathway is part of the Ub system, which mediates selective protein turnover.
The physically related Ubr1 (N-‐recognition) and Ufd4 E3s have substrate binding sites that recognize internal (non-N-terminal) degrons in substrates of the Arg/N-‐end rule pathway that lack N-‐. Physically related Ubr1 (N-recognition) and Ufd4 E3s have substrate-binding sites that recognize internal (non-N-terminal) degrons in substrates of the Arg/N-terminus rule pathway that lack N-degrons.
CHAPTER 2
Using in vitro and in vivo approaches, we show that the Ubr1-‐Ufd4 complex mediates the Arg/N-‐end rule pathway and also part of the UFD pathway. The Arg/N-end rule pathway in the yeast Saccharomyces cerevisiae is mediated by the 225 kDa RING-type Ubr1 E3 Ub ligase (Fig. 1a). Remarkably, the Ubr1/Rad6 Ub ligase of the Arg/N-terminal pathway could also ubiquitously (with low processivity) enter these UFD substrates.
We now report that the Arg/N-end regulatory pathway is mediated by a physical complex of the RING-type Ubr1 E3 and the HECT-type Ufd4 E3, respectively, together with their cognate E2 enzymes Rad6 and Ubc4/Ubc5 ( Fig. 1c . Interestingly, the function of Ufd4 in the Arg/N-end regulatory pathway is broader than that of a. Purified Sic1PY (Fig. S1c) was incubated in the above ubiquitylation assay, followed by SDS-PAGE and immunoblotting with anti-T7- antibody.
ClpS is the recognition component for Escherichia coli substrates of the N-terminus degradation pathway.
CHAPTER 3
We found that the presence of MNNG, an alkylating agent that causes DNA damage, in the yeast growth media contributes to the formation of a complex of Ubr1-‐Ufd4 with Mgt1 and also, remarkably, to the formation of the Ubr1‐‐Ufd4 -complex. yourself. The presence of MNNG in the media had no effect on the binding of Ubr1 and Ufd4 to Mgt1 when expressed independently (Fig. 3.4A, compare lanes 3 and 6). However, when Ubr1 and Ufd4 were overexpressed, the presence of MNNG in the growth media reduced the binding of Ubr1 or Ufd4 to Mgt1 (Fig. 3.4B compare lanes 3 and 6).
We also investigated gel filtration properties of the Ubr1-‐Ufd4 complex in the presence of type-‐1/2 dipeptides. Remarkably, the Ubr1-‐Ufd4 complex apparently dissociates in the presence of Arg-‐Ala and Leu-‐Ala, the complex of Ubr1 and Ufd4 dissociates, with Ubr1 eluting later and Ufd4 eluting earlier than in the presence of the control dipeptides Ala-‐Arg and Ala-‐Leu. Ufd4 and Ubr1 apparently compete for their binding to Mgt1 (Fig. 3.2), suggesting that in the Mgt1-bound Ubr1- ‐Ufd4 complex only one of these E3s.
We also found that the presence of the MNNG alkylating agent in the growth media facilitates the formation of the Ufd4-‐Ubr1 complex (Fig. 3.5). Expression of Ubr1 or Ufd4 independently in the presence or absence of MNNG does not affect binding to Mgt1. Coexpression of Ubr1 and Ufd4 in the presence of MNNG reduces their binding to Mgt1.
The amount of Ubr1 and Ufd4 pulled down with Mgt1 was decreased when cells were grown in the presence of MNNG (compare lanes 3 and 6). The presence of MNNG in the growth media increases the formation of the Ubr1-‐Ufd4 complex. The Ubr1-‐Ufd4 complex apparently dissociated in the presence of ‐1/2‐type dipeptides, as indicated by the absence of overlapping Ubr1 and Ufd4 bands in the elution profile (lower panels).
CHAPTER 4
Previous studies of Na-terminal acetylation (Nt--acetylation) have characterized Nt-acetylated proteins and the Na-terminal acetyltransferases (Nt--. acetylases) that catalyze this cotranslational modification (2-7). A retained N-terminal Met followed by “acetylation-permissive” residues is usually Nt-acetylated (fig. S1A) (5–7). Met-aminopeptidases cleave N-terminal Met if the residue at position 2 has a small enough side chain, resulting in N-terminal Ala, Val, Ser, Thr,.
In mammalian cells, the N-terminal Cys of N-end rule substrates can be oxidized by nitric oxide (NO) and oxygen, and then arginylated by an arginyl-‐. The results (fig. S5A) indicated nearly complete Nt-acetylation of MATα2 (no MATα2 lacking Nt-acetylation could be detected), consistent with Nt-acetylation of other proteins containing the N-terminal Met-Asn. (5–7). In addition, we produced an antibody, termed anti-AcNtMATα2, that recognized the Nt-acetylated N-terminal sequence of MATα2 (Fig. 2C) and was specific for the Nt-acetylated, hemagglutinin (HA)-tagged, MATα2-derived, MN -α23-‐.
Similar patterns were observed in wild-type cells expressing MKα2 (Lys at position 2) or GNα2 (N-terminal Gly) (Figure 2A). Except for the C-terminal Cys and various N-terminal residues, these peptides were identical to the N-terminal region of MATα2. Doa10myc13 bound to the MATα2 peptide with N-terminal AcNtMet, but not to otherwise identical peptides with.
Gly and Pro are therefore (largely) stabilizing in the N-‐terminus (Fig. 1D and fig. S3E) because N-‐terminal Gly and Pro are Nt‐‐acetylated in relatively few proteins (5–7). Doa10 did not bind to N-terminal AcNtMet when followed by Lys at position 2 (Fig. 2E). Of 20 amino acids in the genetic code, 18 are now known to function as destabilizing N-terminal residues in the N-end rule pathway (Fig. 4 and Fig. S1C).
Except for the C-terminal Cys (added for bead crosslinking) and the different identity of the N-terminal residue, these peptides were identical to 11-‐. Both branches target the N-terminal Cys residue (yellow rectangles) by different mechanisms, with the oxidized Cys indicated by an asterisk.
CHAPTER 5
Cog1, a subunit of the Golgi-associated COG complex, also appears to contain an Ac/N degron. To investigate this model and the new field of the Ac/N-end regulatory pathway, we focused on Cog1 and Hcn1. Ac-MDEVLPLFRDS, the Nt-acetylated N-terminal peptide of MD-Cog1wt, was used to produce a rabbit antibody, named anti-Cog1AcNt, that recognized Ac-‐.
Therefore, it is likely that Doa10 can recognize Nt-‐acetylated MD-‐Cog1wt, but may not efficiently target its Ac/N-‐degron. In summary, an unknown E3 N---recognition (the S. cerevisiae genome encodes more than 150 E3s) targets the Ac/N--degron of MD--Cog1wt either by itself or redundantly with Doa10. Both the putative conditionality of Ac/N-‐degrons (95) and electron microscopy of the yeast Cog1-‐Cog4 subcomplex (Figure S1D) (120) suggested that the Ac/N-‐degron of MD-‐Cog1wt may be at least partially sequestered in .
We conclude that the degradation of ML-Hcn1wt is mediated by its Ac/N-degron. The discovery of the Ac/N-end regulatory pathway and Ac/N-degrons ( Figure S1A ) ( 95 ) has revealed the largest class of degradation signals in the proteome, as nearly 90% of human proteins are Nt-acetylated. . Until now, these were only suggestions about possible biological roles of Ac/N degrons, made after the discovery of the Ac/N end rule pathway ( 95 ).
To address the functions of Ac/N-degrons, we chose two unrelated natural Nt-acetylated proteins, Cog1 (MD-Cog1wt) and Hcn1 (ML-Hcn1wt), described in the Introduction. As discussed below, further analyzes of MD-‐Cog1wt and ML-‐Hcn1wt showed that their Ac/N‐‐degrons are regulated by the formation of complexes between these proteins and their cognate protein ligands. Thus, the Ac/N-degrons of MD-‐Cog1wt and ML-‐Hcn1wt are conditional in a physiologically relevant manner, as they can be suppressed by the availability of the previously demonstrated physical interactions between these proteins and their protein ligands.
Specifically, the Nt-acetylated proteins that become long-lived do so after cessation of their initial degradation by the Ac/N-end rule pathway. As a result, most (but not necessarily all) molecules of newly formed MD-‐Cog escape the degradation of the Ac/N-‐end regulatory pathway through the formation of complexes with other COG subunits that protect Ac/N-‐ .
APPENDIX 1
This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. The S-‐alkyl-‐Cys product of AGT does not revert to Cys, so repair proteins of this type can only act once. A functionally severe lesion in double-stranded DNA is O6-methylguanine (O6meG), which is demethylated by O6--alkylguanine--DNA-alkyltransferase (AGT).
This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae (5-8). The resulting S-‐alkyl-‐Cys residue of AGT is not restored back to Cys, so repair proteins of this kind can only act once. In human cells expressing the E6 protein from the human papillomavirus (HPV), MGMT is also targeted for degradation by a complex of the viral E6 protein and E6AP, one of the mammalian HECT-domain E3 Ub ligases (8, 10).
Functions of the N-end rule pathway (Figure 1A) include the sensing of nitric oxide (NO), oxygen, heme, and short peptides; maintenance of high accuracy of chromosome separation;. The UFD pathway was discovered using engineered Ub fusions in which the structure of either the N-terminal part of Ub or its downstream polypeptide junction inhibited cleavage of the Ub fusion by deubiquitylating enzymes (DUBs. Ufd4 is a 168- ‐kDa HECT-‐domain E3 enzyme of UFD pathway that works together with the E2 enzymes Ubc4 or Ubc5.
Several domains of Ufd4 were shown to recognize the N-terminal Ub group of UFD substrates ( 29 ) as well as specific subunits of the 26S proteasome ( 30 , 31 ). Rad4, a nucleotide excision repair protein, is partially stabilized in ufd4Δ cells, suggesting that Rad4 may be a substrate of the UFD pathway ( 33 ). The N-end rule and UFD pathways were the first specific pathways of the Ub system to be discovered ( 14 ).
