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. 2006 Feb 28;103(9):3078-83.
doi: 10.1073/pnas.0511224103. Epub 2006 Feb 21.

Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen

Emmanuelle Graciet  1 , Rong-Gui Hu, Konstantin Piatkov, Joon Haeng Rhee, Erich M Schwarz, Alexander Varshavsky
Affiliations

Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen

Emmanuelle Graciet et al. Proc Natl Acad Sci U S A. .

Abstract

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Primary destabilizing N-terminal residues (Nd(p)) are recognized directly by the targeting machinery. The recognition of secondary destabilizing N-terminal residues (Nd(s)) is preceded by conjugation of an Nd(p) residue to Nd(s) of a polypeptide substrate. In eukaryotes, ATE1-encoded arginyl-transferases (R(D,E,C*)-transferases) conjugate Arg (R), an Nd(p) residue, to Nd(s) residues Asp (D), Glu (E), or oxidized Cys residue (C*). Ubiquitin ligases recognize the N-terminal Arg of a substrate and target the (ubiquitylated) substrate to the proteasome. In prokaryotes such as Escherichia coli, Nd(p) residues Leu (L) or Phe (F) are conjugated, by the aat-encoded Leu/Phe-transferase (L/F(K,R)-transferase), to N-terminal Arg or Lys, which are Nd(s) in prokaryotes but Nd(p) in eukaryotes. In prokaryotes, substrates bearing the Nd(p) residues Leu, Phe, Trp, or Tyr are degraded by the proteasome-like ClpAP protease. Despite enzymological similarities between eukaryotic R(D,E,C*)-transferases and prokaryotic L/F(K,R)-transferases, there is no significant sequelogy (sequence similarity) between them. We identified an aminoacyl-transferase, termed Bpt, in the human pathogen Vibrio vulnificus. Although it is a sequelog of eukaryotic R(D,E,C*)-transferases, this prokaryotic transferase exhibits a "hybrid" specificity, conjugating Nd(p) Leu to Nd(s) Asp or Glu. Another aminoacyl-transferase, termed ATEL1, of the eukaryotic pathogen Plasmodium falciparum, is a sequelog of prokaryotic L/F(K,R)-transferases (Aat), but has the specificity of eukaryotic R(D,E,C*)-transferases (ATE1). Phylogenetic analysis suggests that the substrate specificity of R-transferases arose by two distinct routes during the evolution of eukaryotes.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
The N-end rule pathways and the activities of aa-transferases, the Bpt LD,E-transferase of V. vulnificus (a prokaryotic pathogen) and the ATEL1 RD,E-transferase of P. falciparum (an eukaryotic pathogen). (A) The N-end rule pathway in mammals. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. C* denotes oxidized N-terminal Cys, produced in reactions mediated by NO, with subsequent arginylation of C* by ATE1-encoded isoforms of Arg-tRNA-protein transferase (RD,E,C*-transferase) (11). The type-1 and type-2 primary destabilizing N-terminal residues are recognized by multiple E3 Ub ligases (N-recognins) of the N-end rule pathway that share the UBR motif (12). Through their other substrate-binding sites, these E3 enzymes also recognize internal (non-N-terminal) degradation signals (degrons) in other substrates of the N-end rule pathway, denoted by a larger yellow oval. (B) The E. coli N-end rule pathway (7, 25, 26). Although the purified Aat L/FK,R-transferase is capable of conjugating either L (Leu) or F (Phe), the conjugated residue in vivo (in E. coli) is largely L (26). (C) The V. vulnificus N-end rule pathway, characterized in the present work, including the aa-transferase Bpt (LD,E-transferase). ClpS was demonstrated to be the N-recognin of the E. coli N-end rule pathway (31); its similar function in V. vulnificus is an inferred one at present. (D) The putative N-end rule pathway of P. falciparum and the ATEL1 aa-transferase. A question mark denotes the expected but unproven features of this pathway in P. falciparum, where the only characterized component thus far is ATEL1.
Fig. 2.
Fig. 2.
The N-end rule pathway in V. vulnificus and E. coli. (A) The V. vulnificus aat-bpt operon and its derivatives. The ORF encoding the Bpt aa-transferase begins within the stop codon of the preceding aat ORF. The nucleotide sequence of the bpt ORF (including its start codon, in red) is shown below the diagram. Shown above is the 3′ end of aat ORF, including its TGA stop codon, in red. Also indicated are the lengths of flanking V. vulnificus DNA fragments in the plasmid insert. (B) Relative enzymatic activities of βgal in Aat-lacking E. coli (TS351) expressing S. cerevisiae UBP1, both aa-transferases of V. vulnificus (WT), and either Met-βgal (M), Lys-βgal (K), Arg-βgal (R), Asp-βgal (D), Glu-βgal (E), or Leu-βgal (L), produced from the corresponding Ub fusions. (C) The same as B but in the absence of V. vulnificus Aat. (D) The same as B but in the absence of V. vulnificus Bpt. (E) The same as B but in the absence of V. vulnificus Aat and Bpt. One hundred percent is the (averaged) activity of Met-βgal (M). (F) Determination, through Edman degradation, of the N-terminal sequence of Asp-βgal (derived from Ub-Asp-βgal) from E. coli KPS18 that lacked both Aat and ClpA and expressed V. vulnificus Bpt. The indicated ratio of Leu-Asp-βgal/Asp-βgal refers to the incomplete in vivo leucylation of (overexpressed) Asp-βgal by V. vulnificus Bpt in E. coli, as detected by Edman sequencing.
Fig. 3.
Fig. 3.
Enzymatic specificities of V. vulnificus Bpt and P. falciparum ATEL1 aa-transferases. (A) (Left) 3H-Leu: lane 1, purified V. vulnificus Bpt was incubated with 3H-Leu, other components of the aa-transferase assay, and the 14-kDa bovine α-lactalbumin (bearing N-terminal Glu) as a reporter. Lanes 2–5, the same as lane 1 but with A. tumefaciens Bpt, M. loti Bpt, S. cerevisiae ATE1, and E. coli Aat, respectively. Lanes 6–10, the same as lanes 1–5 but with the 24-kDa bovine α-casein (bearing N-terminal Lys) as a reporter. (Right) 3H-Arg: the same as Left but with 3H-Arg instead of 3H-Leu. Arrows indicate the bands of 3H-lactalbumin and 3H-casein, and numbers to the left indicate molecular masses, in kDa, of protein standards. (B) Relative enzymatic activities of V. vulnificus Bpt in which specific Tyr residues were converted to Phe. Purified mutant and WT V. vulnificus Bpt (0.2 μg each) were assayed in vitro with 3H-Leu as described in Supporting Text, using α-lactalbumin as a reporter. 3H was measured in triplicate samples, and the data were corrected by subtracting 3H incorporation in the control assay lacking Bpt. (C) aa-transferase assay with purified (0.25 μg) P. falciparum ATEL1 enzyme in the presence of either α-casein or α-lactalbumin and either 3H-Leu or 3H-Arg as indicated.
Fig. 4.
Fig. 4.
Parsimony-based phylogenetic analyses of selected Bpt/ATE1 and Aat/ATEL1 aa-transferases. (A) Phylogeny of Bpt and ATE1. (B) Phylogeny of Aat and ATEL1. Numbers by the phylogenetic branchpoints give their statistical strength, with 100 being a maximum score. See the text and Supporting Text for details, including the complete names of the indicated organisms.
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