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

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.

Arginyltransferase, Its Specificity, Putative Substrates, Bidirectional Promoter, and Splicing-derived Isoforms

“…Splicing-derived Isoforms of Mouse ATE1-Work by this laboratory has shown that prokaryotes, in addition to containing L-transferases (encoded by aat genes) (44,45) that are nonsequelogous to the ATE1-encoded eukaryotic R-transferases, also contain a distinct family of bpt-encoded L-transferases that are sequelogs (48) of eukaryotic ATE1 R-transferases (see Introduction) (47). One of the most highly conserved sequences in ATE1 R-transferases is CGYC.…”

“…1A). In this previously described assay (26,36,47), identical amounts of purified mouse ATE1 1A7A , ATE1…”

“…The sets of destabilizing residues in prokaryotic N-end rules contain secondary destabilizing (Nd s ) residues as well. Their identities (Arg and Lys in Escherichia coli; Arg, Lys, Asp, and Glu in some other prokaryotes) are either overlapping with, or distinct from, the eukaryotic Nd s residues (47). The activity of Nd s residues in prokaryotes requires their conjugation to Leu (an Nd p residue) by Leu-tRNA-protein transferases (L-transferases), in contrast to the conjugation of Arg (an Nd p residue in eukaryotes) to N-terminal Asp, Glu, or (oxidized) Cys (Fig.…”

“…Prokaryotic L-transferases are of two distinct types, differing in both amino acid sequence and substrate specificity. Remarkably, L-transferases of one class, encoded by bpt genes, are sequelogs of ATE1-encoded eukaryotic R-transferases, despite the fact that Bpt (prokaryotic) aminoacyltransferases conjugate Leu, rather than Arg, to the N termini of cognate substrates (47). (In this terminology, "sequelog" and "spalog" denote, respectively, a sequence that is similar, to a specified extent, to another sequence and a three-dimensional structure that is similar, to a specified extent, to another three-dimensional structure (48).…”

“…The activity of Nd s residues in prokaryotes requires their conjugation to Leu (an Nd p residue) by Leu-tRNA-protein transferases (L-transferases), in contrast to the conjugation of Arg (an Nd p residue in eukaryotes) to N-terminal Asp, Glu, or (oxidized) Cys (Fig. 1A) (44,47). Prokaryotic L-transferases are of two distinct types, differing in both amino acid sequence and substrate specificity.…”