Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein

Abstract

Protein splicing involves the excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and Cterminal regions. It occurs in mesophilic bacteria, yeast, and thermophilic archaea. The ability to control protein splicing of a thermophilic intein by temperature and pH in a foreign protein context facilitated the study of the mechanism of protein splicing in thermophiles. On the other hand, no direct studies have been done on the mechanism of protein splicing in mesophiles. We examined the splicing of a chimeric protein containing the intein of the vacuolar ATPase subunit (VMA) of Saccharomyces cerevisiae that involves cysteines rather than serines at the reaction center. The steps in the splicing process were deduced by analyzing intermediates and side products that accumulated as a result of amino acid substitutions and were found to be analogous to those occurring in thermophiles. Moreover, appropriate amino acid replacements allowed us to develop the first mesophilic in vitro protein splicing system as well as strategies for modulating the rate of protein splicing and for converting the splicing reaction to an efficient protein cleavage reaction at either splice junction.Protein splicing is a novel mode of gene expression that has been described in mesophilic bacteria and yeast and in extremely thermophilic archaea (1-6). It is a process in which a single gene directs the synthesis of two separate proteins by the precise excision of an internal protein segment, the intein, from a precursor protein and the concomitant ligation of the flanking N-and C-terminal regions, the exteins, to yield two new proteins (7). Some of the excised inteins are homing endonucleases that catalyze lateral transfer of their DNA coding sequences by an intein homing mechanism (8 -11), whereas the ligated exteins are usually enzymes with a specific cellular function.Efficient protein splicing also occurs when inteins are transferred into heterologous proteins, suggesting that all structural and catalytic elements needed for the splicing reaction reside in the inteins plus the first C-extein residue (4,(12)(13)(14). The protein splicing function of inteins is independent of their homing endonuclease activity (15) and depends on highly conserved amino acid residues at both splice junctions. A hydroxyl-or thiol-containing residue (Ser, Thr, or Cys) is always present at the positions that immediately follow the two splice junctions, and the sequence His-Asn is invariant at the intein C terminus. Substitution of any of these conserved residues retards or abolishes protein splicing (2,12,13,15).The fact that the thermophilic archaeal inteins in a foreign protein context undergo efficient splicing only at elevated temperatures (25-65°C) opened the way for the development of an in vitro system to study the mechanism of protein splicing (14). We constructed a fusion protein containing the intein from the thermostable DNA polymerase of Pyroccocus sp. GB-D, which could be expressed i...

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

“…The proposed mechanism for the S. cerevisiae Vma intein is shown in Figure 3 (see refs. 24,25). Splicing is initiated by an N-S acyl shift that converts a peptide bond into a thioester at the N-extein junction site.…”

Protein splicing: its chemistry and biology

“…The third step of the splicing reaction yields two immediate products, the excised 50 kDa VDE with a C-terminal aminosuccinimide residue and the spliced MT polypeptide linked by a thioester bond. Though the rate of hydrolysis of the terminal succinimide residue has been determined as slow, the rate of rearrangement of thioester of the Cys455 residue by S→N acyl shift was proved to be much faster, with the equilibrium favouring the amide form, thus autocatalytically yielding a normal peptide bond (Chong et al 1996).…”

“…The chemical framework for protein splicing in the molecule of the VMA1 protein has been thoroughly explained (Chong et al 1996;Kawasaki et al 1996). However, the structural context which enables the exact splicing between the two reactive cysteine residues separated by 454 amino acids is still in question (see Fig.…”

Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction

“…In the papain family, highly conserved Asn is essential for catalysis, forming the Cys-His-Asn catalytic triad. The residues Cys, His, and Asn are also important for protein splicing: mutations of Cys 284 at the N-terminal splice site, His 736 , Asn 737 and Cys 738 at the C-terminal splice site result in defects of protein splicing [5][6][7][8][9][10]. The His 362 residue apart from the N-terminal splice site is also crucial for protein splicing [12].…”

“…In the yeast Saccharomyces cerevisiae, the 120 kDa nascent translation product of the VMA1 gene, termed Vmal protozyme [1], autocatalytically excises out the 50 kDa DNA endonuclease (VDE; FM^7-derived endonuclease, see [2]) and splices the two external polypeptides to form the 70 kDa catalytic subunit of the vacuolar H + -ATPase [3,4] Substitution of amino acids around the splice junctions has shown that the structure around the cleavage sites of the substrate is important for the splicing reaction [5][6][7][8][9][10]. Mutational analysis has demonstrated the crucial requirement of the thiol-or hydroxyl-containing residues at the two splice junctions (Cys 284 and Cys 738 as for the Vmal protozyme) and the asparagine residue immediately preceding the C-terminal junction (Asn 737 ).…”