Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein.
Protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein involves four highly coordinated reactions that result in precise cleavage and formation of peptide bonds. In this study, we investigated the roles of the last N-extein residue (-1 residue) and the intein penultimate residue in modulating splicing reactions. Most of the 20 amino acid substitutions at the -1 position had no effect on overall protein splicing but could lead to significant accumulation of thioester intermediates when splicing was blocked by mutation. A subset of -1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein containing all wild-type catalytic residues. Substitutions involving the intein penultimate residue allowed modulation of the branch resolution and C-terminal cleavage reaction. Our data suggest that the N-S acyl rearrangement, which initiates splicing, may also serve as the rate-limiting step. Through appropriate amino acid substitutions, we were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and deletion were tolerated in the central region of the intein although splicing or structure of the intein may have been affected.
Chong
S
S
New England Biolabs, Inc., Beverly, Massachusetts 01915, USA.
Williams
K S
KS
Wotkowicz
C
C
Xu
M Q
MQ
D019943:Amino Acid Substitution; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology*; D016254:Mutagenesis, Insertional; D019154:Protein Splicing; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*
D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases