Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein

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 C-terminal 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.

[1]  H. Paulus,et al.  Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process. , 1996, Biochemistry.

[2]  Jack Benner,et al.  Activation of Glycosylasparaginase , 1996, The Journal of Biological Chemistry.

[3]  J. Tam,et al.  Peptide synthesis using unprotected peptides through orthogonal coupling methods. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  T. Stevens,et al.  Protein splicing: self-splicing of genetically mobile elements at the protein level. , 1995, Trends in biochemical sciences.

[5]  H. Paulus,et al.  Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. , 1995, Biochemistry.

[6]  Stephen C. Ekker,et al.  The product of hedgehog autoproteolytic cleavage active in local and long-range signalling , 1995, Nature.

[7]  J. J. Lee,et al.  Autoproteolysis in hedgehog protein biogenesis. , 1994, Science.

[8]  F. Perler,et al.  Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. , 1994, The EMBO journal.

[9]  S. Pietrokovski,et al.  Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins , 1994, Protein science : a publication of the Protein Society.

[10]  Neil D. Clarke.  A proposed mechanism for the self-splicing of proteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Tam,et al.  Chemical Ligation Approach To Form a Peptide Bond between Unprotected Peptide Segments. Concept and Model Study , 1994 .

[12]  N. Neff,et al.  Protein splicing elements: inteins and exteins--a definition of terms and recommended nomenclature. , 1994, Nucleic acids research.

[13]  M. Colston,et al.  Evidence of selection for protein introns in the recAs of pathogenic mycobacteria. , 1994, The EMBO journal.

[14]  Reed J. Harris,et al.  Identification of succinimide sites in proteins by N‐terminal sequence analysis after alkaline hydroxylamine cleavage , 1994, Protein science : a publication of the Protein Society.

[15]  Francine B. Perler,et al.  In vitro protein splicing of purified precursor and the identification of a branched intermediate , 1993, Cell.

[16]  T. Stevens,et al.  Protein splicing of the yeast TFP1 intervening protein sequence: a model for self‐excision. , 1993, The EMBO journal.

[17]  J. Xu,et al.  Peptide splicing in the vacuolar ATPase subunit A from Candida tropicalis. , 1993, The Journal of biological chemistry.

[18]  M. Belfort,et al.  Introns as mobile genetic elements. , 1993, Annual review of biochemistry.

[19]  P. S. Nelson,et al.  Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone. , 1992, Nucleic acids research.

[20]  S. Sedgwick,et al.  Protein splicing in the maturation of M. tuberculosis RecA protein: A mechanism for tolerating a novel class of intervening sequence , 1992, Cell.

[21]  H. Goodrich-Blair,et al.  Protein introns: A new home for endonucleases , 1992, Cell.

[22]  R. Hirata,et al.  Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing. , 1992, Biochemical and biophysical research communications.

[23]  B. Slatko,et al.  Intervening sequences in an Archaea DNA polymerase gene. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Thorner,et al.  Homing of a DNA endonuclease gene by meiotic gene conversion in Saccharomyces cerevisiae , 1992, Nature.

[25]  S. Kent,et al.  Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease. , 1992, Science.

[26]  S. Sedgwick,et al.  Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product , 1991, Journal of bacteriology.

[27]  P. Kane,et al.  Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. , 1990, Science.

[28]  P. D. van Poelje,et al.  Pyruvoyl-dependent enzymes. , 1990, Annual review of biochemistry.

[29]  W. D. de Jong,et al.  Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate. , 1988, The Journal of biological chemistry.

[30]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[31]  W. Lennarz,et al.  Fatty acylation of proteins during development of sea urchin embryos. , 1984, The Journal of biological chemistry.

[32]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[33]  E. Gross.  [27] The cyanogen bromide reaction , 1967 .

[34]  W. Jencks,et al.  The free energy of thiol ester hydrolysis. , 1960, The Journal of biological chemistry.

[35]  P. Gallop,et al.  Analysis of hydroxamic acids and hydrazides; preparation and properties of dinitrophenyl derivatives of hydroxamic acids, oximes, hydrazides, and hydrazones. , 1960, The Journal of biological chemistry.