Efficient secretion in yeast based on fragments from K1 killer preprotoxin.
The alpha and beta components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox). The beta N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of beta la secretion resulted. Over-expression of Kex2 failed to increase beta la secretion while a kex2-null mutation reduced secretion by 98%. beta la secretion in a Kex+ strain was not enhanced by inactivation of the alpha toxin component or by deletion of most of its central hydrophobic segments. However SP-beta la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta la activity and the precursor was now secreted with similar efficiency in a kex2-null strain. Fusions of beta la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains. Since these beta la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP-beta la was the most efficient construct tested; secreted beta la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF alpha 1-encoded prepro alpha-factor, suggesting potential for the production of foreign proteins in yeast.
Cartwright
C P
CP
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
Zhu
Y S
YS
Tipper
D J
DJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D006056:Golgi Apparatus / Q000378:metabolism; D055646:Killer Factors, Yeast; D008969:Molecular Sequence Data; D009154:Mutation; D009183:Mycotoxins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D043484:Proprotein Convertases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics*; D013381:Subtilisins; D014158:Transcription, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D055646:Killer Factors, Yeast; D009183:Mycotoxins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D001618:beta-Lactamases