Secretion of killer toxin encoded on the linear DNA plasmid pGKL1 from Saccharomyces cerevisiae.
By the kar1-mediated cytoduction, linear double-stranded DNA plasmids pGKL1 and pGKL2, encoding killer toxin complex, have been successfully transferred to the recipient strains with about 30% frequency. The killer toxin was found to be secreted through the normal yeast secretory pathway by introducing pGKL plasmids into the several Saccharomyces cerevisiae sec mutants and examining the secretion of killer toxin. S. cerevisiae cells, harboring newly isolated deletion plasmid pGKL1D, expressed only the 28K protein among three killer subunits, and secreted the 28K subunit at a level of zero to 20% efficiency of the cells containing intact pGKL1 plasmid. These data indicated that subunit interaction (cosecretion) of killer proteins is required for the efficient secretion of 28K subunit. The 28K precursor protein was found to translocate across the canine pancreatic endoplasmic reticulum membrane under the direction of its own signal peptide in vitro without any other subunits. From kex2 mutant cells harboring pGKL1 plasmid, the 97K subunit, and its precursor 128K protein were not secreted, however, the 28K subunit was secreted in the same amount as that secreted from KEX2 cells. These lines of evidence suggest that the final assembly of killer toxin complex after KEX2 site of Golgi apparatus is not essential for the secretion of 28K subunit, and therefore, that putative interaction between 128K protein and 28K subunit for the transport between endoplasmic reticulum and Golgi apparatus may be required for the efficient secretion of 28K subunit.
Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
M
M
Tokunaga
A
A
Kawamura
K
K
Kitada
F
F
Hishinuma
D000818:Animals; D002872:Chromosome Deletion; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D055646:Killer Factors, Yeast; D046911:Macromolecular Substances; D008970:Molecular Weight; D009154:Mutation; D009183:Mycotoxins / Q000096:biosynthesis / Q000235:genetics*; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; C527157:KHR1 protein, S cerevisiae; D055646:Killer Factors, Yeast; D046911:Macromolecular Substances; D009183:Mycotoxins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins