Cell cycle studies on the mode of action of yeast K28 killer toxin

Abstract

The virally encoded K28 killer toxin of Sacchsmmyces cerewisiae kills sensitive cells by a receptor-mediated process. DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells eventually arrest, apparently in the S phase of the cell cycle with a medium-sized bud, a single nucleus in the mother cell and a pre-replicated (In) DNA content. Cytoplasmic microtubules appear normal, and no spindle is detectable. Arrest of a sensitive haploid yeast strain by a-factor a t START gave complete protection for a t least 4 h against a toxin concentration that killed non-arrested cells a t the rate of one log each 2.5 h. Cells released from a-factor arrest were killed by toxin a t a similar rate; arrest occurred with medium-sized buds within the same cell cycle. Cells arrested by hydroxyurea, with unreplicated DNA, or by the spindle poison methylbenzimidazol-2yl-carbamate, with unseparated chromosomes, both arrest at the checkpoint at the C2/M boundary; these arrested cells were not protected against toxin, losing about one log of viability every 4 h. Following release from the cell cycle block, a majority of these toxin-exposed cefls progressed through the Celt cycle and arrested in the following S-phase, again with medium-sized buds. Killing by K28 toxin apparently requires entry into the nuclear division and bud cycles, but can result from inhibition of either early or late events in these cycles. Morphogenesis in moribund cells is uniformly blocked in early S-phase with an immature bud. Toxin action causes either independent blockage of both DNA synthesis and the budding cycle, or inhibits some unknown step required for both events. M. J. S C H M I T T a n d O T H E R S that retained its hypersensitivity. Cells that had been arrested in different stages of the cell cycle were exposed to more rapidly lethal concentrations of K28 toxin and phenotypes were determined following release from the cell cycle block in the continued presence o r absence of toxin. We demonstrated that tuxin-treated cells arrest in the budded phase of the cell cycle with an unreplicated (Gl) content of DNA in a single nucleus located in the mother cell. While cells arrested with a-factor at START were protected against toxin action, cells arrested at the G2/M checkpoint because of inhibition of DNA synthesis or microtubular function remained sensitive ; following removal of the cell-cycle inhibitor, these moribund cells progressed through the cell cycle to arrest again in the following S-phase, even in the absence of additional toxin. toxins and the outer 1,3-~-linked mannose residues of cell wall mannoproteins in the case of K28 killer toxin (Hutchins 8r Bussey, 1983; Schmitt Br Radler, 1988).Synthesis and assembly oE these primary cell-wall binding sites requires a whole set of chromosomal KRE and MiVN genes, the products of which are involved in glucan and mannop ro tein synthesis , respectively (Busse y, 1991; Tipper & Schmitt, 1991). Mutations within these nuclear genes lead to failure in cell-wall binding of toxin and a s...

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

“…In natural killer strains of S. cerevisiae, the virally encoded K28 pptox represents the unprocessed precursor of a secreted protein toxin (K28) that kills sensitive yeast cells in a receptor-mediated fashion by causing a cell cycle arrest at the G 1 /S boundary followed by a rapid inhibition of DNA synthesis in the nucleus (26,27,28). The 38-kDa pptox consists of an N-terminal leader peptide necessary for pptox import into the ER lumen followed by the ␣ and ␤ subunits of the mature toxin (10.5 and 11.0 kDa, respectively) which are separated from each other by a potentially N-glycosylated ␥ sequence (28).…”

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis

“…Several killer toxins (K1, PMKT) seem to be poreforming-related toxins (Breinig et al, 2002;Santos & Marquina, 2004b). K28, from S. cerevisiae, has been found to cause sensitive yeasts to arrest proliferation as unbudded cells, suggesting that it blocks completion of the cell cycle (Schmitt et al, 1996), whereas zymocin, from Kluyveromyces lactis, exerts a tRNase activity (Butler et al, 1991;Studte et al, 2008). Finally, HM-1, from Hansenula mrakii, kills sensitive cells, presumably by interfering with b-(1A3)-glucan synthesis (Takasuka et al, 1995).…”

Pichia anomalaDBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity

“…In the second step, which has been far less characterized, the killer toxin is assumed to interact with receptors on the cell membrane and to kill the sensitive cells via different mechanisms. These include cell membrane permeabilization, cell cycle perturbation, inhibition of DNA synthesis, and inhibition of ␤-1,3-glucan synthetase activity (7,14,15,41,49,53).As the spectrum of action of some toxins has extended to microbial pathogens of clinical interest, killer toxins and/or killer toxin-like antibodies and mimotopes are of great relevance to medicine (8,25). Other toxins that exert a killing action on spoilage yeasts have interesting applications in the fermentative (46) and food and feed industries (10,11,12,23,32,44), where they can be used as "natural" food antimicrobials.…”