Role of the γ component of preprotoxin in expression of the yeast K₁ killer phenotype

Abstract

K1 killer strains of Saccharomyces cerevisiae secrete a polypeptide toxin to which they are themselves immune. The alpha and beta components of toxin comprise residues 45-147 and 234-316 of the 316-residue K1 preprotoxin. The intervening 86-residue segment is called gamma. A 26-residue signal peptide is removed on entry into the endoplasmic reticulum. The Kex2 protease excises the toxin components from the 290-residue glycosylated protoxin in a late Golgi compartment. Expression of a cDNA copy of the preprotoxin gene confers the complete K1 killer phenotype on sensitive cells. We now show that expression of immunity requires the alpha component and the N-terminal 31 residues of gamma. An additional C-terminal extension, either eight residues of gamma or three of four unrelated peptides, is also required. Expression of preprotoxin terminating at the alpha C-terminus, or lacking the gamma N-terminal half of gamma causes profound but reversible growth inhibition. Inhibition is suppressed in cis by the same 31 residues of gamma required for immunity to exocellular toxin in trans, but not by the presence of beta. Both immunity and growth inhibition are alleviated by insertions in alpha that inactivate toxin. Inhibition is not suppressed by kex2, chc1 or kre1 mutations, by growth at higher pH or temperature, or by normal K1 immunity. Inhibition, therefore, probably does not involve processing of the alpha toxin component at its N-terminus or release from the cell and binding to glucan receptors. Some insertion and substitution mutations in gamma severely reduce toxin secretion without affecting immunity. They are presumed to affect protoxin folding in the endoplasmic reticulum and translocation to the Golgi.

Yeast Virus Propagation Depends Critically on Free 60S Ribosomal Subunit Concentration

“…2B). Comparison of the same strains lacking L-A and M 1 but making toxin from an M 1 cDNA clone, pP-T 316 (59), showed toxin production essentially the same in the three strains (Fig. 2C).…”

“…Plasmids pTIC19 (MAK11) (22) and YCp50-1C (MAK16) (53) were used to transform the respective mutant strains. p596 is Bluescript KSϩ with the 1.3-kb PstI-SalI fragment of pP-T 316 (59) carrying M 1 sequences inserted between the PstI and SalI sites.…”

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

“…Enzyme dilutions of purified FX7 (33 nM, final concentration) and Ec AAO (18 nM, final concentration) were prepared in such a way that aliquots of 20 l gave rise to a linear response in kinetic mode. The optimum temperature for activity was estimated in prewarmed 96-well reading plates (Labnet VorTemp 56 Shaking Incubator; Labnet International, USA) with 100 mM sodium phosphate (pH 6.0) containing 1 mM p-methoxybenzyl alcohol at various corresponding temperatures (25,30,40,50, 60, 70, 80, 90, and 99°C), followed by incubation in an Eppendorf Thermomixer Comfort apparatus (Thermo Fisher Scientific). Reactions were performed by triplicate and p-methoxybenzyl alcohol oxidation, followed by aldehyde production at 285 nm.…”

“…In the present study, the native signal peptide of AAO was replaced by two different signal sequences to drive its functional expression in Saccharomyces cerevisiae: (i) the signal prepro-leader of the mating ␣-factor of S. cerevisiae, which has been used widely to evolve different ligninases (18)(19)(20)(21)(22)(23), and (ii) the signal prepro(␦)-leader and the ␥-spacer segment of the K 1 killer toxin, which have been seen to be useful in boosting ␤-lactamase secretion in yeast (24,25). For the first time, chimeric versions of these leaders were designed by combining the different pre-and pro-regions, and these constructs were subjected to conventional and focused-directed evolution using a very sensitive dual highthroughput screening (HTS) assay based on the Fenton reaction.…”

Evidence that the SKI antiviral system of Saccharomyces cerevisiae acts by blocking expression of viral mRNA.

“…When M1 was maintained by the L-A virus, the toxin produced was increased in the ski2:-1HIS3 strain, as expected (65). When M1 was supported by the L-A cDNA clone, even though the copy number of To test whether this result could be an effect of the SKI system on the processing or secretion of the K1 killer toxin, we tested the effect of the sAi2::HIS3 mutation on toxin production from either of two cDNA clones of M1: pP-T316, in which the preprotoxin gene is controlled by the PGKI promoter (74), or pVT100-U/KT, in which its transcription is driven by the ADHI promoter (68). In each case, there was no detectable effect of the sh2 mutation (Table 4) (38,50) from an rDNA promoter was affected by a shi2::HIS3 mutation (Table 5).…”