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M double-stranded RNA (MdsRNA) plasmid mutants were obtained by mutagenesis and screening of a diploid killer culture partially heat cured of the plasmid, so that a high proportion of the cells could be expected to have only one M plasmid. Mutants with neutral (nonkiller [K-], immune [R+]) or suicide (killer [K+], sensitive [R-]) phenotypes were examined. All mutants became K-Rsensitives on heat curing of the MdsRNA plasmid, and showed cytoplasmic inheritance by random spore analysis. In some cases, M plasmid mutations were indicated by altered mobility of the MdsRNA by agarose gel electrophoresis or by altered size of in vitro translation products from denatured dsRNA. Neutral mutants were of two types: nonsecretors of the toxin protein or secretors of an inactive toxin. Of three neutral nonsecretors examined, one (NLP-1), probably a nonsense mutation, made a smaller protoxin precursor in vitro and in vivo, and two made full-size protoxin molecules. The in vivo protoxin of 43,000 molecular weight was unstable in the wild type and kinetically showed a precursor-product relationship to the processed, secreted 11,000-molecular-weight toxin. In one nonsecretor (N1), the protoxin appeared more stable in a pulse-chase experiment, and could be altered in a recognition site required for protein processing.Killer strains of the yeast Saccharomyces cerevisiae contain M double-stranded RNA (MdsRNA) plasmids encapsidated in a virus-like particle. This MdsRNA codes for a protein toxin and an immunity component. The toxin is secreted and kills sensitive yeast cells devoid of the plasmid (see references 5, 7, and 28 for recent reviews). The toxin appears to be membrane acting (9); it interacts initially with a cell wall receptor (8) and then, in an energy-dependent reaction, causes membrane damage (23). In vitro, in a phospholipid bilayer system, the toxin inserts into the bilayer and forms a pore (B. Kagan, personal communication), and, as in the case of the membrane-acting colicin K (21), this is likely to be the mechanism of action in vivo.The killer toxin system is a useful model for secretion and for events in the interaction of a protein with the cell surface. The existence of mutants in the MdsRNA plasmid that affect toxin or immunity function permits us to make a genetic approach to these problems and to extend work done with chromosomal mutants (1). Although MdsRNA mutations have been described previously (24-26), none but the suppressive sensitive deletions (10) have been examined in detail. Here we examine nine mutants, six defective in toxin activity or secretion and three partially defective in the immunity component. MATERIALS AND METHODSYeast stains and media. The diploid killer strain T158C/S14a (8) was used as the wild type for the production of mutants and as the killer strain in phenotype tests. Sensitive strains used were S6, K19.10, and A8207NK. Neutral mutants Ni and N2 were also used (24).Growth medium was the minimal medium of Halvorson (11) plus 0.5% yeast extract, 0.5% peptone, and 2% dextrose [YEPD ...

Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (secl). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In secl8 the protoxin was stable after a chase; but in sec7 and seci the protoxin was unstable, and in secl 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-L-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and secl cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-secl8 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kexi but unstable in kex2.Protoxin stability in kexi kex2 double mutants indicated the order kexi -* kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEXI-and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.

3S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes krel or kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant krel-1. This implied that binding to krel-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 106 M-' and a receptor number of 1.1 x 107 per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 3S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from krel-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 107 per cell) wall receptor appeared necessary for toxin action, as few as 2.8 x 104 toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics of killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.