Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

Abstract

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.

Expression of a cDNA derived from the yeast killer preprotoxin gene: implications for processing and immunity.

“…Strain K12-1 (a ade arg) contains Ml-dsRNA and is the standard type K1 killer used in previous studies (3,4,6,8). The diploid strain T158C/S14a has also been described previously (7). Low-phosphate (low-Pi) and high-phosphate (high-Pi) media (12,13) were employed for growth of yeast strains under repressed (high-Pi) or derepressed (low-Pi) conditions for PH05 and were used in standard patch assays (14) for toxin and immunity.…”

“…The secreted toxin is encoded by Ml-dsRNA (3) and is composed oftwo dissimilar, 9.5-and 9.0-kDa, disulfide-linked, nonglycosylated protein subunits, denoted a and A, respectively (4). An intracellular 43-kDa glycosylated precursor, or protoxin, is precipitated from extracts ofpulse-labeled killer cells by anti-toxin IgG (5,6) and is processed, with a half-life of about 25 min, into the 19-kDa exocellular toxin (7). In vitro translation of denatured Ml-dsRNA produces Ml-P1, a 35-kDa product (3,4).…”

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

“…Work with a-factor and Kl killer toxin indicates that the Kex2p cleavage event occurs in a post-sec18 manner (4,21) and potentially in a pre-sec7 compartment. Modification to Kexlp N-linked glycosylation can be used to indicate the localization of the protein in the cell.…”

“…Earlier work showed that kexl mutations abolish the production of active Kl killer toxin (4,39). In addition, such mutants produce less active ax-factor pheromone than KEXI strains (7).…”

KEX2 mutations suppress RNA polymerase II mutants and alter the temperature range of yeast cell growth.

“…Alternatively, the KEX2 protease could act on some proteins in the endoplasmic reticulum even though it appears to process ot-factor and killer toxin in the Golgi (7,17). If so, the protease could process the proposed protein in the endoplasmic reticulum before transport of the protein to the nuclear membrane.…”