Secretion ofSaccharomyces cerevisiaeKiller Toxin: Processing of the Glycosylated Precursor

Abstract

Killer toxin secretion was blocked at the restrictive temperature inSaccharomyces cerevisiae secmutants with conditional defects in theS. cerevisiaesecretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in allsecmutants at the restrictive temperature. Insec18the protoxin was stable after a chase; but insec7andsec1the protoxin was unstable, and insec111K 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 insec7andsec1cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18and 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 twokexmutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable inkex1but unstable inkex2. Protoxin stability inkex1 kex2double mutants indicated the orderkex1→kex2in the protoxin processing pathway. TPCK did not block protoxin instability inkex2mutants. This suggested that theKEX1- andKEX2-dependent steps preceded thesec7Golgi block. We attempted to localize the protoxin inS. cerevisiaecells. 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.