Two criteria suggest that most of the proteinase of
Streptococcus lactis
is localized in the cell wall. (i) Intact cells possess proteinase activity when incubated with a high-molecular-weight substrate. (ii) Most of the cell-bound proteinase activity is released during spheroplast formation under conditions which result in the release of only 1% of the intracellular enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase. The solubilized cell wall, plasma membrane, and cytoplasm fractions contained 84, 0, and 16%, respectively, of the total proteinase activity with casein as substrate. The physiological role of a surface-bound proteinase in this organism is discussed.
Gene fusions were constructed between a yeast expression plasmid and a Cellulomonas fimi DNA fragment encoding an endo-1,4-beta-D-glucanase or carboxymethylcellulase. Yeast transformed with the recombinant plasmids secreted carboxymethylcellulase activity. Secretion of active enzyme was greatly increased when the leader of a secreted yeast protein, the Kl toxin, was inserted immediately upstream of and in frame with the bacterial cellulase sequence. This is the first step in constructing a functional cellulase complex in Saccharomyces cerevisiae. It also provides an excellent system for the detailed examination of the determinants of protein secretion because of the ease with which secreted cellulase can be detected.
Complementary DNA (cDNA) copies of the M1‐1, toxin‐coding region of the yeast M1 double‐stranded RNA (dsRNA) have been cloned and sequenced. These sequences, in combination with the known terminal sequence of M1‐1 dsRNA, identify a translation reading frame for a 316 amino acid protein of 34.7 kd, similar in size to the preprotoxin produced from M1 dsRNA by in vitro translation. Potential glycosylation sites in the preprotoxin peptide are identified. Based on its methionine content the extracellular yeast toxin appears to be contained within the C‐terminal region of the precursor.
Two cDNA clones of the segment of Saccharomyces cerevisiae M1 double‐stranded RNA, which codes for the yeast killer toxin, have been expressed in yeast using the expression vector pYT760. Toxin expression and secretion depended upon the presence of a yeast promoter. Transformants not only contain an authentic preprotoxin precursor, as determined by precipitation of intracellular proteins with antitoxin antisera, but also display an immunity phenotype. The evidence is that the immunity protein is part of the preprotoxin and may act by masking toxin binding sites. Neither cDNA clone had a complete 5′ terminus and the preprotoxin translational start was missing. The promoter and the initiator ATG were supplied by the expression vector. One clone with a full‐length preprotoxin but altered N‐terminal amino acids gave a normal glycosylated intracellular precursor. A clone with an N‐terminal nine amino acid deletion gave a precursor which was not glycosylated but toxin was still secreted.
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