K1 preprotoxin is the 316 residue precursor of the K1 killer toxin secreted by the yeast Saccharomyces cerevisiae. The SP beta la reporter consists of the mature, secreted form of beta-lactamase (beta la) fused to S and P, two fragments of preprotoxin. S is the N-terminal 34 residues, including the secretion signal. P, a 67 residue 'processing' segment with three sites for N-glycosylation, terminates in a Lys Arg site for cleavage by the Kex2 protease. Expression of SP beta 1a in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing pro beta la. Kex2 cleavage of pro beta la in the lumen of a late Golgi compartment releases beta la, which accumulates stably in culture media buffered at pH 5.8-7. The half-life of secretion is 11 min at 30 degrees C; 10-12% of the total activity in exponential-phase cells is intracellular, mostly in the form of pro beta la, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting. We have used SP beta la expression in single- and multi-copy vectors to compare the PGK, GAL1, GAL10, PHO5 and CUP1 promoters under varying nutritional conditions. In exponential-phase cells, secretion of beta la over a 40-fold range and up to several micrograms/ml was proportional to transcript level, demonstrating that SP beta la can be employed as a convenient secreted reporter of promoter function in yeast.

• PMID: 7941736
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Journal Article | Research Support, U.S. Gov't, Non-P.H.S. | Research Support, U.S. Gov't, P.H.S.

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Cartwright CP, Li Y, Zhu YS, Kang YS, Tipper DJ

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