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Use of β‐lactamase as a secreted reporter of promoter function in yeast
Author(s) -
Cartwright Charles P.,
Li Yu,
Zhu YunSong,
Kang YoonSe,
Tipper Donald J.
Publication year - 1994
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.320100409
Subject(s) - biology , yeast , function (biology) , reporter gene , microbiology and biotechnology , genetics , gene , gene expression
K 1 preprotoxin is the 316 residue precursor of the K 1 killer toxin secreted by the yeast Saccharomyces cerevisiae . The SPβla reporter consists of the mature, secreted form of β‐lactamase (βla) 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 SPβla in yeast results in efficient secretion, processing by signal peptidase and glycosylation in the endoplasmic reticulum, producing proßla. Kex2 cleavage of proßla in the lumen of a late Golgi compartment releases βla, which accumulates stably in culture media buffered at pH 5·8–7. The half‐life of secretion is 11 min at 30°C; 10–12% of the total activity in exponential‐phase cells is intracellular, mostly in the form of proßla, indicating that transit from the endoplasmic reticulum to the Golgi is rate limiting. We have used SPβla 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 βla over a 40‐fold range and up to several μg/ml was proportional to transcript level, demonstrating that SPβla can be employed as a convenient secreted reporter of promoter function in yeast.