Use of beta-lactamase as a secreted reporter of promoter function in yeast.
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.
Cartwright
C P
CP
University of Massachusetts Medical School, Department of Molecular Genetics and Microbiology, Worcester 01655.
Li
Y
Y
Zhu
Y S
YS
Kang
Y S
YS
Tipper
D J
DJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002241:Carbohydrates / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D055646:Killer Factors, Yeast; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000097:blood* / Q000235:genetics; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D010710:Phosphates / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000378:metabolism
D001426:Bacterial Proteins; D002241:Carbohydrates; D005656:Fungal Proteins; C089359:K1 killer toxin; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D010446:Peptide Fragments; D010710:Phosphates; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D001618:beta-Lactamases