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An MF α1-SUC2 (α-factor-invertase) gene fusion for study of protein localization and gene expression in yeast

Emr, Scott D. and Schekman, Randy and Flessel, Monica C. and Thorner, Jeremy (1983) An MF α1-SUC2 (α-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proceedings of the National Academy of Sciences of the United States of America, 80 (23). pp. 7080-7084. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:20120709-104828314

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Abstract

The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.


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http://www.pnas.org/content/80/23/7080.abstractPublisherUNSPECIFIED
Additional Information:© 1983 National Academy of Sciences. Communicated by Jesse C. Rabinowitz, August 1, 1983. We are extremely grateful for the generosity and cooperation of both Marian Carlson and Tony Brake, who freely provided plasmids, strains, and advice. The work was supported by an award from the Miller Institute for Basic Research in Science of the University of California, Berkeley, to S.D.E., by National Institutes of Health Research Grant GM26755 to R.S., by U.S. Public Health Service Predoctoral Traineeship GM07232 to M.C.F., and by National Institutes of Health Research Grant GM21841 to J.T. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S.C. §1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
University of California Berkeley Miller Institute for Basic Research in Science UNSPECIFIED
NIHGM26755
U.S. Public Health Service Predoctoral TraineeshipGM07232
NIHGM21841
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Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120709-104828314
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ID Code:32307
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:09 Jul 2012 20:08
Last Modified:25 Mar 2014 17:19

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