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Intracellular Targeting and Import of an F1-ATPase ß-subunit-ß-galactosidase Hybrid Protein into Yeast Mitochondria

Douglas, Michael G. and Geller, Bruce L. and Emr, Scott D. (1984) Intracellular Targeting and Import of an F1-ATPase ß-subunit-ß-galactosidase Hybrid Protein into Yeast Mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 81 (13). pp. 3983-3987. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:DOUpnas84

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Abstract

The gene coding for the yeast mitochondrial F1-ATPase ß subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase ß subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, ß-galactosidase (ß-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The ß-subunit-ß -galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid pß Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the pß Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the ß-subunit-ß-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the ß-subunit-ß-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.


Item Type:Article
Additional Information:Copyright © 1984 by the National Academy of Sciences Communicated by Ronald W. Estabrook, March 9, 1984 We are grateful to Marjorie Britten and Mac Biggs for expert technical assistance and to Connie Katz for typing the manuscript. S.D.E. especially thanks R. Schekman for his support and advice and for providing laboratory space to carry out part of this work. This investigation was supported by the National Institutes of Health Grant GM-26713 and Grant AQ-814 from the Robert A. Welch Foundation to M.G.D. and by an award from the Miller Institute for Basic Research in Science of the University of California, Berkeley, to S.D.E. 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.
Subject Keywords:gene fusion; mitochondrial biogenesis; protein import; targeting signals
Record Number:CaltechAUTHORS:DOUpnas84
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:DOUpnas84
Alternative URL:http://www.pnas.org/cgi/content/abstract/81/13/3983
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:5826
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:03 Nov 2006
Last Modified:26 Dec 2012 09:15

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