ABSTRACT
When overexpressed in Saccharomyces cerevisiae, beta-galactosidase fusion proteins directed to the mitochondria are toxic, preventing growth of yeast cells on non-fermentable carbon sources (Emr, S. D., Vassarotti, A., Garrett, J., Geller, B. L., Takeda, M., and Douglas, M. G. (1986) J. Cell Biol. 102, 523-533). We show that such fusion proteins interfere with the assembly of respiratory complexes in the mitochondrial inner membrane, without blocking protein translocation. The gene YME1, encoding an ATP-dependent metalloprotease of the mitochondrial inner membrane, acts as a suppressor of this defect; a 3-fold overexpression of Yme1p is sufficient to restore respiratory complex assembly and mitochondrial function. Detailed knowledge of the topology and effect of the toxic beta-galactosidase fusion proteins will permit the identification and characterization of components that control protein sorting and protein assembly within the mitochondrial inner membrane.
Subject(s)
Intracellular Membranes/drug effects , Mitochondria/drug effects , Proton-Translocating ATPases/toxicity , Recombinant Fusion Proteins/toxicity , Saccharomyces cerevisiae Proteins , beta-Galactosidase/toxicity , ATP-Dependent Proteases , Adenosine Triphosphatases , Biological Transport , Fungal Proteins/biosynthesis , Macromolecular Substances , Oxygen Consumption/drug effects , Proton-Translocating ATPases/genetics , Saccharomyces cerevisiae , Suppression, Genetic , beta-Galactosidase/geneticsABSTRACT
The yeast plasma membrane ATPase gene PMA1 was cloned into Escherichia coli using the high expression tac and T7 promoters. The gene product is toxic to the bacterial cell leading to very low expression levels and arrested growth of the host cell within minutes of induction. The expressed protein is immunologically cross-reactive with the yeast ATPase, comigrates with the original protein in sodium dodecyl sulfate-polyacrylamide gels, and is isolated in the E. coli membrane fraction. The partially purified protein exhibits ATPase activity.
