A SINGLE BASE-PAIR CHANGE (ATG-]ATC) NULLIFIES THE ACTIVITY OF CYTOSOLIC FUMARASE IN SACCHAROMYCES-CEREVISIAE

A respiratory defective pet mutant (W303 del FUM1) of Saccharomyces cerevisiae deficient in fumarase was transformed with the plasmid construct pG5/ST7. This plasmid contains the entire FUM1 gene with only a single base pair change (ATG --> ATC) confined to the putative second in-frame translation initiation codon. While transformation of the fumarase deficient mutant with pG5/ST7 resulted in an elevation of fumarase activity in the mitochondria of the transformed strain, fumarase activity in the cytosol remained negligible. This result indicated that the cytosolic fumarase isoenzyme is synthesized exclusively from the second translation initiation codon of FUM1. Results of RACE-PCR of the 5' ends of FUM1 transcripts confirmed that the FUM1 gene synthesizes two distinct species of transcripts. These data provide strong evidence for the synthesis and targeting of two fumarase isomers. (C) 1995 Academic Press, Inc.