We have previously proposed that a single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24) has been substituted, yet the protein is processed efficiently and retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.
COOL Cloning
Insertion of short DNA sequences into plasmids is a widely applied technique, but it can be complicated by lack of precision and the need to screen and separate clones in order to identify the insert of interest. Blachinsky et al. (p. 933) describe a straightforward procedure for introducing oligonucleotides into plasmids in the desired number, orientation, and order. The protocol entails the sequential insertion of sequences in separate ligation reactions, each of which restores the original restriction sites of the plasmid. The procedure requires only basic cloning skills, can be used with any combination of restriction enzymes, is applicable to inserts of any length (with some caveats), and, although demonstrated here in yeast, can be used in any system. The authors dub their method Controlled and Ordered Oligonucleotides Ligations (COOL).
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