GENETIC-ANALYSIS OF YEAST ISO-1-CYTOCHROME-C STRUCTURAL REQUIREMENTS - SUPPRESSION OF GLY6 REPLACEMENTS BY AN ASN52-]ILE REPLACEMENT

Gly6 (vertebrate numbering system) is an evolutionarily invariant amino acid located in an electron-dense region of cytochrome c. Serine, cysteine, and aspartic acid replacements of Gly6 abolished yeast iso-1-cytochrome c function, presumably by destabilizing the mature forms of the altered proteins (1). Here we report that genetic reversion analysis of these mutants has uncovered a single base-pair substitution, encoding an Asn52 → Ile replacement, that suppresses all three position 6 defects, as well as a Gly6 ... Gly29 → Ser6 ... Ser29 double replacement. In each case the suppressor restored at least partial function to the altered iso-1-cytochromes c, with the Ser6 ... Ile52 protein being nearly indistinguishable from the normal protein. The suppressor also affected otherwise normal iso-1-cytochrome c, enhancing the in vivo amount of the protein by about 20%. While this work was in progress, Das et al. (1989, Proc. Natl. Acad. Sci. USA 86, 496-499) uncovered Ile52 as a suppressor of single Gly29 and His33 replacements in iso-1-cytochrome c. The ability of IIe52 to suppress amino acid replacements at three different sites, and its effect in isolation from the primary mutations, defines Ile52 as a global suppressor of specific iso-1-cytochrome c structural defects. These data suggest that position 52 plays a critical role in the folding and/ or stability of iso- 1-cytochrome c. © 1991.