Saccharomyces cerevisiae is facultative aerobic, and respiratory mutations can be promptly identified on media containing non-fermentable carbon sources.  There are, approximately 500 proteins located in yeast mitochondria, and most of them feature a typical N-terminal addressing sequence. In order to identify new yeast genes involved in respiration we performed a whole genome searches using algorithms that analyses the yeast open reading frames (ORFs) N-terminal sequence. This "in silico" analysis generated a list of one hundred ORFs coding for putative mitochondrial proteins, which were not yet studied. In this ORFs list is present YPR116W, which codes for a protein of 277 aminoacids with homologues only in other fungi. After raising antibodies against Ypr116wp we were able to detect its presence in the mitochondrial inner membrane, facing the matrix side. A strain containing a null mutation of YPR116W is incapable of growing on glycerol and ethanol as carbon sources, indicating a respiratory function of Ypr116wp. Interestingly, the null mutant cannot maintain the mitochondrial DNA (mtDNA), which can be explained due to a wide range of problems, such as a direct mtDNA replication failure, general mitochondrial translation defect, or even an improper ATP synthase assembly.  In any case, the mtDNA instability argues in favor of a direct role of Ypr116wp on respiratory capacity, but difficult a straightforward study of its fuction. Therefore, in order to characterize YPR116W we had to obtained conditional alleles of YPR116W sensitive to temperature, that can maintain mtDNA in a permissive temperature.  We isolated with success an allele with four mutations: E82G, D113G, F150I and N251K and the strain harboring this allele is being used to search for genetic suppressors of YPR116W null mutant phenotype. Two sub-cloned alleles of YPR116W were also constructed. In the first sub-cloning it was removed site the 39 C-terminal residues of the protein, and in the second an internal KpnI fragment coding for 18 residues was removed. Both sub-cloned alleles constructions restore the respiratory defect of the null mutant, even when present in single copy. The specific function of Ypr116wp still has to be elucidated but sequence comparison studies indicates that it might be a FAD binding protein, and we are going to verify it with the purification of Ypr116wp with a "his tag" which it was already constructed and showed to be functional.

Supported by FAPESP,  CNPq