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In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named  TAR1  for  T ranscript  A ntisense to  R ibosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the  authentic  product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the  Saccharomyces cerevisiae TAR1  product, we detected the endogenous Tar1p polypeptides in  S. cerevisiae  ( Sc ) and  Kluyveromyces lactis  ( K l) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous  Sc  Tar1p protein sub-localizes in the inner membrane in a N in -C out  topology. Plasmid-versions of 5′ end or 3′ end truncated  TAR1  ORF were used to demonstrate that neither the N-terminus nor the C-terminus of  Sc  Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous  Sc  Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous  Sc TAR1  transcripts were also found low abundant and consistently 5′ flanking regions of  TAR1  ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous  Sc TAR1  transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene  TAR1  constitutes a functional transcription unit within the nuclear rDNA repeats.

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins