Relocation of the unusual VAR1 gene from the mitochondrion to the nucleus.

Abstract

The Var1 protein (Var1p) is an essential, stoichiometric component of the yeast mitochondrial small ribosomal subunit, and it is the only major protein product of the mitochondrial genetic system that is not part of an energy transducing complex of the inner membrane. Interestingly, no mutations have been reported that affect the function of Var1p, presumably because loss of a functional mitochondrial translation system leads to an instability of mtDNA. To study the structure, function and synthesis of Var1p, we have engineered yeast strains for the expression of this protein from a nuclear gene, VAR1U, in which 39 nonstandard mitochondrial codons were converted to the universal code. Immunoblot analysis using an epitope-tagged form of Var1Up showed that the nuclear-encoded protein was expressed and imported into the mitochondria. VAR1U was tested for its ability to complement a mutation in mtDNA, PZ206, which disrupts '3-end processing of the VARI mRNA, causing greatly reduced synthesis of Var1p and a respiratory-deficient phenotype. Respiratory growth was restored in PZ206 mutants by transformation with a centromere plasmid carrying VAR1U under ADH1 promoter control, thus proving that VAR1 function can be relocated from the mitochondrion to the nucleus. Moreover, epitope-tagged Var1Up co-sedimented specifically with small ribosomal subunits in high salt sucrose gradients. The relocation of VAR1 from the mitochondrion to the nucleus provides an excellent system for the molecular genetic analysis of structure-function relationships in the unusual Var1 protein.