Relocation of the unusualVAR1 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.

Yeast PPR proteins, watchdogs of mitochondrial gene expression

“…38 In this screen, the Var1 ribosomal protein was encoded in the nucleus by a relocated construct producing a mitochondrially targeted version of Var1 that could replace the endogenous mitochondrial Var1 and maintain mitochondrial translation. 39 However, the sov1 mutants were not only unable to synthesize Arg8 m from the VAR1 5' UTR, they also failed to accumulate the chimeric VAR1::ARG8 m mRNA as well as the wild-type VAR1 mt-mRNA. In these strains, the tRNA Ser -VAR1 precursor level was wild-type, so Sov1 appears to be required for stability of the mature VAR1 mt-mRNA rather than processing of the precursor.…”

Translational control of endogenous and recoded nuclear genes in yeast mitochondria: Regulation and membrane targeting

“…If there are translational activators specific for the VAR1 mRNA, then mutations in their genes would have gone undetected in screens for nuclear mutants, since blocking Varlp synthesis would destroy mitochondrial protein synthesis entirely, leading to instability of rho + mtDNA [52]. However, a gene encoding Varlp in the universal genetic code has recently been synthesized [53]. Expression of this gene, VAR1 u, in the nucleus provides a cytoplasmically synthesized protein that is imported into mitochondria where it functions in mitochondrial ribosomes.…”

Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity?

“…Additional fungal taxa exist that also possess only this one code disparity but have not transferred A9; however, this cannot be considered contrary to the code disparity hypothesis because A9 is evidently rather hard to transfer for some other reason (such as hydrophobicity, addressed below), being mtDNA-encoded in all species that retain more than 15 assigned, non-endonuclease, proteincoding genes and having resisted functional transfer in S. cerevisiae in the laboratory. (28) RpS3 was apparently the (29) and S. pombe (30) after code correction, so should be absent from the mtDNA in any fungal lineage that (for whatever reason) loses this codedisparity barrier. Evidence available to date is consistent with the hypothesis.…”