Deletion of the nuclear gene encoding the mitochondrial citrate transport protein from Saccharomyces cerevisiae.
The nuclear gene encoding the mitochondrial citrate transport protein (i.e., CTP1) has been deleted from a haploid yeast strain. The stable yeast deletion strain was constructed by homologous recombination of the HIS3 gene at the CTP1 gene locus. Deletion of the CTP was confirmed by PCR. Immunoblot analysis provided the first quantitative estimate of the level of the CTP in wild-type yeast mitochondria and indicated the absence of expressed CTP in mitochondria isolated from the deletion strain. Deletion of CTP1 did not lead to a phenotype on any carbon source tested, indicating that CTP1 is not an essential gene. This suggests that either known alternative pathways are able to produce sufficient acetyl-CoA to support biosynthetic reactions, or there exists a second CTP gene. The ability of the deletion strain to serve as a host for the correct targeting and overexpression of a mutated CTP was then demonstrated. These studies provide a system which permits the use of site-directed mutagenesis to examine both CTP targeting to mitochondria, as well as the molecular basis underlying CTP function. Moreover, this system will not only facilitate the study of the yeast CTP, but also CTPs expressed from the cDNAs of higher eukaryotes.
Kaplan
R S
RS
Department of Pharmacology, University of South Alabama, Mobile 36688, USA.
Mayor
J A
JA
Kakhniashvili
D
D
Gremse
D A
DA
Wood
D O
DO
Nelson
D R
DR
D002352:Carrier Proteins / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006238:Haploidy; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D002352:Carrier Proteins; C020723:citrate-binding transport protein