title: Activity of mitochondrially synthesized reporter proteins is lower than that of imported proteins and is increased by lowering cAMP in glucose-grown Saccharomyces cerevisiae cells.
abstract: We selected for increased phenotypic expression of a synthetic cox2::arg8m-G66S reporter gene inserted into Saccharomyces cerevisiae mtDNA in place of COX2. Recessive mutations in ras2 and cyr1, as well as elevated dosage of PDE2, allowed cox2::arg8m-G66S to support Arg prototrophy. Each of these genetic alterations should decrease cellular cAMP levels. The resulting signal was transduced through redundant action of the three cAMP-dependent protein kinases, TPK1, TPK2, and TPK3. ras2 had little or no effect on the level of wild-type Arg8p encoded by cox2::ARG8m, but did increase Arg8p activity, as judged by growth phenotype. ras2 also caused increased fluorescence in cells carrying the synthetic cox3::GFPm reporter in mtDNA, but had little effect on the steady-state level of GFP polypeptide detected immunologically. Thus, decreased cAMP levels did not affect the synthesis of mitochondrially coded protein reporters in glucose-grown cells, but rather elevated activities in the matrix that promote efficient folding. Furthermore, we show that when Arg8p is synthesized in the cytoplasm and imported into mitochondria, it has greater activity than when it is synthesized in the matrix. Thus, mitochondrially synthesized proteins may not have the same access to matrix chaperones as cytoplasmically synthesized proteins emerging from the import apparatus.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Demlow
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Fox
mesh_terms: D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism*; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D005656:Fungal Proteins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose