title: Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development.
abstract: Genomic sequencing has revealed a large number of evolutionary conserved genes of unknown function. In the absence of characterized functional domains, the discovery of the role of these genes must rely on experimental approaches. We have selected 30 Dictyostelium discoideum genes of unknown function that showed high similarity to uncharacterized human genes and were absent in the complete proteomes from Saccharomyces cerevisiae and S. pombe. No putative functional motifs were found in their predicted encoded proteins. Eighteen genes were successfully knocked-out and three of them showed obvious phenotypes. A detailed analysis of one of them, midA, is presented in this report. Disruption of midA in Dictyostelium leads to pleiotropic defects. Cell size, growth rate, phagocytosis and macropinocytosis were affected in the mutant. During development, midA- cells showed an enhanced tendency to remain at the slug stage, and spore viability was compromised. The expression of MidA fused to GFP in midA- strain rescued the phenotype and the fused protein was located in the mitochondria. Although cellular oxygen consumption, mitochondrial content and mitochondrial membrane potential were similar to wild type, the amount of ATP was significantly reduced in the mutant suggesting a mitochondrial dysfunction. Metabolomic analysis by natural-abundance 13C-nuclear magnetic resonance has shown the lack of glycogen accumulation during growth. During starvation, mutant cells accumulated higher levels of ammonia, which inhibited normal development. We hypothesize that the lack of MidA reduces mitochondrial ATP synthetic capacity and this has an impact in some but not all energy-dependent cellular processes. This work exemplifies the potential of Dictyostelium as a model system for functional genomic studies.
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Torija
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Vicente
authors.forename: Tiago B
authors.initials: TB
authors.lastname: Rodrigues
authors.forename: Alicia
authors.initials: A
authors.lastname: Robles
authors.forename: Sebastián
authors.initials: S
authors.lastname: Cerdán
authors.forename: Leandro
authors.initials: L
authors.lastname: Sastre
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Calvo
authors.forename: Ricardo
authors.initials: R
authors.lastname: Escalante
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004272:DNA, Mitochondrial / Q000235:genetics; D004023:Dictyostelium / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D016681:Genome, Fungal; D018503:Genome, Protozoan; D006801:Humans; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins; D015800:Protozoan Proteins