Functional genomics inDictyostelium: 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.

Vacuole membrane protein 1, autophagy and much more

“…Vmp1, as explained above, complies with all these characteristics and was selected from a collection of mutants generated in Dictyostelium by our group. 13,15 This is the first lossof-function mutation of Vmp1 analyzed in a model system and our studies reveal unexpected functions for this new protein beyond autophagy.…”

“…Fortunately, the Dictyostelium genome contains many of those genes, being the simplest model system that can be used to address their function. 13,14 Some of these genes do not allow a prediction of their function due to the lack of characterized functional motifs. Vmp1, as explained above, complies with all these characteristics and was selected from a collection of mutants generated in Dictyostelium by our group.…”

MidA is a putative methyltransferase that is required for mitochondrial complex I function

“…The social amoeba Dictyostelium discoideum is a useful model for the study of biological issues that are relevant to human disease, including mitochondrial dysfunction (Annesley and Fisher, 2009a;Barth et al, 2007;Bokko et al, 2007;Chida et al, 2004;Kotsifas et al, 2002;Torija et al, 2006b;Williams et al, 2006). Dictyostelium cells feed on bacteria and remain in the form of individual cells while food is present.…”

“…Recently, we described the identification and initial characterization of a new mitochondrial protein conserved between Dictyostelium and humans that we named MidA (for mitochondrial dysfunction protein A) (Torija et al, 2006a;Torija et al, 2006b). This protein belongs to an uncharacterized conserved protein family (DUF185 or COG1565).…”

Legionella pneumophilamultiplication is enhanced by chronic AMPK signalling in mitochondrially diseased Dictyostelium cells

“…However, the mechanisms that underlie such increased susceptibility have not been studied and are not understood. Here, we use the established Dictyostelium discoideum model for mitochondrial disease (Wilczynska et al, 1997;Kotsifas et al, 2002;Chida et al, 2004;Torija et al, 2006;Barth et al, 2007) and for Legionella pneumophila pathogenesis (Hägele et al, 2000;Solomon et al, 2000;Skriwan et al, 2002;Hilbi et al, 2007) to show that intracellular proliferation of the bacterial pathogen is supported better by mitochondrially diseased cells than by healthy cells.…”