Functional and Regulatory Profiling of Energy Metabolism in Fission Yeast

Abstract

Background: The control of energy metabolism is fundamental for cell growth and function and anomalies in it are implicated in complex diseases and ageing. Metabolism in yeast cells can be manipulated by supplying different carbon sources: yeast grown on glucose rapidly proliferates by fermentation, analogous to tumour cells growing by aerobic glycolysis, whereas on non-fermentable carbon sources metabolism shifts towards respiration. Results: We screened deletion libraries of fission yeast to identify over 200 genes required for respiratory growth. Growth media and auxotrophic mutants strongly influenced respiratory metabolism. Most genes uncovered in the mutant screens have not been implicated in respiration in budding yeast. We applied gene-expression profiling approaches to compare steady-state fermentative and respiratory growth and to analyse the dynamic adaptation to respiratory growth. The transcript levels of most genes functioning in energy metabolism pathways are coherently tuned, reflecting anticipated differences in metabolic flows between fermenting and respiring cells. We show that acetyl-CoA synthase, rather than citrate lyase, is essential for acetyl-CoA synthesis in fission yeast. We also investigated the transcriptional response to mitochondrial damage by genetic or chemical perturbations, defining a retrograde response that involves the concerted regulation of distinct groups of nuclear genes that may avert harm from mitochondrial malfunction.

Identifying genes required for respiratory growth of fission yeast

“…The GO term ‘mitochondrion’ showed an enrichment of 37% in the merged list (Figure 1E). Substantial overlaps were also evident with independently identified genes affecting S. pombe growth on glycerol (FYPO: 0001934 PomBase 11 ), as well as with genes resulting from our previous screen using an older version of the Bioneer deletion library (version 2.0; http://www.bioneer.com/) and a different prototroph library 5 (Figure 1F). We also compared our screen hits with orthologous genes that affect respiratory growth in budding yeast ( Saccharomyces genome database; www.yeastgenome.org) , where the genes resulting from the new screen showed a stronger overlap than the genes from our previous screen (Figure 1F).…”

“…We also report quite large variations among independently repeated screens, which highlights the importance to perform biological repeats. The genes implicated in respiration in the previous screen 5 and in the new screen reported here compare as follows: 149 genes have only been identified in the previous screen, 55 genes have been identified in both screens, and 166 genes have only been identified in the new screen. These substantial differences not only reflect the differences in the mutant library and methods (see above), but also the large variations in respiratory-deficient phenotypes, as also observed in S. cerevisiae 2 .…”

“…As we have shown before 5 , the genetic background of the deletion library can strongly affect the screen results. The commonly used auxotroph mutant background has shortcomings for investigating energy metabolism in particular, yet also provides complementary insights.…”

“…We compared respiratory growth on glycerol to fermentative growth on glucose (control) to identify mutants that show defects in respiration. This screen differed in three respects from screens we previously reported 5 : 1) we used the latest version 5 of the gene-deletion library, which features over 400 more mutants (3420 vs 3003) and corrections of erroneous deletions; 2) we generated a prototrophic library of this latest version using an improved protocol that minimizes contamination by wild-type cells and optimizes library quality; 3) we determined colony sizes at densities of only 384 colonies per plate (instead of 1536 colonies), since larger colonies provide a higher dynamic range and sensitivity that outperforms data from higher density screens despite the reduced number of technical repeats; and 4) we applied a different data analysis and only used glycerol as a non-fermentable carbon source, while the previous study also measured respiratory growth on galactose 5 .…”

“…Much of our fundamental knowledge on energy metabolism stems from research with Saccharomyces cerevisiae , including systematic screens for genes involved in cellular respiration 2, 3 , while Schizosaccharomyces pombe provides a potent complementary model system 4 . We have recently applied large-scale functional and regulatory analyses on energy metabolism in fission yeast 5 . The data presented here provide additional, complementary insights into genes that are required for respiration.…”