Starch degradation in chloroplasts requires b-amylase (BAM) activity, which is encoded by a multigene family. Of nine Arabidopsis (Arabidopsis thaliana) BAM genes, six encode plastidic enzymes, but only four of these are catalytically active. In vegetative plants, BAM1 acts during the day in guard cells, whereas BAM3 is the dominant activity in mesophyll cells at night. Plastidic BAMs have been difficult to assay in leaf extracts, in part because of a cytosolic activity encoded by BAM5. We generated a series of double mutants lacking BAM5 and each of the active plastidic enzymes (BAM1, BAM2, BAM3, and BAM6) and found that most of the plastidic activity in 5-week-old plants was encoded by BAM1 and BAM3. Both of these activities were relatively constant during the day and the night. Analysis of leaf extracts from double mutants and purified BAM1 and BAM3 proteins revealed that these proteins have distinct properties. Using soluble starch as the substrate, BAM1 and BAM3 had optimum activity at pH 6.0 to 6.5, but at high pH, BAM1 was more active than BAM3, consistent with its known daytime role in the guard cell stroma. The optimum temperature for BAM1, which is transcriptionally induced by heat stress, was about 10°C higher than that of BAM3, which is transcriptionally induced by cold stress. The amino acid composition of BAM1 and BAM3 orthologs reflected differences that are consistent with known adaptations of proteins from heat-and coldadapted organisms, suggesting that these day-and night-active enzymes have undergone thermal adaptation.

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8—an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid essential for many core metabolic processes in mitochondria, including oxidative phosphorylation1-3. While lesser appreciated, CoQ also serves as a key membrane-embedded antioxidant throughout the cell4. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of yeast genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally regulate this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids (PUFAs), whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8—an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal new protein machinery central to CoQ trafficking in yeast and lend new insights into the broader interplay between mitochondrial and cellular processes.