Ishino, Y. | bioRxiv Science

bioRxiv2021

Transcriptional regulation of sphingolipid metabolism in budding yeast

Global control for the synthesis of lipids constituting a bilayer of cell membranes is known to be with a small number of transcription factors called master transcriptional regulators, which target a wide range of genes encoding lipid metabolism enzymes and/or their regulators. Although master transcriptional regulators of glycerophospholipids and sterols have been identified in both yeast and mammals, this aspect of sphingolipid metabolism is not yet understood. In the present study, we identified the C2H2-type zinc finger transcription factor, Com2, as a master transcriptional regulator of sphingolipid metabolism in the budding yeast, Saccharomyces cerevisiae. The target of rapamycin complex 2 (TORC2)-activated protein kinase Ypk1 is known to regulate sphingolipid metabolism. Activated Ypk1 stimulates the activity of serine palmitoyl transferase (SPT), the first-step enzyme in sphingolipid biosynthesis, by phosphorylating and inhibiting Orm1/2, a negative regulator of SPT. This regulation of SPT activity is thought to be a major pathway in the regulation of sphingolipid metabolism. In the present study, we found that inhibition of sphingolipid synthesis upregulates the expression of Com2, which in turn leads to the concomitant expression of Ypk1. The upregulation of Ypk1 expression was found to be dependent on a putative Com2-binding site in the YPK1 promoter. Our results also suggested that Com2 senses intracellular sphingolipid levels through a pathway independent of TORC2-Ypk1-mediated sensing of sphingolipids. Our results revealed an additional layer of mechanistic regulation that allows cells to maintain appropriate levels of sphingolipid biosynthesis and to rapidly induce this process in response to environmental stresses. Significance StatementOne of the major regulatory mechanisms involved in the control of lipid metabolism in bilayers of biological membranes is regulation at the transcriptional level by master transcriptional regulators that control the transcription of genes encoding lipid metabolism enzymes and/or their regulators. In the present study, we identified the C2H2-type zinc finger transcription factor Com2 as a master transcriptional regulator in sphingolipid metabolism. We found that Com2 regulates sphingolipid metabolism by transcriptionally controlling the expression of Ypk1, which regulates Orm1/2, a negative regulator of serine palmitoyl transferase, the first-step enzyme in sphingolipid biosynthesis, through phosphorylation. Our study revealed a new layer of regulation that allows the maintenance of an appropriate level of sphingolipid biosynthesis for a rapid response to environmental stresses.

cell biology↗

bioRxiv2021

A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

B-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3-5 exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.

microbiology↗