Rewiring phospholipid biosynthesis reveals robustness in membrane homeostasis and uncovers lipid regulatory players
Intracellular transport of lipids by Lipid Transport Protein (LTP) works alongside vesicular transport to shuttle lipids from their place of synthesis to their destinations. Whereas many LTPs have been identified, it is largely unknown which routes and which LTPs a given lipid utilizes to navigate the multiple membranes of eukaryotic cells. The major and essential phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC) can be produced by multiple pathways and, in the case of PE, also at multiple locations. Here, we present an approach in which we simplify and rewire yeast phospholipid synthesis by redirecting PE and PC synthesis reactions to distinct subcellular locations using chimeric enzymes fused to specific organelle targeting motifs. In rewired conditions, viability is expected to depend on homeostatic adaptation to the ensuing lipostatic perturbations and on efficient interorganelle lipid transport. We therefore performed genetic screens to identify factors involved in both of these processes. The set of genes that we identified is enriched for functions linked to transcriptional regulation of lipid homeostasis, lipid metabolism and transport. In particular, we identify a requirement for Csf1 –an uncharacterized protein harboring an N-chorein lipid transport domain- for survival under certain rewired conditions as well as lipidomic adaptation to cold, implicating it in interorganelle lipid transport and homeostatic adaptation.
This webpage displays interactive plots to browse transposon-based genetic screens on yeast strains with rewired phospholipid synthesis (Fig 1).
Fig 1. A) Schematic depicting the topology of the CDP-DAG -and Kennedy (red) pathways producing PE and PC in wt yeast cells (left panel), or one example of a rewired condition in which PE synthesis is redirected to the MIM and PC synthesis is redirected to peroxisomes (pex) (right panel). Black arrows indicate PS (dashed) and PE (dotted) lipid transport that must occur to enable the sequential enzymatic reactions. B) Outline of the screening procedure. Cells expressing plasmids containing the PE and PC synthesizing enzymes and a plasmid containing the galactose inducible transposase (TPase) and a transposon (TN) disrupting the TRP1 gene were grown for ~56h in galactose-containing medium to induce transposition (step 1). Cells were inoculated in synthetic medium lacking tryptophan in either KennedyON or OFF conditions, grown for several generations (step 2) and harvested for DNA extraction and sequencing of transposon insertion sites (TNs) (step 3). TNs are mapped to the genome to identify genes that become required under certain rewired conditions (step 4). An illustrative example of TN insertions of four libraries visualized in the UCSC genome browser highlights a genetic requirement for DRS2 (green bar) in KennedyOFF conditions. Volcano plot analysis can identify genes that are specifically required or dispensable in a certain test condition with respect to a set of reference conditions (step 5).
Transposon mutagenesis screens have been generated either in KennedyON conditions (supplemented with 10 mM ethanolamine and 10 mM choline) or KennedyOFF conditions in the following ‘rewired’ yeast strains:
The webpage displays two types of analyses.
- Correlation clusterogramHigh correlation and clustering of the transposon insertion profiles of genes may indicate functional relationships between the genes and aid in the identification of gene groups that underlie lipid trafficking and work together to handle lipid imbalances.
- Volcano plot analysisVolcano plots identify genes that are specifically required (devoid for transposons/reads) or dispensable (enriched for transposons/reads) in a certain test condition with respect to a set of reference conditions.
- One rewired combination versus all other rewired combinations using either:
- KennedyON and OFF conditions of one rewired combination combined (test set) versus all other rewired combinations (KennedyON and OFF all, reference set).
- The KennedyON condition of one rewired combination (test set) versus other rewired combinations (KennedyON and OFF all, reference set).
- The KennedyOFF condition of one rewired combination (test set) versus all other rewired combinations (KennedyON and OFF all, reference set).
- Pooled conditions with enzymes expressed at a certain organelle (e.g. all Pmt-MIM libraries, test set) versus all other rewired conditions (reference set).
- Rewired conditions grown in KennedyOFF (test set) versus rewired conditions grown in KennedyON conditions (reference set).