Functional characterization of phosphatidylinositol transfer proteins in yeast and Arabidopsis and their potential use to increase aluminum tolerance in plants
Zusammenfassung der Projektergebnisse
Our work has shown novel insights into the mechanism of lipid regulation by SEC14 proteins. A directed evolution approach has enabled us to obtain ‘gain of function’ alleles of SEC14 hommolog SFH1 for which the mechanism of phospholipid (PL) exchange could be addressed in detail. A combination of high resolution crystallography, molecular dynamics simulations and functional analyses has helped us to identify transient interactions that are required for conformational transitions and protein function. With this approach we have been able to identify substructures that propagate conformational energy from within the hydrophobic pocket to a helical substructure which gates access to the pocket. To our knowledge these findings provide the most detailed description so far of the mechanics of how SEC14 proteins mediate lipid exchange. In the context of this work we have also been able to solve high resolutions structures for the first true apo-SEC14 and novel SFH1* activation mutants without or in complex with various phospholipids that we hope will provide an even better understanding of Sec14-mediated lipid exchange in future work. We have also made substantial progress on the question of how AtSFH5 and SFH1 mediate Altolerance in yeast. A combination of structural, lipidomic and genetic analyses suggest PtdEtn binding is critical for AtSFH5/SFH1-mediated Al-tolerance in yeast. Our lipidomic analyses also show that inositol phosphoryl ceramid (IPC) and mannosyl-IPC (MIPC) are robustly increased in SFH1-expressing cells and that an increase in mannosyl-IPC (MIPC) increases Al-tolerance independent of SFH1 suggesting a causal relation between SFH1-mediated Al-tolerance and the increase in complex sphingolipids. Our genome wide interaction screen also revealed that the cell wall integrity pathway (CWI) is required for SFH1-mediated Al-tolerance. In this screen we have identified 3 strong CWI-related candidate yeast strains (rom2Δ; wsc1Δ; fpk1Δ) in which SFH1 is not able to mediate Altolerance and three weaker CWI-related candidates (mid2Δ, tus1Δ and sac7Δ) where ectopic expression of SFH1 only results in a mild increase in Al-tolerance. We could furthermore show that ectopic expression of the cell wall receptor WSC1 (the gene deleted in one of the CWI candidates) increases Al-tolerance independent of SFH1 further suggesting that this pathway is critically involved in Al-tolerance. A third line of evidence that SFH1 regulates this pathway comes from the observation that ectopic expression of SFH1 also rescues stt4ts-associated growth defects at restrictive temperatures (independent of Al). STT4 is a membrane localized PtdIns 4-OH kinase that provides the substrate PtdIns(4)P for the kinase MSS4 which is the only kinase in yeast producing PtdIns(4,5)P2. PtdIns(4,5)P2 has been shown to be a potent activator of the CWI pathway by recruitment of the GDP/GTP exchange factor ROM2 (via its pleckstrin homology domain) to the plasma membrane. Unexpectedly, SFH1 - albeit complementing stt4ts-associated growth defects - does not increase PtdIns(4)P or PtdIns(4,5)P2 levels. Our current hypothesis is that Al leads to a growth arrest in yeast by blocking the CWI pathway (most likely by binding to PtdIns(4,5)P2) and that SFH1 rescues this defects by activating the synthesis of either a transient pool of PtdIns(4,5)P2 and/or sphingolipids that bypass the requirement for phosphoinositides in activating the CWI pathway. We further hypothesize that similar to SEC14, SFH1 mediates a heterotypic lipid exchange during which one of the lipids becomes accessible to enzymatic modification. Preliminary evidence suggests that PtdEtn or PtdCho most likely serves as a counter lipid for efficient presentation of either PtdIns4P or/and the not yet identified sphingolipid precursor. We think our observations in yeast have not only important implications for our understanding of the function of Sec14 proteins but also for our current model of phosphoinositide signaling. The identification of SFH1-independent strategies to overcome Al-toxicity in yeast (ectopic expression of WSC1 and increase in specific sphingolipids) provides us with further tools to attempt increasing Altolerance in plants that we can now test in detail. These analyses will hopefully also provide a better understanding of phosphoinositide signaling and the interplay with sphingolipids in plants. We have furthermore characterized novel Sec14 inhibitors as potential future antifungal drugs and have described the biosynthesis and functional role of inositol pyrophosphates in Arabidopsis.
Projektbezogene Publikationen (Auswahl)
- (2010). Sphingolipid metabolism in trans-Golgi/endosomal membranes and the regulation of intracellular homeostatic processes in eukaryotic cells. Adv Enzyme Regul 50(1):339-48
Mousley CJ, Trettin KD, Tyeryar K, Ile KE, Schaaf G, Bankaitis VA
- (2010). The Sec14 superfamily and mechanisms for crosstalk between lipid metabolism and lipid signaling. Trends Biochem Sci, 35: 150-60
Bankaitis VA, Mousley CJ, Schaaf G
- (2011). A blueprint for functional engineering: Single point mutations reconstitute phosphatidylinositol presentation in a pseudo-Sec14 protein. Commun Integr Biol, 4:674-8
Winklbauer EM, de Campos MKF, Dynowski D, and Schaaf G
- (2011). Crystallization and Preliminary X-ray Diffraction Analysis of Sfh3, a Member of the Sec14-protein Superfamily. Acta Cryst F67:1239-1243
Ren J, Schaaf G, Bankaitis VA, Ortlund EA, and Pathak MC
- (2011). Resurrection of a functional phosphatidylinositol transfer protein from a pseudo-Sec14 scaffold by directed evolution. Mol Biol Cell 22:892-905
Schaaf G, Dynowski D, Mousley CJ, Shah SD, Yuan P, Winklbauer EM, de Campos MKF, Trettin K, Quinones MC, Smirnova T, Yanagisawa LL, Ortlund EA, and Bankaitis VA
- (2012). Thoughts on Sec14-like nanoreactors and phosphoinositide signaling. Adv Biol Regul, 52:115-21
Bankaitis VA, Ile KE, Nile AH, Ren J, Ghosh R, Schaaf G
- (2015). Pro-Metabolites of 5-diphospho-myo-inositol pentakisphosphate. Angew Chem Int Ed 54, 9622-6
Pavlovic I, Thakor DT, Bigler L, Wilson MSC, Laha D, Schaaf G, Saiardi A, Jessen HJ
(Siehe online unter https://doi.org/10.1002/anie.201503094) - (2015). Sec14-Nodulin Proteins and the Patterning of Phosphoinositide Landmarks for Developmental Control of Membrane Morphogenesis. Mol Biol Cell 26, 1764-81
Ghosh R, de Campos MK, Huang J, Huh SK, Orlowski A, Yang Y, Tripathi A, Nile A, Lee HC, Dynowski M, Schäfer H, Róg T, Lete MG, Ahyayauch H, Alonso A, Vattulainen I, Igumenova TI, Schaaf G, Bankaitis VA
(Siehe online unter https://doi.org/10.1091/mbc.E14-10-1475) - (2015). VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis. Plant Cell 27, 1082-97
Laha D, Johnen P, Azevedo C, Dynowski M, Weiß M, Capolicchio S, Mao H, Iven T, Steenbergen M, Freyer M, Gaugler P, de Campos MKF, Zheng N, Feussner I, Jessen HJ, Van Wees SC, Saiardi A, and Schaaf G
(Siehe online unter https://doi.org/10.1105/tpc.114.135160) - (2016). Inositol polyphosphate binding specificity of the jasmonate receptor complex. Plant Physiol 171, 2364-70
Laha D, Parvin N, Dynowski M, Johnen P, Mao H, Bitters ST, Zheng N, Schaaf G
(Siehe online unter https://doi.org/10.1104/pp.16.00694)
