Francisca Fernández Gil-Homenaje al bioquímico español Alberto Sols
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This document summarizes research on lipid homeostasis and cold tolerance in fermentation biotechnology laboratories. It shows that at low temperatures, there is an increase in the presence of the kinase Ypk1 in the soluble fraction, and a decrease in production of inositol phosphorylceramides (IPCs) by downregulation of Ypk1. The expression of genes involved in cold tolerance, such as ORM2, is also induced at low temperatures. Additionally, low temperatures decrease the level of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane and increase the amount of inositol hexakisphosphate (1-IP7).
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Francisca Fernández Gil-Homenaje al bioquímico español Alberto Sols
- 1. Fermentation biotechnology laboratory Lipid homeostasis: a hot topic in the cold tolerance
- 4. Sln1-Ypd1 GelLiquid-crystal Membrane rigidification Cold-signalling Time (min) Hog1p 0 5 10 30 60 P-Hog1p 5 NaClCold Hog1p activation upon cold shock Slt2 Rho1 Pkc1 Rom2 Bck1 Mkk1Mkk1 Mkk2Mkk2 Mtl1 Mtl1 Mid2 Mid2 Wsc3Wsc3 GTP Wsc1 Wsc2 Wsc2 -P -P -P -P Rlm1 FKS1FKS1 Swi4 Swi6 FKS2FKS2 Slt2Slt2 Rho1Rho1 Pkc1Pkc1 Rom2 Bck1Bck1 Mkk1Mkk1 Mkk2Mkk2 Mtl1 Mtl1 Mid2 Mid2 Wsc3Wsc3 GTP Wsc1Wsc1 Wsc2 Wsc2 -P -P -P -P Rlm1 FKS1FKS1 Swi4Swi4 Swi6Swi6 FKS2FKS2 Time (min) Anti-HA 0 10 30 60 90 P-Slt2p 120 12ºC 180 slt2D pSLT2-HA Slt2 is phosphorylated at low temperatures
- 6. PI(4,5)P2 level regulates CWI pathway activity but does not determine cold tolerance 30oC 12oCSCD wt slt2 inp51 Inp51slt2 inp51 mutant exhibits low Pho85 activity 30oC 15oC wt inp51 CEN.PK inp51 mutant is cold tolerant
- 7. wt inp51mg/mgofcells(dw) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 DG TAG SE b-galactosidase,U/mgprotein 0 50 100 150 200 250 300 350 400 450 +INO -INO 0 1 2 3 4 5 6 Phospholipid,% PA nem1 30ºC 15ºC wt inp51 inp51 pah1 pah1 inp51 nem1 inp51 ≠ pah1 Pah1Pah1 Pho85 Pkc1 PKA Nem1 P PP
- 8. 30ºC 15ºC 0 1 2 3 4 5 Fluorescenceintensity S0 P0 S3 P3 GFP G6PDH S0 P0 S3 P3 wt inp51 PI(4,5)P2 level at the plasma membrane decreases in response to a drop in temperature 0 5 10 15 20 Pho89-lacZ b-galactosidase,U/mgprotein 30ºC 15ºC Increased amount of 1-IP7 upon cold shock The expression of INO1 is induced at low temperature 3 h1 h Foldinductionto30ºC time at 15ºC 0 2 4 6 8 10 12 Cold regulates the activity of Pah1 PH-PLd-GFP
- 9. Cold regulates the abundance of Pah1 0.35 0.45 0.53 0.65 1.0 Hxk2 Hxk2 Pah1-Myc 15ºC OD600 OD600 Time (h) 3 6 11 22 0 1 2 3 4 5 6 0.35 0.45 0.53 0.65 1 Relativeabundance Time (h) 0 1 2 3 4 0 20 40 OD600nm mss4ts stt4ts elo2 elo3 aur1 csg2 YPD YPD mM Myr wt inp51 CEN.PK inp51 mutant is myriocin tolerant MSS4 Pah1-Myc30ºC Hxk2 Time (min) 40 65 90 150 390 4.0
- 10. Low temperature increases the presence of Ypk1 in the soluble fraction S P S P Hxk2 Ypk1-HA 30ºC 15ºC 2h 0 25 50 75 100 S P Ypk1-HA relativecontent(%) YPC1/ YDC1 qPCR ORM2 2X The expression of ORM2 is induced at low temperature and a dephosphorylated isoform appears 0 2 12 30ºC 15ºC Time (h) Orm2-HA Phos-SDS SDS 0 2 12 30ºC 15ºC Time (h) Hxk2 Orm2-HA Hxk2 Ypk1-HA
- 11. csg2D ipt1D 0 4 8 12 16 DHS PHS pmol107cells 0 0.2 0.4 DHS-1P PHS-1P pmol107cells 0 2 4 6 8 IPC-42 IPC-44 IPC-46 pmol107cells Low temperatures decreases the production of IPC by downregualition of Ypk1
Editor's Notes
- I would like to thank the organizers for the opportunity to present the results of our group in this meeting. My talk will be focused on cold-responses in the model yeast S. cerevisiae and will present advances in cold signaling and regulation of membrane composition and properties. Data have an obvious interest in development of commercial strains, like those used in traditional biotechnological applications, including bread or wine, but also knowledge in membrane biology may open exciting areas of study with unexpected applications.
- Altogether, our results demonstrated the activation of 2 MAPK pathways by cold, the HOG and the CWI. The first is triggered in minutes by a rapid decrease in membrane fluidity, while activation of Slt2 takes place at mid-term, mediated likely by a change in membrane composition. Quite interestingly, the Kennedy’s group recently reported a role of the cell wall integrity pathway in membrane fluidity homeostasis. Again, strains deficient in the core components of the pathway, showed increased sensitivity to palmitoleate (C16:1), which causes hyper-fluidity of the membrane. As I will show here, this is not the sole pathway involved in membrane homeostasis.