1. Orm family proteins mediate sphingolipid homeostasis Presentation by: Gillian Dekkers and Soledad Ordoñez Supervisor: Joost Holthuis David K. Breslow et al. Nature, 2010

2. Sphingolipids Are needed for: – Membrane impermeability – Molecular signalling – Sorting – Cell-cell recognition Block in synthesis compromises cell growth and survival However, intermediates of synthesis are mediators of cell stress pathways Accumulation results in growth arrest and cell death 2

3. How do cells solve the dilemma of sphingolipid regulation? Previously known: 3

4. 1. Sphingolipid synthesis 4 ER Golgi Toxic intermediates Vital spingolipids Figure 1d

5. 2. Saccharomyces cerevisiae Budding yeast with short generation time Easily cultured Entire genome sequenced in 1996 Complete set of deletion mutants is available 5

6. 3. Orm proteins Connection whith chilhood asthma. Humans: ORMDL1/2/3, S. cerevisiae: ORM1/2 Trans membrane protein located in ER membrane. No known functional domains Strongly conserved between organisms 6

7. E-MAPs Epistatic mini-array profiles Finding functional relationships between genes by: – Mapping genetic interactions 7

8. E-maps method Take a knock out of one gene of interest Make double knock outs with a variety of genes Check the growth rate of this double knock outs 8 Gene1Δ x Gene2Δ

9. E-Maps: outcome 9

10. Problem: Essential genes Essential genes cannot be screened Solution: the use of hypomorphic alleles mRNA of gene is destabilized Lower expression 10

11. E-Map of the ER Orm1/2Δ versus 1400 genes of the ER 11

12. A strong anti correlation with LCB1/2 Lcb’s: subunits of LCB synthase Suggesting that ORM2 and LCB1/2 have opposing cellular roles 12 * * * hypormorphic alleles Figure 1a

13. Orm1/2 overexpression Orm expression phenocopies reduced Lcb1/2 expression 13 Figure 1a

14. Orm’s: negative regulator of the sphingolipid pathway LCB Synthase 14 * * Serine palmoyltransferase, SPT Figure 1d

15. Orm proteins control LCB levels ORM1/2 overexpression causes reduced levels of LCB’s and ceramides ORM1/2 deletion causes increased flux throughout the pathway 15 Figure 1b

16. Growth defect in Orm1/2 Δ cells is relieved by myriosin Myriosin: LCB synthase inhibitor 16 Figure 1c

17. Orm proteins form a complex with LCB synthase SPOTS

18. SPOTS is conserved from yeast to humans

19. What is known until here? – Orm proteins are part of a conserved complex called SPOTS. – Orm1/2 negatively regulate LCBs synthesis by acting directly on Lcb1/2. – Orm function is conserved in human cells. Why would cells include negative regulators of LCB Synthase, Orm1/2?

20. What is known until here? – Orm proteins are part of a conserved complex called SPOTS. – Orm1/2 negatively regulate LCBs synthesis by acting directly on Lcb1/2. – Orm function is conserved in human cells. Why would cells include negative regulators of LCB Synthase, Orm1/2? Homeostatic regulation !

21. Orm 1/2 are regulated in response to distribution of sphingolipid synthesis Myriocin = LCB synthase inhibitor

22. Orm 1/2 are regulated in response to distribution of sphingolipid synthesis Orm effect canceled

23. Orm 1/2 undergo phosphorylation and respond to myriocin treatment Orm regulation : Alteration in Orm1/2 expresion levels. X Block of the interaction with Lcb1/2 and Sac1

24. Orm 1/2 undergo phosphorylation and respond to myriocin treatment Orm regulation : Alteration in Orm1/2 expresion levels. X Block of the interaction with Lcb1/2 and Sac1

25. Phosphorylation of Orm proteins is a homeostatic response to disruption of early precursors. Increase Orm phosphorilation. No phosphorilation response.

26. Mapping phosphorylation sites

27. Lcb1 interaction with Orm 1/2 is mantained even without Orm phosphorilation. Blocking phosphorylation does not disrupt the complex Blocking of phosphorilation prevents the change in higher order assembly of the SPOTS complex.

28. Orm phosphorylation removes the negative control of LCB synthase.

29. Orm proteins control sphingolipid homeostasis in the Endoplasmic Reticulum Orm proteins are homeostatic regulators of sphingolipid metabolism. Without Orm mediated brake on SPT there is toxic accumulation of sphingolipids. Normal intermidiate phosphorylation will give chance to a rapid responce.

30. Orm proteins control sphingolipid homeostasis in the Endoplasmic Reticulum SPOTS could act as a coordinating center that couples changes in sphingolipids and nutrients to the activity and localization of key enzymes of lipid metabolism and trafficiking.

31. Article relevance Provides a starting point for studying how protein and lipid synthesis is coordinated during membrane biogenesis. Disregulation of sphingolipid metabolism may contribute to the development of childhood asthma. New Approach, which allow studies in proteins with know function.

32. Questions still to be solved – How do Orm proteins regulate SPT? – Which kinases and phosphatases control Orm phosphorilation? – What is the identity of the sphingolipid intermediate whose levels are sensed to regulate Orm activity.