1. A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum By: Dorothee van Breevoort Panos Athanasopoulos Nika Strokappe

2. Main question How is the shape of the tubular ER formed and maintained? – Which proteins are involved – What is the mechanism behind it.

3. In vitro network formation They used: Membranes from Xenopus eggs. Salt wash.

4. Conclusion Small vesicles (GTP) Large vesicles (High salt wash) Tubuli

5. Measuring Ca 2+ efflux For quantification of the observations

6. Protein modification effect MP: Adds PEG NEM: Adds N-ethyl MB: Adds biotin MN: Adds neutravidin DTT: Has free cysteins

7. Identification of the target protein First add biotin, so proteins can be purified. After adding PEG, some protein disappear. They have a SH group on the surface that is accessible also to PEG

8. Conclusion Protein modification prevents the formation of tubuli. Rtn4a and Rtn4b are the most likely candidates that induce the formation of tubuli.

9. The effect of MB on network formation

10. Measuring Ca 2+ efflux

11. Conclusion The modification of proteins by adding biotin correlates with the efflux of Ca 2+

12. Reticulon 4b Blue: Hydrophobic areas Green: Area to which antibodies were raised

13. Antibody effect on Ca 2+ efflux

14. Antibody effect on network formation

15. Conclusion Antibodies against Rtn4a interfere with the formation of tubuli, antibodies against other ER proteins do not.

16. Localization Is Rtn4a/NogoA localized in the ER, more specific in the peripheral ER? Figure 4 Sec61ß  ER Protein nuclear envelope and reticular network Rtn4a/NogaA  Peripheral ER Rtn4c/NogoC  Reticular isoform(only reticulon domain)  same as Rtn4a/NogaA

17. Localization (Yeast) Is Rtn4a/NogoA localized in the ER, more specific in the peripheral ER? Figure 4 Results Rtn2/Rtn1 Absent for NE Abundant in tubules of peripheral ER Conclusion: Reticulons are restricted to the tubular, peripheral ER, consistent with a role in shaping this organelle.

18. ER structure Figure 5 CLOSE Overexpression Rtna/NogoA ER tubules  longer+ less brached Green = Sec61ß (nuclear envelope and reticular network) Red = Rnt4a/NogoA

19. ER structure Rtn1p Disruption of peripheral ER Nuclear envelope intact Together The reticulons have a strong preference to localize to tubular ER When overexpressed  reticulons appear to induce tubules Figure 5 Figure 4

20. Mutants Do yeast cells lacking the reticulons gave altered ER morphology? Single mutants  Normal Double mutant :Stress  peripheral ER  Membrane sheets Conclusion Reticulons are needed for the maintenance of tubular ER under certain stress conditions, but they cannot be the only component required under normal circumstances

21. Are there additional components involved in shaping the tubular ER? Bindingpartners of Rnt4a/NogoA  DP1 Yop1P (Yeast) Blue: Hydrophobic Green: Area to which antibodies were raised Red: Petide identified by mass spec.

22. localization DP1 Figure 6 DP1  Tubular ER Colocalization Rtn4a/NogoA DP1

23. Is DP1 the missing component for maintaining peripheral ER? Figure 6 ΔreticulonΔyop1  disrupted peripheral ER Some peripheral tubules  minor component Conclusion Rtn1p and Yop1p are the major redundant components required to maintain the tubular ER

24. What is the membrane topology? CLOSE Two long hydrophobic segments (30-35 ) Rnt4c/NogoC Introduces single cysteines  W18 andS180 reach by MPEG  first hydrophobic segments  hairspin (second maybe) DP1 First hydrophobic segment  hairpin Conclusion The reticulon and DP1 share a rather unusual membrane topology of at least there first hydrophobic segment. Figure 7

25. Summary Indication that the reticulons and DP1/Yop1p are ’morphogenic’ proteins that are necessary to form and maintain the tubular ER Rnt4a/NogoA is required for ER tubule formation in vitro. Reticulons and DP1/Yop1p localize almost exclusively to the tubular ER, consistent with a role in shaping ER domain Overexpression of the reticulons leads to long relatively unbranched tubules. The deletion of the reticulons leads to disruption of the peripheral tubular ER in stress situations and the additional deletion of Yop1p leads to similar ER morphology defects.

26. Discussion How can salt concentrations affect tubuli formation?

27. Discussion Is the title of he paper justified by its contents?