A Class of Membrane Proteins Shaping the Tubular Endoplasmic

Reticulum

By: Dorothee van BreevoortPanos AthanasopoulosNika Strokappe

Main question

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

In vitro network formation

They used:• Membranes from

Xenopus eggs.• Salt wash.

Conclusion

Small vesicles

(GTP)

Large vesicles

(High salt wash)

Tubuli

Measuring Ca2+ efflux

For quantification of the observations

Protein modification effect

• MP: Adds PEG• NEM: Adds N-ethyl• MB: Adds biotin• MN: Adds neutravidin• DTT: Has free cysteins

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

Conclusion

• Protein modification prevents the formation of tubuli.

• Rtn4a and Rtn4b are the most likely candidates that induce the formation of tubuli.

The effect of MB on network formation

Measuring Ca2+ efflux

Conclusion

• The modification of proteins by adding biotin correlates with the efflux of Ca2+

Reticulon 4b

• Blue: Hydrophobic areas• Green: Area to which antibodies were raised

Antibody effect on Ca2+ efflux

Antibody effect on network formation

Conclusion

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

LocalizationIs Rtn4a/NogoA localized in the ER, more specific in the peripheral ER?

Figure 4

Sec61ßER Protein nuclear envelope and reticular network

Rtn4a/NogaAPeripheral ER

Rtn4c/NogoC Reticular isoform(only reticulon domain)same as Rtn4a/NogaA

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.

ER structure

Figure 5

Overexpression Rtna/NogoA

ER tubules longer+ less brached

Green = Sec61ß (nuclear envelope and reticular network)

Red = Rnt4a/NogoA

ER structureRtn1p• Disruption of peripheral ER• Nuclear envelope intact

TogetherThe reticulons have a strong

preference to localize to tubular ER

When overexpressed reticulons appear to induce tubules

Figure 5

Figure 4

MutantsDo yeast cells lacking the reticulons gave altered ER

morphology?

• Single mutantsNormal• Double mutant :Stress peripheral

ERMembrane 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

Are there additional components involved in shaping the tubular ER?

Bindingpartners of Rnt4a/NogoA DP1 Yop1P (Yeast)

Blue: HydrophobicGreen: Area to which antibodies were raisedRed: Petide identified by mass spec.

localization DP1

Figure 6

DP1 Tubular ER

Colocalization Rtn4a/NogoADP1

Is DP1 the missing component for maintaining peripheral ER?

Figure 6

ΔreticulonΔyop1 disrupted peripheral ER

Some peripheral tubulesminor component

Conclusion

Rtn1p and Yop1p are the major redundant components required to maintain the tubular ER

What is the membrane topology?

CLOSE

Two long hydrophobic segments (30-35 )

Rnt4c/NogoCIntroduces single cysteinesW18 andS180 reach by MPEGfirst hydrophobic segments hairspin (second maybe)

DP1First hydrophobic segment hairpin

ConclusionThe reticulon and DP1 share a rather unusual membrane topology of at least there first hydrophobic segment.

Figure 7

SummaryIndication 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.

Discussion

How can salt concentrations affect tubuli formation?

Discussion

Is the title of he paper justified by its contents?