a class of membrane proteins shaping the tubular endoplasmic reticulum by: dorothee van breevoort...
Post on 19-Dec-2015
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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?