a class of membrane proteins shaping the tubular endoplasmic reticulum by: dorothee van breevoort...
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![Page 1: A Class of Membrane Proteins Shaping the Tubular Endoplasmic Reticulum By: Dorothee van Breevoort Panos Athanasopoulos Nika Strokappe](https://reader035.vdocuments.net/reader035/viewer/2022062515/56649d2b5503460f949ffe3a/html5/thumbnails/1.jpg)
A Class of Membrane Proteins Shaping the Tubular Endoplasmic
Reticulum
By: Dorothee van BreevoortPanos AthanasopoulosNika Strokappe
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Main question
• How is the shape of the tubular ER formed and maintained? – Which proteins are involved– What is the mechanism behind it.
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In vitro network formation
They used:• Membranes from
Xenopus eggs.• Salt wash.
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Conclusion
Small vesicles
(GTP)
Large vesicles
(High salt wash)
Tubuli
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Measuring Ca2+ efflux
For quantification of the observations
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Protein modification effect
• MP: Adds PEG• NEM: Adds N-ethyl• MB: Adds biotin• MN: Adds neutravidin• DTT: Has free cysteins
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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
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Conclusion
• Protein modification prevents the formation of tubuli.
• Rtn4a and Rtn4b are the most likely candidates that induce the formation of tubuli.
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The effect of MB on network formation
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Measuring Ca2+ efflux
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Conclusion
• The modification of proteins by adding biotin correlates with the efflux of Ca2+
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Reticulon 4b
• Blue: Hydrophobic areas• Green: Area to which antibodies were raised
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Antibody effect on Ca2+ efflux
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Antibody effect on network formation
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Conclusion
• Antibodies against Rtn4a interfere with the formation of tubuli, antibodies against other ER proteins do not.
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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
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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.
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ER structure
Figure 5
Overexpression Rtna/NogoA
ER tubules longer+ less brached
Green = Sec61ß (nuclear envelope and reticular network)
Red = Rnt4a/NogoA
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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
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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
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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.
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localization DP1
Figure 6
DP1 Tubular ER
Colocalization Rtn4a/NogoADP1
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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
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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
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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.
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Discussion
How can salt concentrations affect tubuli formation?
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Discussion
Is the title of he paper justified by its contents?