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Segregation of sphingolipids and

sterols during formation of secretory

vesicles at the trans-Golgi network

Kai Simons group, 2009, J. Cell Biology

Deniz Ugurlar Robbert Kim

Lecturer: Gerrit van Meer

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Secretory Pathway

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Lipid raft

Ordered, highly packed

Sterols, sphingolipids, glycerophospholipids, proteins

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Signal transduction, virus assembly, membrane trafficking

Proposed to be involved in the generation of lipid gradients

Lipid sorting at TGN

Low conc in ER and accumulate toward PM

(Simons and van Meer,1988)

Raft involvement in TGN sorting – no direct evidence!

Lipid raft

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Hypothesis

Raft plays a functional role in the sorting machinery

The secretory vesicles should be selectively enriched in sterols and sphingolipids

Previous Research

Shortcomings of the methodology

insufficient purity of isolated TGN-derived vesicles

not be able to characterize lipid composition

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Experimental Outline

S.cerevisiae

Ergosterol cholesterol in mammals

LDSV transporting FusMidGFP (TGN to PM)

Immunoisolation procedure

Purify secretory vesicles

Late Golgi compartments

Western Blot & EM

Quantitative shotgun lipidomics

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The Bait for Immunoisolation

FusMidGFPLTLM9

FusMidGFP High affinity 9xmyc(M9) tag T TEV Protease site L linker FusMidGFPLTLM9FusMidp

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Expression

Exocyst mutant sec6-4, temperature sensitive

24°C, permissive

37°C, restrictive no PM translocation

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EM & Tomography

intracellular accumulation of

vesicles at 37°C

http://jcb.rupress.org/cgi/

content /full/jcb.200901145/DC1/

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Immunoisolation

Vesicle isolation

cell lysis

differential fractionation

Isopycnic sucrose gradient

Immunoisolation

Mouse anti-myc antibody

Sheep anti-mouse immunoadsorbent

Vesicle recovery

TEV protease

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Purification assessment

Distinct vesicles

Raft protein

Late endosome

ER

Late Golgi

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Morphology

Purified vesicles with EM Homogenous

~100 nm (comparable with the ones in living cells)

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Recap

Isolated FusMidp vesicles Good purification

Spherical vesicles

Homogenous ~100nm

Comparable with living cells

Ready for analysis

Composition

Comparison with donor organelle

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Immunoisolation TGN/E

Distinct vesicles

Raft protein

Late endosome

ER

Late Golgi

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Comparison

Morphology TGN/E Heterogenous population

~100-300 nm and ~40-50 nm

Different from yeast PM

Morphology FusMidp vesicles

Distinct from the donor organelle

Immunoisolation protocol Efficient tool

Suitable different vesicles

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Lipidomics study

Quantitative shotgun lipidomics Mass Spectrometry

83 lipids of 12 classes

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Lipidomics results

Most abundant ones FusMidp

Ergosterol (22.8 mol%)

Sphingolipids

TGN/E

Ergosterol (9.8 mol%)

Sphingolipids

FusMidp

Less PS, PE, PC

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Membrane order assay

C-Laurdan spectrophotometry

Calculate general polarization (GP)

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Conclusions

Nice method for purifying vesicles

Analyzed LDSV compared with TGN/E

High in ergosterol and sphingolipids

Low PS, PE, PC

Higher membrane order

Raft involvement

Article conclusion

Membrane architecture modulated

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Discussion

Trustworthy results Many methods

Complementary results COPI coated vesicles low in sphingomyelin and cholesterol Mammal system Brügger et al, 2000. J. Cell Biol.

Endosomes and lysosomes

No known factors raft clustering Candidates have been proposed (Prozynski et al. 2005)

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