Analysis of sphingolipid signaling at the plasma membrane of Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Florian Fröhlich

Florian Fröhlich

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 12 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München




Analysis of sphingolipid-signaling
at the plasma membrane of
Saccharomyces cerevisiae



vorgelegt von
Florian Fröhlich

2010

Eherenwörtliche Versicherung

Ich versichere hiermit ehrenwörtlich, dass die vorgelegte Dissertation von mir
selbstständig und ohne unerlaubte Hilfe angefertigt ist.

München, den .............................. .............................................................
(Unterschrift)



Erklärung

Hiermit erkäre ich,
Dass die Dissertation nicht ganz oder in wesentlichen Teilen einer anderen
Prüfungskomission vorgelegt worden ist

Dass ich mich anderweitig einer Doktorprüfung ohne Erfolg nicht unterzogen
habe

München, den .............................. .............................................................
(Unterschrift)


Diese Dissertation wurde von Prof. Dr. Stefan Jentsch betreut. Die Dissertation
wurde eingereicht am 12.07.2010
1. Gutachter: Prof. Dr. Stefan Jentsch
2. Gutachter: Prof. Dr. Charles David

Tag der mündlichen Prüfung: 13.08.2010 1. Table of contents
Contents
1.  Table of contents .............................................................................................................. 1 
2.  List of Publications ........................................................................................................... 3 
3.  Abbreviations .................................................................................................................... 5 
4.  Summary ........................................................................................................................... 7 
5.  Introduction ........................................................................................................................ 8 
5.1  Architecture of the plasma membrane ................................................................... 8 
5.1.1   Structure and components of the plasma membrane .................................. 8 
5.1.2  Membranes are two dimensional fluids ........................................................ 12 
5.1.3  Lipid rafts ........................................................................................................... 14 
5.2  Mechanisms of plasma membrane organization ............................................... 16 
5.2.1  Macro-organization of the plasma membrane ............................................ 16 
5.2.2  Micro-organization of the plasma membrane .............................................. 17 
5.2.3  Plasma membrane organization in Saccharomyces cerevisiae ............... 18 
5.3  Sphingolipids ........................................................................................................... 21 
5.3.1  Sphingolipid biosynthesis in Saccharomyces cerevisiae .......................... 21 
5.3.2  Regulation of sphingolipid levels ................................................................... 25 
5.3.3  Cellular processes regulated by sphingolipids ............................................ 27 
5.4 Aims of the thesis ........................................................................................................ 28 
6  Discussion ....................................................................................................................... 30 
7  References ...................................................................................................................... 39 
8  Acknowledgments .......................................................................................................... 46 
9  Curriculum Vitae ............................................................................................................. 47 
 
1 
 10  Declaration of Individual Contributions.................................................................... 49 
11  Reprints of the Publications ...................................................................................... 51 

























2 
 2. List of Publications

Publication 1:
Walther TC*, Aguilar PS*, Fröhlich F, Chu F, Moreira K, Burlingame AL, Walter P.
(2007) Pkh-kinases control eisosome assembly and organization. EMBO Journal
26(24), 4946-55.

Publication 2:
de Godoy LM*, Olsen J.V.*, Cox J*, Nielsen ML*, Hubner NC, Fröhlich F, Walther
TC, Mann M. (2008) Comprehensive mass-spectrometry-based proteome
quantification of haploid versus diploid yeast. Nature 455(7217):1251-4

Publication 3:
Fröhlich F*, Moreira K*, Aguilar PS, Hubner NC, Mann M, Walter P, Walther TC.
(2009) A genome wide screen for genes affecting eisosomes reveals Nce102
function in sphingolipid signalling. Journal of Cell Biology, 185(7):1227-42

Publication 4:
Soufi B, Kelstrup CD, Stoehr G, Fröhlich F, Walther TC, Olsen JV. (2009) Global
analysis of the yeast osmotic stress response by quantitative proteomics. Molecular
BioSystems. 5(11):1337-46 

Publication 5:
Fröhlich F and Walther TC. (2009) Comparing cellular proteomes by mass
spectrometry. Genome Biology 10(10):240 Review

Publication 6:
Aguilar PS*, Fröhlich F*, Rehman M*, Shales M*, Ulitsky I, Olivera-Couto A, Braberg
H, Shamir R, Walter P, Mann M, Ejsing CS, Krogan NJ, Walther TC. (2010) A plasma
membrane E-MAP reveals links between the eisosome, sphingolipid metabolism and
endosomal tafficking. (2010) Nat Struct Mol Biol. [Epub ahead of print]
* These authors contributed equally to this work
3 
 

























4 
 3. Abbreviations

DHS dihydrosphingosine
DHS-P dihydrosphingosine phosphate
E-MAP epistatic mini-array profile
ESR electron spin resonance
FRAP fluorescence recovery after photobleaching
GFP green fluorescent protein
GPI glycosylphophatidylinositol
GUV giant unilamellar vesicle
IPC inositol phosphoceramide
LCB long chain base
L liquid ordered o
L liquid disordered d
MAPK mitogen-activated protein kinase
MCC membrane compartment occupied by Can1
MCP membrane compartment occupied by Pma1
MCT membrane compartment occupied by TORC2
MDCK Madin-Darby canine kidney
MIPC mannose-inositol-phosphoceramide
M(IP)C mannose-(inositol-P) -ceramide 2 2
mTORC2 mammalian target of rapamycin complex 2
PA phosphatidic acid
PC phosphatidyl-choline
PDK1 phosphoinositide dependent kinase 1
PE phosphatidyl-ethanolamine
PH domain pleckstrin homology domain
PHS phytosphingosine
5 
 PHS-P phytosphingosine phosphate
PI phosphatidyl-inositol
PIP phosphoinositide
PI(4,5)P phosphatidylinositol 4,5-bisphosphate 2
PS phosphatidyl-serine
SDPE 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanol-amine
SGA synthetic genetic array
SGK serum glucocorticoid inducible kinase
SILAC stable isotope labeling of amino acids in cell culture
SPT serine palmitoyl transferase
SV40 simian virus 40
TORC2 target of rapamycin complex 2

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 
 4. Summary
 
The protein and lipid composition of eukaryotic plasma membranes is highly dynamic
and regulated according to need. Despite its great plasticity, the plasma membrane
retains some organizational features, such as its lateral organization into distinct
domains. In the yeast, Saccharomyces cerevisiae, large immobile protein clusters,
termed eisosomes, are important for plasma membrane organization. Eisosomes
help to sort proteins into discrete domains, function in endocytosis and are implicated
in cellular signaling. The major eisosome components Pil1 and Lsp1 were first
identified as in vitro targets of the sphingolipid long chain base-regulated Pkh-
kinases. However, it is not known if eisosomes are targets of Pkh-mediated
sphingolipid signaling in vivo. In this thesis, I show that Pkh-kinases regulate
eisosome formation in response to alterations of complex sphingolipid levels in the
plasma membrane. I found that Pkh-kinase-dependent phosphorylation of Pil1
controls