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Lipids as trans acting factors for the transport of integral membrane proteins [Elektronische Ressource] / presented by Marcel Fischer

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Lipids as Trans Acting Factors for theTransport of Integral Membrane ProteinsDissertationSubmitted to thecombined Faculties for the Natural Sciences andfor Mathematics of theRuperta-Carola University of Heidelberg, Germanyfor the degree of Doctor of Natural SciencesPresented byDiplom-BiochemikerMarcel Fischerborn in Stadthagen, GermanyDissertationSubmitted to thecombined Faculties for the Natural Sciences andfor Mathematics of theRuperta-Carola University of Heidelberg, Germanyfor the degree of Doctor of Natural SciencesMarcel Fischer2009Oral Examination: …………DeclarationsI hereby declare that I have written the submitted dissertation myself and in this processhave used no other sources or materials than those expressly indicated.I hereby declare that I have not applied to be examined at any other institution, nor have Iused the dissertation in this or any other form at any other institution as an examinationpaper, nor submitted it to any other faculty as a dissertation.Heidelberg, April 30, 2009Marcel FischerLipids as Trans Acting Factors for theTransport of Integral Membrane ProteinsReferees:PD Dr. Matthias SeedorfProfessor Dr. Blanche Schwappach„Einen Vorgang oder einen Charakter verfremdenheißt zunächst einfach, dem Vorgang oder dem Charakterdas Selbstverständliche, Bekannte, Einleuchtende zu nehmenund über ihn Staunen und Neugierde zu erzeugen.
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Lipids as Trans Acting Factors for the
Transport of Integral Membrane Proteins
Dissertation
Submitted to the
combined Faculties for the Natural Sciences and
for Mathematics of the
Ruperta-Carola University of Heidelberg, Germany
for the degree of Doctor of Natural Sciences
Presented by
Diplom-Biochemiker
Marcel Fischer
born in Stadthagen, GermanyDissertation
Submitted to the
combined Faculties for the Natural Sciences and
for Mathematics of the
Ruperta-Carola University of Heidelberg, Germany
for the degree of Doctor of Natural Sciences
Marcel Fischer
2009
Oral Examination: …………Declarations
I hereby declare that I have written the submitted dissertation myself and in this process
have used no other sources or materials than those expressly indicated.
I hereby declare that I have not applied to be examined at any other institution, nor have I
used the dissertation in this or any other form at any other institution as an examination
paper, nor submitted it to any other faculty as a dissertation.
Heidelberg, April 30, 2009
Marcel FischerLipids as Trans Acting Factors for the
Transport of Integral Membrane Proteins
Referees:
PD Dr. Matthias Seedorf
Professor Dr. Blanche Schwappach„Einen Vorgang oder einen Charakter verfremden
heißt zunächst einfach, dem Vorgang oder dem Charakter
das Selbstverständliche, Bekannte, Einleuchtende zu nehmen
und über ihn Staunen und Neugierde zu erzeugen.“
Bertolt BrechtSummary
Ist2 is an integral, polytopic membrane protein of Saccharomyces cerevisiae. Some of the IST2
mRNA is localized to the bud tip of the daughter cell, where the protein is locally translated at
the cortical endoplasmic reticulum (ER). After its translation in mother cells, Ist2 rapidly
accumulates at domains of the cortical ER. This cortical localization is mediated by a protein
signal localized within the last 69 amino acids of the cytosolic Ist2 C-terminus (cortical sorting
Ist2signal of Ist2, CSS ). In particular, certain hydrophobic and basic amino acids, which form an
Ist2amphipathic helix, are important for Ist2 transport. The CSS is a dominant sorting signal, it
can redirect different Golgi- and ER localized membrane proteins efficiently to domains of the
cortical ER. Ist2 localizes to the cortical ER independently of a functional secretory pathway
(sec). Moreover, Ist2 is accessible to proteases added to intact yeast cells, suggesting a sec-
independent transport mechanism from domains of the cortical ER to the plasma membrane
(PM).
The aim of this thesis was to identify the molecular mechanism responsible for the efficient
Ist2transport of Ist2 to the yeast cortical ER. I was able to show that the soluble CSS in vivo
binds to the yeast PM. This membrane binding could be identified as an interaction between
Ist2 the CSS and lipids of the PM. In vitro binding assays using liposomes of a defined
composition revealed phosphoinositides (PIPs) as the lipid class showing the strongest
Ist2interaction with CSS . This protein-lipid interaction depends on certain basic residues of the
Ist2CSS and its multimerization.
The PIP showing the strongest enrichment at the PM is Phosphatidylinositol[4,5]-bisphosphate
(PtdIns[4,5]P ). Therefore, I replaced the cortical sorting signal of Ist2 by a characterized2
PtdIns[4,5]P binding domain from Phospholipase C-δ . The chimeric protein localized under2 1
all tested conditions like wild type Ist2 and complements cell wall defects of ist2-knockout
strains caused by calcofluor white.
My data show that a binding of PM lipids is necessary and sufficient for the transport of Ist2 to
Ist2the cortical ER. After translation of Ist2 the CSS serves as a lipid-binding domain, which
efficiently anchors the protein at organelle junctions between the ER and the PM.
Fusion proteins of different Golgi- and ER localized membrane proteins and the PtdIns[4,5]P2
binding domain from Phospholipase C-δ are efficiently redirected to the cortical ER. This1
novel sorting mechanism for integral membrane proteins is dominant over signals responsible
for ER export and dominant over the proteasomal degradation of unstable ER proteins. This
demonstrates the efficiency of protein sorting via protein-lipid interactions.Zusammenfassung
Ist2 ist ein integrales, polytopisches Membranprotein in Saccharomyces cerevisiae. Ein Teil
der IST2 mRNA wird zur Spitze der Tochterzelle transportiert, wo das Protein lokal am
kortikalen endoplasmatischen Retikulum (ER) translatiert wird. Nach der Translation in
Mutterzellen akkumuliert Ist2 sehr schnell in Domänen des kortikalen ERs. Für diese kortikale
Lokalisation ist ein Protein-Signal ausreichend, das sich in den letzten 69 Aminosäuren des
cytosolisch orientierten C-Terminus von Ist2 befindet (kortikales Sortierungssignal von Ist2,
Ist2CSS ). Vor allem hydrophobe und basische Aminosäuren, die eine amphipathische Helix
Ist2bilden, sind von Bedeutung für den Transport von Ist2. Das CSS wirkt als dominantes Signal
über andere Proteinsortierungssignale, so kann es verschiedene Golgi- und ER-
Membranproteine effizient in Domänen des kortikalen ERs umleiten. Die Lokalisation von Ist2
im kortikalen ER ist unabhängig von einem funktionellen sekretorischen Weg (sec). Ist2 ist
zugänglich für Proteasen, die zu intakten Hefezellen zugegeben werden. Daher wurde für Ist2
ein sec-unabhängiger Transportweg von Domänen des kortikalen ERs zur Plasmamembran
(PM) vorgeschlagen.
Das Ziel dieser Doktorarbeit bestand in der Aufklärung des molekularen Mechanismus, der für
den effizienten Transport von Ist2 zum kortikalen ER verantwortlich ist. Ich konnte zeigen,
Ist2dass das lösliche CSS in vivo an die Hefe-PM bindet. Diese Membranbindung konnte als
Ist2eine Interaktion des CSS mit Lipiden der PM identifiziert werden. Durch in vitro-Versuche
mit Liposomen definierter Zusammensetzung konnte ich Phosphatidylinositolphosphate (PIPs)
Ist2als die Lipidklasse identifizieren, die die stärkste Interaktion mit CSS aufweist. Diese
Ist2Protein-Lipid-Interaktion hängt von basischen Resten des CSS , sowie dessen
Multimerisierung ab.
Das an der PM am stärksten angereicherte PIP ist Phosphatidylinositol[4,5]-bisphosphat
(PtdIns[4,5]P ). Daher ersetzte ich das kortikale Sortierungssignal von Ist2 durch eine2
charakterisierte PtdIns[4,5]P -Bindungsdomäne aus Phospholipase C-δ . Das chimäre Protein2 1
lokalisiert unter allen getesteten Bedingungen wie Wildtyp-Ist2 und komplementiert durch das
Reagenz Calcofluor weiß hervorgerufene Zellwanddefekte von ist2-knockout-Stämmen.
Diese Daten zeigen, dass für den Transport von Ist2 zum kortikalen ER die Bindung von
Ist2Lipiden der PM notwendig und ausreichend ist. Nach der Translation von Ist2 dient das CSS
als Lipid-Bindungsdomäne, die das Protein effizient an Kontakten zwischen ER und PM
verankert.
Fusionsproteine aus verschiedenen Golgi- und ER-Membranproteinen und der PtdIns[4,5]P -2
Bindungsdomäne aus Phospholipase C-δ werden effizient in das kortikale ER umgeleitet.1
Dieser neu entdeckte Sortierungsmechanismus für integrale Membranproteine ist dominant
über Signale für den Export von Proteinen aus dem ER und dominant über den proteasomalen
Abbau instabiler ER-Proteine, was die Effizienz der Proteinsortierung durch Protein-Lipid-
Interaktionen aufzeigt.Table of Contents
1 Introduction .................................................................................................................. 1
1.1 The endoplasmic reticulum ...................................................................................... 1
1.1.1 ER morphology of Saccharomyces cerevisiae 3
1.1.2 ER - organelle junctions in 5
1.2 Intracellular transport ............................................................................................... 6
1.2.1 The secretory pathway 7
1.2.2 ER export and retrieval signals 10
1.2.3 Unconventional secretion of proteins 12
1.3 Lipids in Saccharomyces cerevisiae ........................................................................14
1.3.1 Lipid content 14
1.3.2 Transport and transfer of lipids 16
1.3.3 Lipids and organelle identity 17
1.3.4 Phosphoinositides 19
1.3.5 Lipid-binding motifs and domains 21
1.4 The Ist2 protein.......................................................................................................23
1.4.1 The TMEM16 superfamily 23
1.4.2 Structure and function of Ist2 24
1.4.3 Localization of Ist2 24
1.4.4 Biosynthesis of Ist2 25
1.4.5 Mapping of the cortical sorting signal of Ist2 27
1.5. Aim of this thesis ...................................................................................................30
2 Results ..........................................................................................................................31
2.1 Genetic identification of factors involved in Ist2 transport to the cell periphery.......31
2.1.1. Results 32
2.1.2. Materials 34
2.1.3. Methods 35
2.2 Biochemical characterization of the cortical sorting signal ......................................38
2.2.1. Results 38
2.2.2. Materials 40
2.2.3. Methods 40
2.3 Results and publications of this cumulative dissertation ..........................................42
2.3.1. A signal comprising a basic cluster and an amphipathic α-helix interacts with lipids and
is required for the transport of Ist2 to the yeast cortical ER. 43
2.3.2. Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to
the cortical ER. 643 Discussion...................................................................................................................103
3.1 Lipid mediated sorting of integral membrane proteins...........................................103
3.1.1 The cortical sorting signal of Ist2 is a lipid-binding motif 103
3.1.2 Transport to subdomains of the ER is a novel transport mechanism 105
3.2 Signals and factors involved in Ist2 sorting ...........................................................108
3.3 Structural properties and multimerization of Ist2 ...................................................112
Ist23.3.1 Domains within the CSS 112
3.3.2 Multimerization of Ist2 114
3.4 Model for Ist2 transport to the cortical ER.............................................................116
3.5 Localization and function of Ist2 ...........................................................................118
3.6 Lipid mediated sorting of eukaryotic membrane proteins.......................................121
3.7 Future perspectives ...............................................................................................123
4 Abbreviation list.........................................................................................................124
5 List of tables and figures............................................................................................127
5.1 List of tables .........................................................................................................127
5.2 List of figures........................................................................................................127
6 References ..................................................................................................................128
7 Acknowledgements ....................................................................................................150