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THESE Présentée pour obtenir le titre de Docteur de l'Université de Strasbourg Discipline: Sciences de la vie et de la santé Spécialité: Aspects moléculaires et cellulaires de la biologie par Matthias Spiess Soutenue le Décembre Evolution des réseaux d'interaction des domaines protéiques SH3 dans la levure fonctions et interactions d'une famille de protéines composée de Lsb1 et Lsb2 Evolution of the SH3 domain protein interaction networks in yeast functions and interactions of the Lsb1 and Lsb2 protein family Devant le jury composé de: Dr B Winsor Directrice de thèse Dr M Geli Rapporteur externe Dr M Knop Rapporteur externe Pr J de Montigny Examinateur interne Dr N Entelis Membre invité Génétique Moléculaire Génomique Microbiologie GMGM CNRS UMR7156

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Niveau: Supérieur, Doctorat, Bac+8
THESE Présentée pour obtenir le titre de Docteur de l'Université de Strasbourg Discipline: Sciences de la vie et de la santé Spécialité: Aspects moléculaires et cellulaires de la biologie par Matthias Spiess Soutenue le 20 Décembre 2010 Evolution des réseaux d'interaction des domaines protéiques SH3 dans la levure; fonctions et interactions d'une famille de protéines composée de Lsb1 et Lsb2 Evolution of the SH3 domain protein interaction networks in yeast; functions and interactions of the Lsb1 and Lsb2 protein family Devant le jury composé de: Dr. B. Winsor Directrice de thèse Dr. M. Geli Rapporteur externe Dr. M. Knop Rapporteur externe Pr. J. de Montigny Examinateur interne Dr. N. Entelis Membre invité Génétique Moléculaire Génomique Microbiologie GMGM, (CNRS, UMR7156)

  • winsor directrice de thèse dr.

  • who helped

  • bedanken möchte

  • freude und die

  • auch noch spät

  • die kulinarisch hochstehenden

  • und allen

  • ich mich


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Published 01 December 2010
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Language English
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THESE

Présentée pour obtenir le titre de

Docteur de l’Université de Strasbourg

Discipline: Sciences de la vie et de la santé
Spécialité: Aspects moléculaires et cellulaires de la biologie

par
Matthias Spiess

Soutenue le 20 Décembre 2010

Evolution des réseaux d’interaction des domaines
protéiques SH3 dans la levure; fonctions et interactions
d’une famille de protéines composée de Lsb1 et Lsb2

Evolution of the SH3 domain protein interaction networks
in yeast; functions and interactions of the Lsb1 and Lsb2
protein family


Devant le jury composé de:

Dr. B. Winsor Directrice de thèse
Dr. M. Geli Rapporteur externe Dr. M. Knop Rapporteur externe Pr. J. de Montigny Examinateur interne
Dr. N. Entelis Membre invité



Génétique Moléculaire Génomique Microbiologie GMGM, (CNRS, UMR7156)


















Acknowledgements


I would like to thank my thesis director Barbara Winsor for giving me the opportunity to do
my thesis in her laboratory, assisting me with the little and big problems of such a work, for
fighting for funding to finish the work and for all the rest. Especially I would like to mention
her support to find and exploit collaborations.

I would like to thank Rudolph Volkmer for accommodating me in his laboratory and
Christiane Landgraf for showing me around and introducing me into the technique SPOT.
Thanks also to Victor Tapia Mancilla who did a great work in the data analysis. Thanks to all
the other lab members as well, I really had a great time in Berlin.

I would like to thank David Drubin for accommodating me in his laboratory and Alphée
Michelot for showing me around and introducing me into the technique of in vitro actin
polymerization. It was a great and fruitful stay in Berkeley.

I would like to thank Danièle Urban-Grimal for accommodating me in Paris and helping me
in the laboratory with ubiquitination experiments and all other lab members of the Rosine
Hagenauer-Tsapis laboratory. I really learned to understand the fascination of and appreciate
the French capital.

I would like to thank the numerous temporal and permanent members of Penelope. Although
it was not all the time the easiest collaboration, I always enjoyed our meetings, had a lot of
fun with you in the rare spare time and looked forward to the next. And Michela did a
fantastic job.

I would like to thank Aline Huber and Audrey Geissler who helped me a lot in the lab, who
were kindly concerned about my security and amused me with their unique alsacian humor.
Thanks to Albert who introduced me into the new environment and the new techniques in the
laboratory with his ability to speak English and thanks to Vincent who helped me to speak French and in return learned that beer can be as good as wine. Many thanks also to the
remaining Winsor lab members Gladys, Guillaume, Lydia and Ray.

I would like to thank Sylvie Friant and her laboratory for discussing and advancing my
project. We passed some journal clubs, some interesting lab meetings and some fantastic food
or Pétanque parties together. Thanks to Johan, Serge, Dimitri and Fanny. Many thanks also to
Joelle Morvan for kindly helping me to correct this work and last but not least I would like to
thank Bruno, who sold me my fist car and helped with his thousand and one tools where he
could.

I would like to thank Cathy and Aline for helping me to get through the jungle of French
administration and for being there for some de-stressing chat.

Bedanken möchte ich mich auch bei Tom und Serge, die nicht nur tagsüber im Labor sondern
auch noch spät Abends für gute Stimmung sorgten. Die kulinarisch hochstehenden
Abendessen waren mir eine wahre Freude und die Diskussionen auf der Terrasse, bei einem
Bier oder einem vom Meistersommelier auserkorenen Wein waren allemal ein kürzere Nacht
wert.

Zum Schluss möchte ich noch all meinen Freunden aus der schönen Schweiz danken, meiner
Familie natürlich und allen voran meinen Eltern Paul und Denise, die all die Jahre über an
mich geglaubt und mich tatkräftig unterstützt haben.
















Table of contents

Preface................................................................................................................................................3 
Abstract...............4 
Résumé................5 
Chapter I – Introduction................................................................................................9 
I. The Budding Yeast Saccharomyces cerevisiae............9 
II. The Actin Cytoskeleton...................10 
II.1 The cytoskeleton...........................................................................................................10 
II.2 The budding yeast actin cytoskeleton.11 
III. Actin.....................................................................................................................13 
II.1 Actin polymerization..................................................................14 
IV. Actin Nucleation...............................16 
IV.1 The Arp2/3 complex.17 
V. Nucleation Promoting Factors......................................................................................................19 
V.1 The WASP family proteins........................21 
VII. Membrane Traffic...........................................................24 
VIII. Endocytosis.....25 
VIII.1 Historical overview of endocytosis in Saccharomyces cerevisiae.......................................26 
VIII.2 Early steps of endocytosis....................................................................................27 
IX. Regulation of Endocytosis by Ubiquitin...................................................30 
X. Objectives.............................................33 
Chapter II ­ Characterization of Lsb1 and Lsb2..................................................................37 
I. Introduction to Lsb1 and Lsb2.......................................37 
II. Lsb1 and Lsb2 and their Homologs.............................37 
III. Characterization of lsb1Δlsb2Δ...39 
III.1 Growth of deletion strains......................................................................................39 
III.2 Functional analysis of LSB1 and LSB2 deletion strains..............................39 
III.3 Biochemical and genetic interactions of LSB1 and LSB2...........................40 
III.4 Analysis of budding polarity..................................................................................................................42 
III.5 Lat‐A sensitivity...........................................43 
III.6 Actin cytoskeleton organization...........44 
III.7 Endocytosis...................................................................................................................45 
IV. Localization of Lsb1 and Lsb2......47 
IV.1 Subcellular Fractionation of Lsb1 and Lsb2....................48 
IV.2 Lsb1 and Lsb2 pellet upon centrifugation.......................................................................................49 
IV.3 Lsb1 is present in a high molecular weight complex..................................50 
V. Colocalization of Lsb1 and Lsb2 with endocytic Proteins...................53 
V.1 Lsb1 and Lsb2 localization is not dependent on actin patch proteins..55 
V.2 Lsb2 and prion formation.........................................................................................................................56 
VI. Ubiquitination of Lsb1 and Lsb2.................................................................58 
VI.1 Lsb1 is stable.................................................59 
VII. Role of Lsb1 and Lsb2 in Actin Polymerization....60 
VII.1 Lsb1 and Lsb2 bind Las17 but do not induce ex vivo actin polymerization....................60 
VII.2 Lsb1 and Lsb2 inhibit Las17 NPF activity in vitro.......................................................................62 
VIII. Lsb1 and Lsb2 stabilize Las17..................................67 
IX. Lsb1 and Lsb2 Overexpression does not affect Endocytosis.............................................68 
1 Chapter III – SH3 Domain Networks......................................................................................70 
I. Introduction.........................................................................70 
I.1 Protein interaction networks...................70 
I.2 SH3 domains....................................................71 
I.3 The peptide array SPOT..............................................................................................74 
II. Production of the SH3 domains for SPOT analysis................................75 
III. The SPOT assay.................................................................................................77 
III.1 Peptide interactions of the type I myosin SH3 domains............................78 
III.2 Peptide interactions of the Hof1 SH3 domains..............................................84 
IV. The AgMyo5 tail recruits the actin polymerization machinery in Saccharomyces 
cerevisiae protein extracts..................................................................................87 
Chapter IV – Discussion and Perspectives...........................................93 
I. Localization of Lsb1 and Lsb2........................................................................93 
II. Lsb1 and Lsb2 Function..................95 
III. SPOT analysis of type I myosins and Hof1...........100 
III.1 Conservation of type I myosin tail function.................................................103 
Chapter V ­ Materials and Methods......................................................................................107 
I. Bacterial Growth Media, Strains, Reagents and Procedures............107 
II. Yeast Growth Media, Strains, Reagents and Procedures..................107 
II.1 Yeast transformation...............................................................................................108 
II.2 Yeast genomic DNA extraction............................................................................108 
II.3 Yeast conjugation and sporulation....108 
II.4 Rapid yeast protein extract...................................................108 
II.5 Yeast protein extract................................................................................................109 
II.6 Yeast strains................................................................................................................109 
III. DNA Techniques and Plasmid Construction........111 
IV. Yeast Growth and Viability Assay............................................................................................113 
V. Lat­A Sensitivity..............................................................114 
VI. Fluorescence Microscopy...........................................................................................................114 
VII. Actin and Calcofluor White staining.....................114 
VIII. FM4­64 and Lucifer Yellow uptake......................115 
IX. Endocytosis of Fur4 GFP.............115 
X. Western Blot....................................................................................................................................115 
XI. Subcellular Fractionation..........116 
XII. FPLC and Immunoprecipitation.............................117 
XIII. SDD­AGE........117 
XIV. Test of Protein Stability...........................................................................................................118 
XV. SPOT Assay.....................................118 
XV.1 SH3 domain production.......................118 
XV.2 Hybridization on SPOT membranes...............................................................................................119 
XVI. Actin Polymerization on Sepharose Beads and Pull Down..........119 
XVII. In vitro Actin Polymerization................................................................120 
Appendix I – List of ScSH3 Domains.....................................................123 
Appendix II – List of SPOT peptides124 
Abbreviations..............................................................................................................................128 
List of Figures and Tables........................................130 
Résumé en français....................................................................................132 
References....................................................................................................142 
2 Preface


All living cells have to deal with a series of challenges during their life span. Being well
adapted to their environment is, not only for humans, but also for all other organisms, a clear
advantage. Some organisms such as parasites have found a constant, sheltered environment. A
human body can be considered as quite a constant environment, exploited by several well
tempered or not so well tempered, pathogenic organisms. Other organisms that could perhaps
be considered more adventurous have populated all the familiar and strange places on earth.
Ways to win the daily struggle for survival may be to infiltrate a host, to get rid of a parasite
or just to resist the nasty alsacian weather. Cellular robustness, flexibility and perhaps even
mobility are of great advantage. The cytoskeleton of a cell is implicated in all these
advantages. The actin cytoskeleton not only helps the cell to be in a good shape and to move
towards the food, it is also implicated in uptake from the extracellular medium. But as every
good thing, the actin cytoskeleton also has a dark side. Malfunction or misregulation can harm
an organism severely and several human diseases have been linked to actin cytoskeleton
defects. Thus, understanding its component parts and underlying principles is an easily
justifiable subject of basic research and may be of help in understanding disease.
As human are not the simplest organisms on earth in many ways, but not so different from
yeast as some would think, I accepted the proposition of studying the role of two fameless
proteins Lsb1 and Lsb2 (Las seventeen binding), of the actin cytoskeleton in the yeast
Saccharomyces cerevisiae. But it would have been a bit narrow minded to do experiments on
budding yeast during the day and drink brewing yeast in the evening. One can always argue
that drinking water would have been a solution but I decided to open my horizon and
participate, in parallel, in the study of protein interaction networks, the clandestine rulers of
cell’s life. Our objective, as part of the multi laboratory European project Penelope, was to
understand the evolution of protein interaction networks through the study of SH3 (Src
homology) protein domains in four yeasts which are close in the evolutionary scale but
populate different environments.



3 Abstract


In the budding yeast Saccharomyces cerevisiae, nutrients, lipids and membrane proteins are
internalized by fluid phase (bulk) endocytosis or by receptor-mediated endocytosis. These
processes require dynamic actin filament assembly. A key factor for actin polymerization is
the nucleating complex Arp2/3 that is activated by nucleation promoting factors (NPF).
During the process of endocytosis, a strong NPF activity is exhibited by Las17, the unique
Saccharomyces cerevisiae homolog of WASP (Wiskott Aldrich syndrome protein) and the
type I myosin, Myo5, among others. Here, we characterize two SH3 domain containing
proteins Lsb1 and Lsb2. We could show that both proteins bind to the proline rich sequence
of the NPF Las17 via their SH3 domains and efficiently inhibit Las17 induced Arp2/3-
dependent actin polymerization in vitro. We also could show that Lsb1 and Lsb2 partially
colocalize with Las17 and that they influence the stability of Las17 in vivo. However, we
could not detect any defect in endocytosis for the single or double deletion mutants of LSB1
and LSB2. In conclusion, we identified two new regulators of the NPF activity of Las17 that
will help us to further understand the process of actin nucleation and endocytosis.
We also present the characterization of the SH3 domain binding specificity of the type I
myosins in four yeast species Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans
and Schizosaccharomyces pombe. A high conservation of the type I myosin specificity during
evolution was found. And the type I myosin tail of Ashbya gosypii like that of Saccharomyces
cerevisiae is able to recruit the actin polymerization machinery in Saccharomyces cerevisiae.
Similar analysis of other SH3 domains, including Lsb1 and Lsb2, is in progress, which will
allow us to predict new protein-protein interactions and to gain insights into the evolution of
protein interaction networks.

Key Words: Endocytosis, Actin polymerization, Las17, SH3 domain




4
Résumé


Chez la levure Saccharomyces cerevisiae, les nutriments, les lipides et les protéines
membranaires sont internalisés par endocytose. Un assemblage des filaments d’actine
dynamique est requis pour ce processus. Un facteur clé pour la polymérisation de l’actine est
le complexe nucléateur Arp2/3. Arp2/3 est activé par des facteurs de promotion de la
nucléation (NPF). Pendant l’endocytose, la polymérisation de l’actine est initiée entre autre
par le NPF Las17, le seul membre de la famille WASP (Wiskott-Aldrich syndrome protéine)
chez la levure, et les myosines de type I. Nous présentons la caractérisation de deux protéines
à domaine SH3, Lsb1 et Lsb2. Nous montrons qu’elles lient Las17 par leurs domaines SH3 et
qu’elles inhibent la polymérisation de l’actine dépendante de Las17 et du complexe Arp2/3 in
vitro. D’autre part, Lsb1 et Lsb2 colocalisent avec Las17, Abp1 et Sla1 et elles influencent la
stabilité de Las17 in vivo. Cependant nous n‘avons pas pu montrer un defaut d’endocytose
pour les souches avec simple ou double délétions du LSB1 et LSB2. Nous avons ainsi identifié
deux nouveaux régulateurs de l’activité NPF de Las17 ce qui permettra de mieux comprendre
son rôle dans la polymérisation de l’actine et l’endocytose.
Nous présentons également la caractérisation des spécificités de liaison des domaines SH3 des
myosines de type I des levures Saccharomyces cerevisiae, Ashbya gossypii, Candida albicans
et Schizosaccharomyces pombe par la technique SPOT. Une conservation de la spécificité a
été montrée et nos résultats validés par une analyse bioinformatique. De plus, nous montrons
que la queue de la myosine de type I d’Ashbya gossypii comme celui de Saccharomyces
cerevisiae est capable de recruter la machinerie de polymérisation de l’actine dans un extrait
protéique brut de Saccharomyces cerevisiae. Nous faisons actuellement des analyses
similaires pour d’autres protéines à domaine SH3, entre autres Lsb1 et Lsb2, ce qui nous
permettra de prédire des interactions protéines-protéines ainsi de mieux comprendre
l’évolution des réseaux d’interaction protéique.

Mots clés: Endocytose, Polymérisation de l’actine, Las17, Domaine SH3


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