ER-associated protein degradation (ERAD) [Elektronische Ressource] : an unexpected function of Yos9 and the discovery of Mnl2, a new component of the pathway / Elena Martínez Benítez. Betreuer: Dieter H. Wolf
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ER-associated protein degradation (ERAD) [Elektronische Ressource] : an unexpected function of Yos9 and the discovery of Mnl2, a new component of the pathway / Elena Martínez Benítez. Betreuer: Dieter H. Wolf

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ER-associated protein degradation (ERAD): An unexpected function of Yos9 and the discovery of Mnl2, a new component of the pathway von der Fakultät Chemie der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. Rer. Nat.) genehmigte Abhandlung vorgelegt von Elena Martínez Benítez aus Barcelona (Spanien) Hauptberichter: Prof. Dr. Dieter H. Wolf Mitberichter: PD. Dr. Wolfgang Hilt Tag der mündlichen Prüfung: 16.09.2011 Institut für Biochemie der Universität Stuttgart 2011 1 Hiermit versichere ich, dass ich die Arbeit selbst verfasst und dabei keine anderen als die hier angegebenen Quellen und Hilfsmittel verwendet habe. Stuttgart, den 20.07.2011 Elena Martínez Benítez 2 Acknowledgments I am grateful to Prof. Dr. Dieter H. Wolf for the fun I had working in his group (except for the last few months, that I had a horrible time trying to write this thesis). Thanks to the UbiRegulators Network for giving me the possibility to travel and attend many courses and conferences. To all members of the IBC between February 2007 and September 2011, thank you. It has been awesome to share time with you not only for the science, but also for the friendships we have build. To my friends that highly motivated me by asking: so, are you almost done now? Or, are you stressed?

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Published 01 January 2011
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Language English
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ER-associated protein degradation (ERAD): An
unexpected function of Yos9 and the discovery of
Mnl2, a new component of the pathway


von der Fakultät Chemie der Universität Stuttgart zur
Erlangung der Würde eines Doktors der Naturwissenschaften
(Dr. Rer. Nat.) genehmigte Abhandlung


vorgelegt von
Elena Martínez Benítez
aus Barcelona (Spanien)


Hauptberichter: Prof. Dr. Dieter H. Wolf
Mitberichter: PD. Dr. Wolfgang Hilt
Tag der mündlichen Prüfung: 16.09.2011







Institut für Biochemie der Universität Stuttgart
2011
1
















Hiermit versichere ich, dass ich die Arbeit selbst
verfasst und dabei keine anderen als die hier angegebenen
Quellen und Hilfsmittel verwendet habe.
Stuttgart, den 20.07.2011




Elena Martínez Benítez
2

Acknowledgments

I am grateful to Prof. Dr. Dieter H. Wolf for the fun I
had working in his group (except for the last few months,
that I had a horrible time trying to write this thesis).
Thanks to the UbiRegulators Network for giving me the
possibility to travel and attend many courses and
conferences.
To all members of the IBC between February 2007 and
September 2011, thank you. It has been awesome to share
time with you not only for the science, but also for the
friendships we have build.
To my friends that highly motivated me by asking: so,
are you almost done now? Or, are you stressed? A big thank
you ;)
To my family I want to show my gratitude for their
support from the moment I decided to stay in Germany for
the Ph. D. and their words of encouragement in every phone
call. Especially to Marco for his patience, calmness and
understanding in the difficult moments of these last
months, thank you.



3

Table of contents

Abbreviations ............................................ 8
Zusammenfassung ......... 11
Abstract ................ 15
1. Introduction ........ 18
1.1. Saccharomyces cerevisiae as cell model ............ 18
1.2. The secretory pathway ............................. 19
1.3. Degradation of proteins in eukaryotes ............. 22
1.4. Quality control in the endoplasmic reticulum ...... 23
1.4.1. Endoplasmic reticulum associated degradation .... 29
1.5. Model substrates .................................. 34
1.5.1. Cystic fibrosis transmembrane conductance regulator
(CFTR) 34
1.5.2. Carboxypeptidase Y mutants ...................... 35
1.6. Aim of this work .. 36
2. Materials and methods ............................... 38
2.1. Materials ......................................... 38
2.1.1. Saccharomyces cerevisiae strains ................ 38
2.1.2. Plasmids ........ 42
2.1.3. Primers ......... 43
2.1.4. Antibodies ...... 45
2.1.5. Kits, enzymes, chemicals and media .............. 46
2.1.5.1. Yeast media ................................... 48
4

2.1.5.2. E.coli media .................................. 49
2.1.6. Laboratory equipment ............................ 50
2.2. Methods ........... 51
2.2.1. S. cerevisiae and E.coli growth ................. 51
2.2.1.1. E.coli cell cultures .......................... 52
2.2.1.2. Preparation of competent DH5α E.coli cells .... 52
2.2.1.3. Yeast cell cultures ........................... 53
2.2.1.4. Yeast growth test ............................. 54
2.2.2. Molecular biology ............................... 54
2.2.2.1. Calculation of E. coli cell competence ........ 54
2.2.2.2. E. coli heat shock transformation ............. 55
2.2.2.3. Plasmid amplification and mini-prep ........... 55
2.2.2.4. Generation of strains by homologous recombination
56
2.2.2.5. Generation of strains by mating ............... 58
2.2.2.6. Transformation of S. cerevisiae cells ......... 59
2.2.3. Protein and DNA biochemistry .................... 60
2.2.3.1. Preparation of yeast cell lysates ............. 60
2.2.3.1.1. TCA precipitation ........................... 60
2.2.3.1.2. Denaturing glass-bead lysis ................. 60
2.2.3.2. SDS-PAGE ...................................... 61
2.2.3.3. Western blotting .............................. 63
2.2.3.4. Immnunodetection 64
2.2.3.5. Chromosomal DNA isolation from yeast .......... 65
5

2.2.3.6. Agarose gel electrophoresis ................... 66
2.2.3.7. Southern blotting ............................. 66
2.2.3.8. Cycloheximide decay experiments ............... 69
2.2.3.9. Pulse-chase experiments ....................... 69
3. Results ............................................. 72
3.1. Degradation studies on the human ERAD substrate CFTR
in yeast ................ 72
3.1.1. Systematic studies of CFTR degradation .......... 75
3.1.1.1. UPS components of the cytosol ................. 77
3.1.1.2. Observations on possible vacuolar degradation of
CFTR 82
3.1.1.3. Quality control of CFTR folding in the ER ..... 83
3.1.1.4. Conclusion .................................... 85
3.2. Non-glycosylated and glycosylated substrates in ERAD
88
3.2.1. Degradation studies on non-glycosylated CPY*0000 88
3.2.1.1. The vacuole is not required for CPY*0000
degradation. ............................................ 88
3.2.1.2. CPY*0000 is an ERAD-L substrate. .............. 89
3.2.1.3. ERAD-L requirements for CPY*0000 91
3.2.1.3.1. Lectins of the ER and quality control of
CPY*0000 91
3.2.1.3.1.1. The mannosidase Mnl1/Htm1 has no influence on
CPY*0000 degradation .................................... 92
6

3.2.1.3.1.2. The degradation efficiency of CPY*0000 in
YOS9 deleted cells is increased and is independent of the
MRH domain. 93
3.2.1.3.2. The degradation efficiency of CPY*0000
decreases in DER1 and USA1 deleted cells ................ 95
3.2.2. Mnl2, a novel component of the ER quality control
of misfolded glycoproteins. ............................. 97
3.2.2.1. Involvement of Mnl2 in ERAD ................... 99
3.2.2.2. Mnl2 is involved in glycan processing ........ 102
4. Discussion ......................................... 105
4.1. New function of the lectin Yos9 .................. 105
4.1.1. CPY*0000 is an ERAD-L substrate ................ 105
4.1.2. ERAD-L requirements for CPY*0000 ............... 107
4.2. Mnl2 is a novel putative α1-2 mannosidase of the ER
111
4.2.1. Mnl2 is involved in ERAD ....................... 112
4.3. Prospect ......................................... 115
5. References 116
Curriculum Vitae ....... 137





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Abbreviations

AAA ATPase associated with a variety of
cellular activities
Ac Acetate
Amp Ampicillin
AMP Adenosine monophosphate
APS Ammonium persulphate
ATP Adenosine triphosphate
ATPase Adenosintriphospatase
BSA Bovine serum albumine
cAMP Cyclic adenosine monophosphate
CHX Cycloheximide
CM Complete minimal medium
CPY Carboxypeptidase yscY (Gen PRC1)
CPY* Mutated CPY (allele prc1-1)
CPY*0000 Unglycosylated CPY*
ddH2O Double deionised water
DMF Dimethylformamide
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
Dnase Deoxyribonuclease
DTT Dithiotheitol
E. coli Escherichia coli
ECL Enhanced chemiluminescence
EDTA Ethylenediamine tetraacetic acid
ER Endoplasmic reticulum
ERAD ER associated degradation
Fig Figure
g Gram
h Hour
8

HA Haemaglutinin
Hect Homologous to E6-AP c-terminous
HRP Horse radish peroxidase
IB Immunoblot
IgG Immunoglobulin G
IP Immunoprecipitation
IPTG Isopropyl-β-D-thiogalactopyranoside
Kan Kanamycin
Kb Kilobase pair
l Liter
LB Luria Brooth
M Molar
min Minute
NEM N-ethiylmaleimide
OD Optical density
ORF Open reading frame
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffer saline
PBS-T -Tween20
PCR Polymerase chain reaction
PEG Polyethileneglycol
PGK 3-Phosphoglycerate kinase
PMSF Phenylmethylsulphonylfluoride
RING Really interesting new gene
rpm Revolutions per minute
RT Room temperature
S.cerevisiae Saccharomyces cerevisiae
SDS Sodium dodecylsulphate
T4 ligase Bacteriophage T4 ligase
TAE Tris acetate EDTA
TCA Trichloroacetic acid
TE Tris EDTA
9

TEMED Tetramethylethydiamine
Tris Tris (hydroxymethyl)aminomethane
TritonX-100 Akylphenylpolyethylenglycol
Tween 20 Polyoxyethylensorbitolmonolaurate
Ub Ubiquitin
UBA Ubiquitin associated domain
UBL Ubiquitin like domain
UV Ultraviolet
V Volts
v/v Volume/volume
w/v Weight/volume
WT Wild type
X-gal 5-Bromo-4-chloro-3-indolyl-β-D-
galactopyranoside














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