Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Köln
2019
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 110 Seiten Illustrationen 21 cm |
Internformat
MARC
| LEADER | 00000nam a2200000 c 4500 | ||
|---|---|---|---|
| 001 | BV046260824 | ||
| 003 | DE-604 | ||
| 005 | 00000000000000.0 | ||
| 007 | t | ||
| 008 | 191119s2019 gw a||| m||| 00||| eng d | ||
| 015 | |a 19,H12 |2 dnb | ||
| 016 | 7 | |a 1192916581 |2 DE-101 | |
| 020 | |c Broschur | ||
| 035 | |a (OCoLC)1128834089 | ||
| 035 | |a (DE-599)DNB1192916581 | ||
| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
| 044 | |a gw |c XA-DE | ||
| 049 | |a DE-355 | ||
| 084 | |a 570 |2 sdnb | ||
| 100 | 1 | |a Schuster, Ramona |d 1989- |e Verfasser |0 (DE-588)1191306542 |4 aut | |
| 245 | 1 | 0 | |a Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |c vorgelegt von Ramona Schuster |
| 264 | 1 | |a Köln |c 2019 | |
| 300 | |a 110 Seiten |b Illustrationen |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität zu Köln |d 2019 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1192916581/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031638830&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-031638830 | ||
Datensatz im Suchindex
| _version_ | 1804180695579361280 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
1
INTRODUCTION
..............................................................................................
1
1.1
T
HE
UBIQUITIN
-
PROTEASOME
SYSTEM
...........................................................................................................................
1
1.1.1
UBIQUITIN
AND
UBIQUITYLATION
..................................................................................................................................
1
1.1.2
UBIQUITIN-SPECIFIC
PEPTIDASES
...............................................................................................................................
5
1.2
T
HE
UBIQUITIN
-
SPECIFIC
CHAPERONE
C
DC
48
.............................................................................................................
7
1.3
M
ITOCHONDRIA
......................................................................................................................................................................
8
1.3.1
MITOCHONDRIAL
DYNAMICS
.......................................................................................................................................
10
1.3.1.1
LARGE
DYNAMIN-RELATED
GTPASES
IN
MITOCHONDRIAL
DYNAMICS
...................................................................
11
1.3.1.2
MITOCHONDRIAL
FISSION
...........................................................................................................................................
12
1.3.1.3
MITOCHONDRIAL
FUSION
............................................................................................................................................
13
1.3.1.4
THE
YEAST
MITOFUSIN
FZO1
..................................................................................................................................
15
1.4
A
IM
..........................................................................................................................................................................................
17
2
RESULTS
....................................................................................................
20
2.1
M
ITOCHONDRIAL
TUBULATION
DEPENDS
ON
C
DC
48
................................................................................................
20
2.2
C
DC
48
LOSS
-
OF
-
FUNCTION
MUTATION
DOES
NOT
RESULT
IN
MT
DNA
LOSS
...................................................
23
2.3
M
ITOCHONDRIAL
PHENOTYPES
OF
CDC
48-2
MUTANT
CELLS
CAN
BE
RESCUED
BY
EXPRESSION
OF
FUNCTIONAL
CDC48
................................................................................................................................................................
24
2.4
C
DC
48
SPECIFICALLY
AFFECTS
UBIQUITYLATED
F
ZO
1
...........................................................................................
26
2.5
C
DC
48
SUPPORTS
UBIQUITIN
-
DEPENDENT
TURNOVER
OF
U
BP
12
....................................................................
28
2.6
C
DC
48
REGULATES
F
ZO
1
VIA
U
BP
12
..........................................................................................................................
31
2.7
UBP12
AND
UBP2
HAVE
OPPOSING
EFFECTS
ON
FZO1
..........................................................................................
34
2.8
U
BP
2
IS
A
SUBSTRATE
OF
UBP12
..................................................................................................................................
35
2.9
B
OTH
U
BP
1
2
SUBSTRATES
,
F
ZO
1
AND
U
BP
2,
ARE
UBIQUITYLATED
WITH
K48-
LINKED
UBIQUITIN
CHAINS
36
2.10
UBP12
IS
AN
UNSPECIFIC
DUB
THAT
CLEAVES
BOTH
K48
AND
K63-LINKED
UBIQUITIN
CHAINS
............
37
2.11
FZO1
REGULATION
BY
UBP2
AND
UBP12
IS
GOVERNED
BY
COC48
.................................................................
38
2.12
C
HARACTERIZATION
OF
THE
CONSERVED
UBIQUITYLATION
PATTERN
OF
F
ZO
1
............................................
39
2.13
M
UTATION
OF
F
ZO
1
-K398
RENDERS
MITOCHONDRIA
FUSION
-
INCOMPETENT
..............................................
44
2.14
T
HE
UBIQUITYLATION
PATTERN
OF
THE
K398R
MUTANT
OF
F
ZO
1
HARBORS
K48-
LINKED
UBIQUITIN
CHAINS,
TARGETING
THE
PROTEIN
FOR
PROTEASOMAL
DEGRADATION
....................................................................
47
2.15
T
HE
CONSERVED
UBIQUITYLATION
PATTERN
ON
F
ZO
1
IS
REQUIRED
FOR
FUSION
BEYOND
F
ZO
1
STABILITY
....................................................................................................................................................................................
48
2.16
LOSS
OF
WILD-TYPE
UBIQUITYLATION
PATTERN
RENDERS
FZO1
INSENSITIVE
TO
THE
DUB
UBP2
.......
50
2.17
K398
MUTATION
OF
FZO1
DOES
NOT
IMPAIR
FZO1
COMPLEX
FORMATION
....................................................
52
2.18
CDC48
STILL
BINDS
FZO1
K398R
....................................................................................................................................
53
2.19
D
YNAMIC
CHANGES
IN
HELIX
A
5
OF
FZO1
CAN
COMPENSATE
FOR
THE
UBIQUITYLATION
DEFECT
IN
FZO1
K398R
.....................................................................................................................................................................................
54
3
DISCUSSION
.............................................................................................
59
3.1
T
HE
ROLE
OF
C
DC
48
IN
MITOCHONDRIAL
DYNAMICS
...............................................................................................
59
3.2
C
DC
48
REGULATES
F
ZO
1
VIA
REGULATION
OF
A
DUB
CASCADE
.......................................................................
59
3.3
N
PL
4/U
FD
1
AND
DO
AL
ARE
INVOLVED
IN
C
DC
48-
DEPENDENT
REGULATION
OF
F
ZO
1
................................
60
3.4
FZO1
IS
DUALLY
REGULATED
BY
TWO
DIFFERENT
KINDS
OF
UBIQUITYLATION
PATHWAYS
............................
61
3.5
UBP1
2
AND
UBP2
HAVE
OPPOSING
FUNCTIONS
ON
FZO1
AND
BEYOND
...........................................................
61
3.6
UBP2
AND
UBP12
ARE
REGULATED
BY
UBIQUITYLATION
........................................................................................
62
3.7
UBP12
SPECIFICALLY
REMOVES
STABILIZING
UBIQUITYLATION
FROM
SUBSTRATES
......................................
62
3.8
FZO1
SLOWER
MIGRATING
FORMS
CONSIST
OF
DI-UBIQUITIN
CHAINS
.................................................................
63
3.9
A
DDRESSING
THE
SHIFTED
SLOWER
MIGRATING
UBIQUITYLATED
FORMS
OF
F
ZO
1
.......................................
64
3.10
LOW
LEVELS
OF
FZO1
K398R
ARE
NOT
THE
REASON
FOR
MITOCHONDRIAL
FRAGMENTATION
....................
66
3.11
T
HE
K398R
MUTATION
OF
F
ZO
1
RENDERS
YEAST
FUSION
INCOMPETENT
UPON
HEAT
STRESS
............
66
3.12
T
HE
CONTRADICTORY
FUNCTION
OF
K48
UBIQUITIN
CHAINS
ON
F
ZO
1
...........................................................
66
3.13
CDC48
STILL
RECOGNIZES
FZO1
K398R
.......................................................................................................................
67
3.14
FZO1
K398R
IS
STILL
ABLE
TO
FORM
HIGHER
OLIGOMER
COMPLEXES
................................................................
68
3.15
H
ELIX
A
5
OF
F
ZO
1
IS
IMPORTANT
FOR
MITOCHONDRIAL
FUSION
......................................................................
68
3.16
A
MODEL
FOR
MITOCHONDRIAL
FUSION
.....................................................................................................................
69
3.17
F
INAL
REMARKS
...............................................................................................................................................................
70
4
MATERIAL
AND
METHODS
...........................................................................
72
4.1
M
ATERIAL
.............................................................................................................................................................................
72
4.1.1
YEAST
STRAINS
.............................................................................................................................................................
72
4.1.2
PLASMIDS
......................................................................................................................................................................
74
4.1.3
PRIMERS
........................................................................................................................................................................
76
4.1.4
ANTIBODIES
USED
IN
THIS
STUDY
............................................................................................................................
76
4.2
M
ETHODS
.............................................................................................................................................................................
79
4.2.1
YEAST
GENETICS
..........................................................................................................................................................
79
4.2.1.1
MEDIA
AND
GROWTH
CONDITIONS
............................................................................................................................
79
4.2.1.2
PLASMID
TRANSFORMATION
.....................................................................................................................................
81
4.2.1.3
HIGH
EFFICIENCY
TRANSFORMATION
FOR
GENE
DELETION
.........................................................................................
81
4.2.1.4
MATING,
SPORULATION
AND
PLASMID
DISSECTION
.................................................................................................
82
4.2.1.5
SPOT
TEST
................................................................................................................................................................
82
4.2.1.6
ALKALINE
LYSIS
FOR
WHOLE
CELL
EXTRACTS
...............................................................................................................
82
4.2.1.7
ISOLATION
OF
CRUDE
MEMBRANES
.........................................................................................................................
83
4.2.1.8
IMMUNOPRECIPITATION
(OF
FZO1)
........................................................................................................................
83
4.2.1.9
CYCLOHEXIMIDE
AND
MG
132
CHASE
..................................................................................................................
83
4.2.1.10
MICROSCOPY
.........................................................................................................................................................84
4.2.1.11
DUB
PURIFICATION:
UBP12
PURIFICATION
BY
IMMUNOPRECIPITATION
............................................................... 84
4.2.1.12
DUB
PURIFICATION
BY
AFFINITY
CHROMATOGRAPHY
AND
SIZE
EXCLUSION
CHROMATOGRAPHY
...........................84
4.2.1.13
DUB
ASSAY
.........................................................................................................................................................
85
4.2.1.14
MATING
ASSAY
.....................................................................................................................................................86
4.2.1.15
RNASE/DNASE
ASSAY
......................................................................................................................................
87
4.1.2.16
DEPHOSPHORYLATION
ASSAY
...............................................................................................................................87
4.1.2.17
ACETYLATION
ASSAY
.............................................................................................................................................87
4.1.2.18
GLYCOSYLATION
ASSAY
........................................................................................................................................
87
4.2.2
PROTEIN
BIOCHEMISTRY.
............................................................................................................................................
88
4.2.2.1
SDS
PAGE
-
TRIS/GLYCINE
..................................................................................................................................
88
4.2.2.2
SDS
PAGE-
TRIS/TRICINE
.....................................................................................................................................
88
4.3.2.3
WESTERN
BLOT
.........................................................................................................................................................
89
4.2.2.4
IMMUNOBLOTTING
....................................................................................................................................................89
4.2.3
MOLECULAR
BIOLOGY
..................................................................................................................................................
90
4.2.3.1
E.COLI
MEDIA
AND
GROWTH
CONDITIONS
................................................................................................................90
4.2.3.2
E.COLI
PLASMID
TRANSFORMATION
...........................................................................................................................90
4.2.3.3
SITE-DIRECTED
MUTAGENESIS
................................................................................................................................
90
4.2.3.4
AMPLIFICATION
OF
DELETION
CASSETTE
...................................................................................................................
91
4.2.3.5
STRAIN
CONFIRMATION
BY
COLONY
PCR
................................................................................................................
92
4.2.3.6
MTDNA
MEASUREMENT
BY
QRT-PCR
...............................................................................................................
93
4.2.4
STRUCTURAL
BIOLOGY
...................................................................................................................................................
93
4.2.4.1
STRUCTURAL
MODELING
OF
FZO1
..............................................................................................................................
93
5
REFERENCES
.............................................................................................
95
6
LIST
OF
ABBREVIATIONS
..........................................................................
103
7
LIST
OF
FIGURES
.......................................................................................
104
8
KURZZUSAMMENFASSUNG
.....................................................................
105
9
ACKNOWLEDGEMENTS
.............................................................................
107
10
EIDESSTATTLICHE
ERKLARUNG
................................................................
109
11
CURRICULUM
VITAE
................................................................................
110
|
| any_adam_object | 1 |
| author | Schuster, Ramona 1989- |
| author_GND | (DE-588)1191306542 |
| author_facet | Schuster, Ramona 1989- |
| author_role | aut |
| author_sort | Schuster, Ramona 1989- |
| author_variant | r s rs |
| building | Verbundindex |
| bvnumber | BV046260824 |
| ctrlnum | (OCoLC)1128834089 (DE-599)DNB1192916581 |
| discipline | Biologie |
| format | Thesis Book |
| fullrecord | <?xml version="1.0" encoding="UTF-8"?><collection xmlns="http://www.loc.gov/MARC21/slim"><record><leader>01337nam a2200349 c 4500</leader><controlfield tag="001">BV046260824</controlfield><controlfield tag="003">DE-604</controlfield><controlfield tag="005">00000000000000.0</controlfield><controlfield tag="007">t</controlfield><controlfield tag="008">191119s2019 gw a||| m||| 00||| eng d</controlfield><datafield tag="015" ind1=" " ind2=" "><subfield code="a">19,H12</subfield><subfield code="2">dnb</subfield></datafield><datafield tag="016" ind1="7" ind2=" "><subfield code="a">1192916581</subfield><subfield code="2">DE-101</subfield></datafield><datafield tag="020" ind1=" " ind2=" "><subfield code="c">Broschur</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(OCoLC)1128834089</subfield></datafield><datafield tag="035" ind1=" " ind2=" "><subfield code="a">(DE-599)DNB1192916581</subfield></datafield><datafield tag="040" ind1=" " ind2=" "><subfield code="a">DE-604</subfield><subfield code="b">ger</subfield><subfield code="e">rda</subfield></datafield><datafield tag="041" ind1="0" ind2=" "><subfield code="a">eng</subfield></datafield><datafield tag="044" ind1=" " ind2=" "><subfield code="a">gw</subfield><subfield code="c">XA-DE</subfield></datafield><datafield tag="049" ind1=" " ind2=" "><subfield code="a">DE-355</subfield></datafield><datafield tag="084" ind1=" " ind2=" "><subfield code="a">570</subfield><subfield code="2">sdnb</subfield></datafield><datafield tag="100" ind1="1" ind2=" "><subfield code="a">Schuster, Ramona</subfield><subfield code="d">1989-</subfield><subfield code="e">Verfasser</subfield><subfield code="0">(DE-588)1191306542</subfield><subfield code="4">aut</subfield></datafield><datafield tag="245" ind1="1" ind2="0"><subfield code="a">Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1</subfield><subfield code="c">vorgelegt von Ramona Schuster</subfield></datafield><datafield tag="264" ind1=" " ind2="1"><subfield code="a">Köln</subfield><subfield code="c">2019</subfield></datafield><datafield tag="300" ind1=" " ind2=" "><subfield code="a">110 Seiten</subfield><subfield code="b">Illustrationen</subfield><subfield code="c">21 cm</subfield></datafield><datafield tag="336" ind1=" " ind2=" "><subfield code="b">txt</subfield><subfield code="2">rdacontent</subfield></datafield><datafield tag="337" ind1=" " ind2=" "><subfield code="b">n</subfield><subfield code="2">rdamedia</subfield></datafield><datafield tag="338" ind1=" " ind2=" "><subfield code="b">nc</subfield><subfield code="2">rdacarrier</subfield></datafield><datafield tag="502" ind1=" " ind2=" "><subfield code="b">Dissertation</subfield><subfield code="c">Universität zu Köln</subfield><subfield code="d">2019</subfield></datafield><datafield tag="655" ind1=" " ind2="7"><subfield code="0">(DE-588)4113937-9</subfield><subfield code="a">Hochschulschrift</subfield><subfield code="2">gnd-content</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">B:DE-101</subfield><subfield code="q">application/pdf</subfield><subfield code="u">https://d-nb.info/1192916581/04</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="856" ind1="4" ind2="2"><subfield code="m">DNB Datenaustausch</subfield><subfield code="q">application/pdf</subfield><subfield code="u">http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031638830&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA</subfield><subfield code="3">Inhaltsverzeichnis</subfield></datafield><datafield tag="999" ind1=" " ind2=" "><subfield code="a">oai:aleph.bib-bvb.de:BVB01-031638830</subfield></datafield></record></collection> |
| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV046260824 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T08:39:51Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-031638830 |
| oclc_num | 1128834089 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | 110 Seiten Illustrationen 21 cm |
| publishDate | 2019 |
| publishDateSearch | 2019 |
| publishDateSort | 2019 |
| record_format | marc |
| spelling | Schuster, Ramona 1989- Verfasser (DE-588)1191306542 aut Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 vorgelegt von Ramona Schuster Köln 2019 110 Seiten Illustrationen 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2019 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1192916581/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031638830&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schuster, Ramona 1989- Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |
| subject_GND | (DE-588)4113937-9 |
| title | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |
| title_auth | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |
| title_exact_search | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |
| title_full | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 vorgelegt von Ramona Schuster |
| title_fullStr | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 vorgelegt von Ramona Schuster |
| title_full_unstemmed | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 vorgelegt von Ramona Schuster |
| title_short | Regulation of mitochondrial outer membrane fusion by ubiquitylation of Fzo1 |
| title_sort | regulation of mitochondrial outer membrane fusion by ubiquitylation of fzo1 |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1192916581/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=031638830&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schusterramona regulationofmitochondrialoutermembranefusionbyubiquitylationoffzo1 |
Es ist kein Print-Exemplar vorhanden.
Inhaltsverzeichnis