Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | German |
| Veröffentlicht: |
1998
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | München, Univ., Diss., 1998 |
| Beschreibung: | 125 S. graph. Darst. |
Internformat
MARC
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| 001 | BV012500482 | ||
| 003 | DE-604 | ||
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| 008 | 990323s1998 gw d||| m||| 00||| ger d | ||
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| 035 | |a (OCoLC)246454132 | ||
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| 100 | 1 | |a Rojo, Elena |d 1968- |e Verfasser |0 (DE-588)12060275X |4 aut | |
| 245 | 1 | 0 | |a Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |c vorgelegt von Elena Rojo |
| 264 | 1 | |c 1998 | |
| 300 | |a 125 S. |b graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a München, Univ., Diss., 1998 | ||
| 650 | 0 | 7 | |a Mitochondrium |0 (DE-588)4124939-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Protein-Sortierung |0 (DE-588)4533502-3 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Membranproteine |0 (DE-588)4130026-9 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Mitochondrium |0 (DE-588)4124939-2 |D s |
| 689 | 0 | 1 | |a Membranproteine |0 (DE-588)4130026-9 |D s |
| 689 | 0 | 2 | |a Protein-Sortierung |0 (DE-588)4533502-3 |D s |
| 689 | 0 | |5 DE-604 | |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-008486381 | |
Datensatz im Suchindex
| _version_ | 1807681675764695040 |
|---|---|
| adam_text |
CONTENTS
1.
INTRODUCTION
1
1.1.
PROTEIN
TRANSLOCATION
SYSTEMS
IN
THE
CELL
1
1.2.
STRUCTURE
AND
FUNCTION
OF
MITOCHONDRIA
3
1.3.
MITOCHONDRIAL
IMPORT
SIGNALS
4
1.4.
IMPORT
PATHWAYS
4
1.4.1.
THE
GENERAL
IMPORT
PATHWAY
4
1.4.2.
THE
IMPORT
PATHWAY
FOR
MEMBERS
OF
THE
CARRIER
FAMILY
8
1.5.
SORTING
TO
THE
INNER
MEMBRANE
8
1.5.1.
CONSERVATIVE
SORTING
9
1.5.2.
ARREST
IN
THE
INNER
MEMBRANE
DURING
IMPORT
10
1.5.3.
THE
SPECIAL
CASES
OF
CYTOCHROME
I 2
CYTOCHROME
CI
AND
NADH-CYTOCHROME
HS-REDUCTASE:
THE
BIPARTITE
TARGETING
SEQUENCE
11
1.5.4.
D-LACTATE
DEHYDROGENASE
12
1.6.
PROTEIN
SORTING
IN
PROKARYOTES
13
1.6.1.
THE
BACTERIAL
SORTING
MACHINERIES
13
1.6.2.
TOPOLOGICAL
RULES
IN
.REC-INDEPENDENT
N-TERMINAL
EXPORT
AND
DETERMINATION
OF
MEMBRANE
TOPOLOGY
IN
PROKARYOTES
14
1.7.
AIM
OF
THIS
STUDY
16
2.
MATERIAL
AND
METHODS
18
2.1.
MATERIAL
18
2.1.1.
STRAINS
USED
IN
THIS
STUDY
18
2.1.1.1
E.
COLI
STRAINS
18
2.1.1.2.
S.
CEREVISIAE
STRAINS
18
2.1.2.
CHEMICALS
AND
ENZYMES
18
2.1.3.
MEDIA
20
2.1.3.1.
E.
COLI
20
2.1.3.2.
S.
CEREVISIAE
20
2.1.4.
EQUIPMENT
21
2.2.
METHODS
22
2.2.1.
MOLECULAR
BIOLOGICAL
METHODS
22
2.2.1.1.
PLASMID
DNA
PREPARATION
22
-
SMALL
SCALE
PREPARATION
(MINIPREPARATION)
22
-
MEDIUM
SCALE
PREPARATION
(MIDIPREPARATION)
23
2.2.1.2.
STORAGE
OF
E.
COLI
AND
5.
CEREVISIAE
STRAINS
AND
TRANSFORMANTS
23
2.2.1.3.
AMPLIFICATION
OF
DNA
WITH
THE
POLYMERASE
CHAIN
REACTION
(PCR)
24
2.2.1.4.
MODIFICATION
OF
DNA
FOR
CLONING
25
-
RESTRICTION
DIGESTION
25
-
DEPHOSPHORYLATION
25
-
FILL-IN
OF
DNA
OVERHANGING
ENDS
WITH
THE
KLENOW
ENZYME
25
-
LIGATION
26
2.2.1.5.
PURIFICATION
AND
ANALYSIS
OF
DNA
26
-
DETERMINATION
OF
THE
DNA
CONCENTRATION
26
-
PHENOL-EXTRACTION
AND
ETHANOL
PRECIPITATION
26
-
SEPARATION
OF
DN
A
FRAGMENTS
BY
GEL
ELECTROPHORESIS
27
AGAROSE
GEL
ELECTROPHORESIS
27
TAE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(TAE-PAGE)
27
-
ISOLATION
OF
DNA
FRAGMENTS
FROM
ELECTROPHORESIS
GELS
27
-
DNA
SEQUENCING
28
2.2.1.6.
PCR
SITE-DIRECTED
MUTAGENESIS
28
2.2.1.7.
PREPARATION
OF
ELECTROCOMPETENT
E.
COLI
CELLS
30
2.2.1.8.
TRANSFORMATION
OF
E.
COLI
CELLS
BY
ELECTROPORATION
30
2.2.1.9.
CONSTRUCTION
OF
THE
PLASMIDS
USED
IN
THIS
STUDY
31
-
FOR
THE
STUDY
OF
THE
SORTING
OF
THE
SUBUNIT
9
OF
THE
FO-ATPASE
OF
N.
CRASSA
31
-
FOR
THE
STUDY
OF
THE
SORTING
OF
THE
D-LACTATE
DEHYDROGENASE
OF
S.
CEREVISIAE
32
-
PRIMERS
USED
IN
THIS
STUDY
34
2.2.2.
PROTEIN
BIOCHEMISTRY
METHODS
37
2.2.2.1.
PURIFICATION
OF
RECOMBINANT
PROTEINS
FROM
E.
COLI
37
-
IPTG-INDUCIBLE
OVERPRODUCTION
OF
PROTEINS
IN
E.
COLI
37
-
PURIFICATION
OF
(HISG)-TAGGED
PROTEINS
BY
NI-NTA
CHROMATOGRAPHY
37
2.2.2.2.
DETERMINATION
OF
PROTEIN
CONCENTRATION
BY
THE
METHOD
OF
BRADFORD
38
2.2.2.3.
TCA
PRECIPITATION
OF
PROTEINS
38
2.2.2.4.
SYNTHESIS
OF
MITOCHONDRIAL
PRECURSOR
PROTEINS
IN
VITRO
38
-
IN
VITRO
TRANSCRIPTION
38
-
IN
VITRO
TRANSLATION
39
2.2.3.
METHODS
OF
CELL
BIOLOGY
40
2.2.3.1.
S.CEREVISIAE
CULTURES
40
2.2.3.2.
ISOLATION
OF
MITOCHONDRIA
FROM
S.
CEREVISIAE
40
2.2.3.3.
IMPORT
OF
IN
VITRO
SYNTHESIZED
PRECURSOR
PROTEINS
INTO
ISOLATED
MITOCHONDRIA
41
2.2.3.4.
INHIBITION
OF
THE
MITOCHONDRIAL
PROCESSING
PEPTIDASE
(MPP)
BY
METAL
CHELATORS
AND
ITS
REACTIVATION
41
2.2.3.5.
ATP
DEPLETION
42
-
DEPLETION
OF
THE
MITOCHONDRIAL
MATRIX
ATP
42
-
DEPLETION
OF
THE
ATP
OUTSIDE
THE
MITOCHONDRIA
42
2.2.3.6.
PREPARATION
OF
MITOPLASTS
BY
OSMOTIC
SHOCK
(SWELLING)
42
2.2.3.7.
CROSS-LINKING
43
2.2.3.8.
DIGITONIN
FRACTIONATION
43
2.2.3.9.
ASSAY
FOR
FOLDING
OF
IMPORTED
AND
ENDOGENOUS
PROTEINS
THROUGH
TRITON
X-100
LYSIS
AND
PROTEASE
TREATMENT
44
2.2.3.10.
SUBFRACTIONATION
OF
MITOCHONDRIA
BY
SONICATION
44
2.2.3.11.
GEL
FILTRATION
ANALYSIS
44
2.2.4.
ANALYSIS
OF
PROTEINS
45
2.2.4.1.
SDS-POLYACRYLAMIDE
ELECTROPHORESIS
(SDS-PAGE)
45
2.2.4.2.
COOMASSIE
STAINING
OF
PROTEIN
ACRYLAMIDE
GELS
46
2.2.4.3.
TRANSFER
OF
ELECTROPHORETICALLY
SEPARATED
PROTEINS
TO
A
NITROCELLULOSE
MEMBRANE
46
2.2.4.4.
AUTORADIOGRAPHY,
FLUOROGRAPHY
AND
DENSITOMETRY
46
2.2.5.
IMMUNOLOGICAL
METHODS
47
2.2.5.1.
COUPLING
OF
A
PEPTIDE
TO
A
CARRIER
PROTEIN
47
2.2.5.2.
PREPARATION
OF
RABBIT
ANTISERA
47
2.2.5.3.
IMMUNOPRECIPITATION
AND
CO-IMMUNOPRECIPITATION
48
2.2.5.4.
WESTERN
BLOT
48
2.2.5.5.
COVALENT
COUPLING
OF
ANTIBODIES
TO
PROTEIN
A-SEPHAROSE
49
3.
RESULTS
50
3.1.
BIOGENESIS
OF
SUBUNIT
9
OF
THE
FIFO-ATPASE
50
3.1.1.
PRESU9(L-U2)-DHFR
IS
CORRECTLY
SORTED
IN
YEAST
MITOCHONDRIA
50
3.1.2.
THE
PRESENCE
OF
THE
HYDROPHOBIC
DOMAIN
IN
PRESU9(L-L
12)-DHFR
RENDERS
MT-HSP70
DISPENSABLE
FOR
PRESEQUENCE
STABILIZATION
DURING
IMPORT
52
3.1.3.
THE
IMPORT
AND
EXPORT
STEPS
OF
PRESU9(L-L
12)-DHFR
ARE
TWO
DISTINCT
EVENTS
54
3.1.4.
MAINTENANCE
OF
THE
PRESEQUENCE
PREVENTS
EXPORT
OF
PRESU9(L-L
12)-DHFR
FROM
THE
MATRIX
54
3.1.5.
EXPORT
OF
PRESU9(L-L
12)-DHFR
FROM
THE
MATRIX
REQUIRES
APH
AND
MATRIX
ATP
HYDROLYSIS
55
3.1.6.
PRESU9(L-L
12)-DHFR
CAN
BE
SHOWN
TO
INTERACT
WITH
THE
TIM
MACHINERY,
OXALP
AND
TIML
1
58
3.2.
SORTING
REQUIREMENTS
OF
PRESU9(L-112)-DHFR:
ROLE
OF
THE
CHARGES
FLANKING
THE
HYDROPHOBIC
CORE
63
3.2.1.
REPLACEMENT
OF
THE
N-TERMINAL
POSITIVELY-CHARGED
RESIDUE
IN
PRESU9
(1-1
12)-DHFR
BY
AN
UNCHARGED
ONE
RESULTS
IN
MORE
EFFICIENT
SORTING
TO
THE
INTERMEMBRANE
SPACE
63
3.2.2.
AN
N-TERMINAL
TAIL
BEARING
A
NET
POSITIVE
CHARGE
IS
RETAINED
IN
THE
MATRIX
66
3.2.3.
ROLE
OF
THE
NET
POSITIVE
CHARGE
C-TERMINAL
TO
THE
TRANSMEMBRANE
SEGMENT
68
3.2.4.
REVERSING
THE
NET
CHARGE
FLANKING
THE
HYDROPHOBIC
DOMAIN
RESULTS
IN
A
PARTIAL
INVERSION
OF
THE
TOPOLOGY
OF
PRESU9(
1
-11
2)-DHFR
71
3.2.5.
DEPLETION
OF
MATRIX
ATP
LEADS
TO
A
REVERSAL
OF
TOPOLOGY
OF
PRESU9(L-L
12)-DHFR
BUT
DOES
NOT
AFFECT
THE
SORTING
OF
PRESU9(L-L
12)
E7OK,E77K,R111D,N112D-DHFR
74
3.3.
BIOGENESIS
OF
THE
D(+)-LACTATE
DEHYDROGENASE
(DLD)
75
3.3.1.
DETERMINATION
OF
THE
TOPOLOGY
OF
DLD
75
3.3.2.
IMPORTED
RADIOLABELLED
PREDLD
ATTAINS
THE
CORRECT
ORIENTATION
IN
VITRO
75
3.3.3.
SORTING
SIGNAL
REQUIREMENTS
FOR
DLD
77
3.3.4.
ROLE
OF
HYDROPHOBICITY
FOR
THE
SORTING
OF
DLD
80
3.3.5.
EXTERNAL
ATP
AND
MATRIX
ATP/MT-HSP70
REQUIREMENTS
FOR
IMPORT
AND
SORTING
OF
DLD
81
3.3.6.
IMPORTED
DLD
FOLDS
IN
THE
INTERMEMBRANE
SPACE
TO
ITS
NATIVE
CONFORMATION
82
3.3.7.
IMPORTED
DLD
FORMS
A
HIGH
MOLECULAR
MASS
COMPLEX
87
4.
DISCUSSION
89
4.1.
SORTING
OF
SU9
OF
THE
FIFO-ATPASE
89
4.1.1.
THE
SORTING
OF
SU9
OCCURS
VIA
A
CONSERVATIVE
PATHWAY
89
4.1.2.
DO
ALL
MITOCHONDRIAL
INNER
MEMBRANE
PROTEINS
WHICH
UNDERGO
EXPORT
FROM
THE
MATRIX
HAVE
THE
SAME
REQUIREMENTS
FOR
THE
EXPORT
STEP?
91
4.1.3.
REQUIREMENTS
FOR
EXPORT:
THE
"POSITIVE
INSIDE
RULE"
APPLIES
TO
MITOCHONDRIA
92
4.1.4.
INFLUENCE
OF
THE
FLANKING
CHARGES
C-TERMINAL
TO
THE
TRANSMEMBRANE
DOMAIN
ON
THE
EXPORT
OF
THE
N-TERMINAL
TAIL
93
4.1.5.
AN
EXPORT
MACHINERY?
94
4.1.6.
ARE
ALL
INNER
MEMBRANE
PROTEINS
SORTED
THROUGH
AN
EXPORT
STEP?
96
4.1.7.
REVERSAL
OF
THE
NET
CHARGE
BETWEEN
N
AND
C-TERMINI
OF
PRESU9(
1-11
2)-DHFR
LEADS
TO
A
PARTIAL
REVERSAL
OF
THE
TOPOLOGY
97
4.2.
SORTING
OF
DLD
98
4.2.1.
THE
HYDROPHOBIC
CORE
OF
DLD
ACTS
AS
SORTING
SIGNAL
AND
MEMBRANE
ANCHOR
98
4.2.2.
A
ROLE
FOR
THE
CHARGES
FLANKING
THE
HYDROPHOBIC
DOMAIN
IN
THE
GENERATION
OF
THE
NI
N
-C
O
UT
TOPOLOGY?
99
4.2.3.
RECOGNITION
OF
THE
COMPOSITE
SORTING
SIGNAL
OF
DLD?
101
4.2.4.
ROLE
OF
THE
HYDROPHOBICITY
IN
THE
SORTING
OF
DLD
102
4.2.5.
WHAT
ARE
THE
ENERGETIC
REQUIREMENTS
FOR
ATTAINING
THE
NI
N
-C
OU
T
TOPOLOGY?
103
4.2.6.
FOLDING
IN
THE
IMS:
A
PHENOMENON
LOOKING
FOR
A
MECHANISM
103
4.2.7.
OLIGOMERIZATION
AS
DRIVING
FORCE
FOR
IMPORT?
104
4.2.8.
A
MODEL
FOR
THE
SORTING
OF
DLD?
105
4.3.
PERSPECTIVES
107
5.
SUMMARY
109
6.
REFERENCES
111 |
| any_adam_object | 1 |
| author | Rojo, Elena 1968- |
| author_GND | (DE-588)12060275X |
| author_facet | Rojo, Elena 1968- |
| author_role | aut |
| author_sort | Rojo, Elena 1968- |
| author_variant | e r er |
| building | Verbundindex |
| bvnumber | BV012500482 |
| ctrlnum | (OCoLC)246454132 (DE-599)BVBBV012500482 |
| format | Book |
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| indexdate | 2024-08-18T00:06:24Z |
| institution | BVB |
| language | German |
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| open_access_boolean | |
| owner | DE-703 |
| owner_facet | DE-703 |
| physical | 125 S. graph. Darst. |
| publishDate | 1998 |
| publishDateSearch | 1998 |
| publishDateSort | 1998 |
| record_format | marc |
| spelling | Rojo, Elena 1968- Verfasser (DE-588)12060275X aut Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins vorgelegt von Elena Rojo 1998 125 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 1998 Mitochondrium (DE-588)4124939-2 gnd rswk-swf Protein-Sortierung (DE-588)4533502-3 gnd rswk-swf Membranproteine (DE-588)4130026-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Mitochondrium (DE-588)4124939-2 s Membranproteine (DE-588)4130026-9 s Protein-Sortierung (DE-588)4533502-3 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008486381&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rojo, Elena 1968- Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins Mitochondrium (DE-588)4124939-2 gnd Protein-Sortierung (DE-588)4533502-3 gnd Membranproteine (DE-588)4130026-9 gnd |
| subject_GND | (DE-588)4124939-2 (DE-588)4533502-3 (DE-588)4130026-9 (DE-588)4113937-9 |
| title | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |
| title_auth | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |
| title_exact_search | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |
| title_full | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins vorgelegt von Elena Rojo |
| title_fullStr | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins vorgelegt von Elena Rojo |
| title_full_unstemmed | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins vorgelegt von Elena Rojo |
| title_short | Mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |
| title_sort | mechanisms of sorting and determination of topology of mitochondrial inner membrane proteins |
| topic | Mitochondrium (DE-588)4124939-2 gnd Protein-Sortierung (DE-588)4533502-3 gnd Membranproteine (DE-588)4130026-9 gnd |
| topic_facet | Mitochondrium Protein-Sortierung Membranproteine Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008486381&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT rojoelena mechanismsofsortinganddeterminationoftopologyofmitochondrialinnermembraneproteins |