Interplay Between Nuclear Import and Cell Cycle Control Open Access
Pulliam, Kanika Faye (2009)
Published
Abstract
The bi-directional transport of proteins across the nuclear envelope via nuclear pore complexes is a highly regulated process that involves many different factors. The classical nuclear import pathway is the most studied mechanism for the transport of proteins into the nucleus. During classical nuclear import, proteins are shuttled into the nucleus by an internal targeting sequence termed the classical nuclear localization signal (cNLS) via interactions with nuclear import receptors. The cNLS receptor, importin- alpha recognizes the cNLS-containing protein or cargo in the cytoplasm as the initial step in the transport process. In an effort to investigate how the classical nuclear transport mechanism affects a fundamental process in biology, we examined the interplay between nuclear transport and progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. In eukaryotes, a subset of proteins are transported across the nuclear envelope in a cell cycle dependent manner. Previous studies support a link between nuclear transport and the cell cycle but these studies did not demonstrate a direct role of the nuclear transport machinery in cell cycle regulation. We first demonstrated that the cNLS cargo binding affinity for importin- alpha dictates the rate of import of a cNLS cargo into the nucleus, suggesting that cargo binding to the receptor regulates the import process. We next hypothesized that temporally regulated interactions between specific cNLS cargoes and importin alpha are required for progression through the cell cycle. Results of our study exploiting importin alpha mutants with defects in cargo binding and release showed that a defect in cNLS cargo recognition causes a profound delay in progression through the G1/S transition of the cell cycle. In conclusion, cNLS cargo recognition by the classical nuclear import receptor, importin- alpha is required for efficient transition through the G1/S stage of the cell cycle. This is the first study to demonstrate a direct role of importin- alpha in regulation of the G1/S phase of the cell cycle. In conclusion, this work defines the rate-limiting step in the classical nuclear import process and then reveals that the G1/S stage of the cell cycle is strongly dependent on classical protein import.
Table of Contents
CHAPTER 1.
GENERAL
INTRODUCTION..............................................................
1
1.1 The Nucleus and Cell
Compartmentalization..............................................................
1
1.2 The Nuclear Pore Complex and Nuclear Pore
Proteins............................................... 2
1.3 Recognition of Nuclear Targeting
Signals...................................................................
4
1.4 Identification of Transport Factors
..............................................................................
6
1.5 Nuclear Transport Receptors
.......................................................................................
8
1.6 Ran-GTPase
Cycle.....................................................................................................
10
1.7 A Model of Classical Nuclear
Import........................................................................
12
1.8 Classical Nuclear Transport Factors
..........................................................................
14
1.8a The Classical Adapter Receptor: Importin alpha
................................................. 14
1.8b The Classical Carrier Receptor: Importin
beta.................................................. 18
1.8c FG-Nucleoporins:
Nup2/Nup50......................................................................
18
1.8d Classical Export Receptor:
CAS/Cse1............................................................
19
1.9 Regulation of cNLS Cargo Delivery into the Nucleus
.............................................. 19
1.9a Recognition of cNLS Cargo in the Cytoplasm
................................................. 20
1.9b Release of cNLS Cargo
....................................................................................
21
1.10 Regulation of Nuclear Transport
.............................................................................
22
1.11 General Overview of the Eukaryotic Cell cycle
...................................................... 23
1.11a Regulation of the Eukaryotic Cell
Cycle........................................................
28
1.12 Interplay of Nuclear Transport and Cell Cycle
Progression.................................... 29
1.12a Cell Cycle Regulated Proteins
.......................................................................
31
1.12b Nuclear Transport, Cell Cycle, and Cancer
.................................................. 31
1.13 S. cerevisiae as a Model to Study Nuclear Transport and
the Cell Cycle ............... 32
1.14 Dissertation Overview
.............................................................................................
33
CHAPTER 2. Nuclear Signal-Receptor Affinity Localization
Correlates with in vivo Localization
..........................................................................................34
Abstract.............................................................................................................................
35
2.1
Introduction................................................................................................................
36
2.2 Material and Methods
................................................................................................
39
CONSTRUCTION OF PLASMIDS
.........................................................................................
39
MICROSCOPY
..................................................................................................................
41
NLS-GFP IMPORT
ASSAY................................................................................................
41
OVEREXPRESSION OF NLS
CARGO...................................................................................
42
2.3
Results........................................................................................................................
43
MEASUREMENTS OF IN VIVO IMPORT RATES
......................................................................
43
ADDITION OF AN NES TO INCREASE THE DYNAMIC RANGE OF THE
ANALYSIS ...................... 48
UPPER LIMIT TO FUNCTIONAL NLS
AFFINITY....................................................................
50
2.4
Discussion..................................................................................................................
53
2.5
Acknowledgments......................................................................................................
56
CHAPTER 3. The Classical Nuclear Localization Signal Receptor,
Importin
alpha, is
Required for Efficient Transition through the G1/S Stage of the
Cell Cycle in
Saccharomyces cerevisiae
.........................................................................59
Abstract.............................................................................................................................
60
3.1
Introduction.................................................................................................................
61
3.2 Materials and
Methods...............................................................................................
62
YEAST STRAINS AND
PLASMIDS..........................................................................................
62
IN VIVO FUNCTIONAL ANALYSIS
........................................................................................
63
IMMUNOBLOT
ANALYSIS...................................................................................................
64
NLS-GFP IMPORT
ASSAY.................................................................................................
64
MICROSCOPY
..................................................................................................................
65
HIGH COPY SUPPRESSOR
ANALYSIS...................................................................................
65
CELL CYCLE ARREST AND RELEASE
...................................................................................
66
FLOW CYTOMETRY ANALYSIS
............................................................................................
66
PLASMID
LOSS.................................................................................................................
67
STATISTICAL ANALYSIS
.....................................................................................................
67
3.3
Results........................................................................................................................
67
FUNCTIONAL ANALYSIS OF SRP1 MUTANTS
........................................................................
68
NLS-GFP IMPORT ASSAY
................................................................................................
71
IMPORTIN
ALPHA VARIANTS DIFFER IN THEIR IN VIVO MOLECULAR
DEFECTS.............................. 72
MUTANTS OF IMPORTIN
ALPHA AFFECT CELL CYCLE
PROGRESSION........................................... 74
BIPARTITE CNLS CANDIDATES INVOLVED IN G1/S OF THE CELL CYCLE
.............................. 85
3.4
Discussion..................................................................................................................
89
3.5
Acknowledgements....................................................................................................
93
CHAPTER 4. GENERAL
DISCUSSION....................................................................
97
4.1 cNLS Cargo Binding Affinity for Importin alpha Dictates Nuclear
Import.................... 97
Validity of a Predicted cNLS cargo
..........................................................................
99
A Lower Limit for cNLS Cargo Binding Affinity for Importin
alpha
............................ 100
Is There an Upper Limit for cNLS Cargo Binding Affinity for
Importin alpha
............ 100
A New Quantitative Model for Classical Nuclear Transport
................................. 103
Examination of Targeting Sequences not Imported into the Nucleus
via the Classical
Nuclear Transport Machinery
................................................................................
105
4.2 Classical Nuclear Transport Regulates Cell Cycle Transitions
............................... 106
Importin
alpha mutants with Defects in Cargo Binding and
Cargo Release................ 106
Identification of Candidate Bipartite cNLS Cargoes involved in Cell
Cycle
Progression.............................................................................................................
109
A Role for Importin
alpha at the G1/S and G2/M Stages of the
Cell Cycle ................... 110
Limitations of using S. cerevisiae as model system to study
nuclear transport and the
cell
cycle..................................................................................................................
112
4.3 GENERAL
CONCLUSIONS..................................................................................
113
CHAPTER 5.
REFERENCES
....................................................................................
116
List of Figures and Tables
Figure 1. Structure of the Nuclear Pore
Complex..............................................................
3
Figure 2. Ran GTPase System.
........................................................................................
11
Figure 3. Model of Classical Nuclear Import.
.................................................................
13
Figure 4. Domain structures of Importin alpha, Importin beta, Nup2,
and Cse1....................... 15
Figure 5. Binding Affinity of Importin alpha for cNLS
cargo............................................... 17
Figure 6. The Eukaryotic Cell Cycle, Cell Cycle Regulators, and
Checkpoints. ............ 25
Figure 7. The yeast S. cerevisiae cell cycle.
....................................................................
27
Figure 8. Localization of NLS
Variants...........................................................................
45
Figure 9. A kinetic assay for NLS reporter import in wild type
cells.............................. 47
Figure 10. Effect of an NES on reporter localization.
..................................................... 49
Figure 11. Overexpression of NLS cargo.
.......................................................................
52
Figure 12. Functional analysis of importin alpha mutants in
vivo. ........................................ 69
Figure 13. High copy suppressor analysis.
......................................................................
73
Figure 14. Analysis of cell cycle defects in srp1
mutants................................................ 75
Figure 15. Analysis of cell cycle progression in cells synchronized
in G1 phase of the cell
cycle.
.................................................................................................................................
77
Figure 16. Analysis of cells synchronized in S phase of the cell
cycle. .......................... 79
Figure 17. Analysis of plasmid
loss.................................................................................
83
Figure 18. Candidate bipartite proteins implicated in G1/S of the
cell cycle are not
efficiently targeted to the nucleus in importin alpha mutants.
................................................ 86
Figure 19. An in vitro Energy Scale for Nuclear Localization.
101
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