Interplay Between Nuclear Import and Cell Cycle Control Open Access

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

Figure 20. A Role for Importin alpha in Regulating Transition through G1/S stage of the Cell Cycle 115 Table 1. NLS variants and their affinity for delta-IBB-importin alpha.....57 Table 2. Yeast strains and plasmids used in study...........58 Table 3. S. cerevisiae strains used in this study......94 Table 4. List of plasmids...........95 Table 5. List of bipartite cNLS candidates involved in G1/S........96

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	PhD
Submission	thesis Dissertation
Language	English
Research field	Biology, Genetics Biology, Molecular Biology, Cell
Keyword	nuclear import cell cycle budding yeast importin alpha classical nuclear localization signal
Committee Chair / Thesis Advisor	Corbett, Anita, Emory University
Committee Members	Fridovich-Keil, Judith, Emory University Pallas, David C, Emory University Peng, Junmin, Emory University Boss, Jeremy, Emory University

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