The Protein-Protein Interactome of Saccharomyces cerevisiae ABC

178 Pages·2012·4.19 MB·English

The Protein-Protein Interactome of Saccharomyces cerevisiae
ABC Transporters Nft1p, Pdr10p, Pdr18p and Vmr1p
by
Asad Hanif
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Molecular Genetics
University of Toronto
© Copyright by Asad Hanif 2012
The Protein-Protein Interactome of Saccharomyces cerevisiae ABC Transporters Nft1p,
Pdr10p, Pdr18p and Vmr1p

Asad Hanif
Master of Science
Graduate Department of Molecular Genetics
University of Toronto
2012

Abstract

The Membrane Yeast Two-Hybrid (MYTH) technology was used in this study to find
protein-protein interactors of Saccharomyces cerevisiae ATP binding cassette (ABC)
transporters Nft1p, Pdr10p, Pdr18p and Vmr1p. There were 23 interactors for Nft1p, 22
interactors for Pdr10p, 4 interactors for Pdr18p and 1 interactor for Vmr1p. The 43 unique
interactors belong to a wide variety of functional categories. There were 11 interactors involved
in metabolism, 9 interactors involved in transport, 8 interactors with unknown function, 4
interactors involved in trafficking and secretion, 3 interactors involved in protein folding, 2
interactors involved in stress response, and 1 interactor in each of the following categories: cell
wall assembly, cytoskeleton maintenance, nuclear function, protein degradation, protein
modification and protein synthesis. Follow up experiments also showed that Pdr15p and Pdr18p
play an important role in zinc homeostasis because deletion of these ABC transporters results in
sensitivity to zinc shock.

ii
Acknowledgments
I would like to thank my supervisor, Dr. Igor Stagljar for his help and support over the
years. I would also like to thank my supervisory committee members, Dr. Leah Cowen and Dr.
Christine Bear for their help and support towards my research project. I also want to thank Dr.
Charlie Boone, Dr. Reinhart Reithmeier and Dr. Andy Fraser for being a part of my examination
committee for my Master’s defense exam.
I would also like to thank everyone in the Stagljar lab for their support. I would like to
especially thank Dr. Jamie Snider for helping me with my project and research, and answering
countless questions. I also want to thank Analyn Yu, Victoria Wong, Dr. Julia Petschnigg,
Mehrab Ali and Dr. Mandy Lam for helping me with various aspects of my research project. I
want to thank Mike Cox from Dr. Andrews’s lab for helping me with confocal microscopy. I
want to thank Christoph Kurat from Dr. Andrews’s lab for helping me with qRT-PCR
experiments and proving reagents for my experiments. I want to thank Bryan-Joseph San Luis
from Dr. Boone’s lab for proving single and double deleted mutant strains for my experiments. I
want to thank Simon Alfred from Dr. Nislow’s lab for helping me setup Tecan growth
experiments.
Finally, I want to thank my parents, brothers and family for their support. This was not
possible without your encouragement.

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Table of Contents
Abstract .......................................................................................................................................... ii
Acknowledgments ........................................................................................................................ iii
List of Tables ................................................................................................................................ ix
List of Figures ............................................................................................................................... xi
List of Appendices ...................................................................................................................... xiii
Abbreviations .............................................................................................................................. xiv
Chapter 1: Introduction .................................................................................................................. 1
1.1: Research Objectives .......................................................................................................................... 2
1.2: ABC Proteins .................................................................................................................................... 2
1.2.1: Function of ABC Proteins .......................................................................................................... 2
1.2.2: Structure of ABC Transporters .................................................................................................. 4
1.2.3: ABC Transporters, Human Diseases and Multi-drug Resistance .............................................. 7
1.2.4: Human ABC Subfamilies ........................................................................................................... 9
1.3: ABC Transporters in S. cerevisiae .................................................................................................. 12
1.3.1: ABCC (MRP/CFTR) Subfamily in S. cerevisiae ..................................................................... 13
1.3.1.1: Nft1p ................................................................................................................................. 14
1.3.1.2: Vmr1p ............................................................................................................................... 15
1.3.2: ABCG (PDR5) Subfamily in S. cerevisiae .............................................................................. 18
1.3.2.1: Pdr10p ............................................................................................................................... 19
1.3.2.2: Pdr15p ............................................................................................................................... 23
1.3.2.3: Pdr18p ............................................................................................................................... 24
1.3.3: Other ABC Subfamilies in S. cerevisiae .................................................................................. 26
1.4: Protein-Protein Interactions, Model Organism, and Research Tools .............................................. 28
1.4.1: Importance of Protein-Protein Interactions .............................................................................. 28
1.4.2: S. cerevisiae as a Model System .............................................................................................. 29
1.4.3: Yeast Two-Hybrid (Y2H) ........................................................................................................ 31
1.4.4: Membrane Yeast Two-Hybrid (MYTH) .................................................................................. 34
1.4.5: Tandem Affinity Purification (TAP) and Mass Spectrometry ................................................. 37
1.5: Zinc Homeostasis in S. cerevisiae ................................................................................................... 38
1.5.1: Importance of Zinc ................................................................................................................... 39
1.5.2: Zap1p Transcription Factor ...................................................................................................... 40
1.5.3: Zinc Transporters ..................................................................................................................... 42
iv
1.6: Thesis Rationale .............................................................................................................................. 46
Chapter 2: Materials and Methods ............................................................................................... 48
2.1: General Experimental Protocols ..................................................................................................... 49
2.1.1: PCR Amplification ................................................................................................................... 49
2.1.2: DNA-Agarose Gel Electrophoresis .......................................................................................... 49
2.1.3: Standard Lithium Acetate Yeast Transformation ..................................................................... 50
2.1.4: DTT Method for Yeast Transformation ................................................................................... 51
2.1.5: Yeast Integration Transformation ............................................................................................ 51
2.1.6: Yeast and E. coli Miniprep ....................................................................................................... 52
2.1.7: Yeast and E. coli Miniprep in a 96-well Format ...................................................................... 53
2.1.8: Genomic DNA ......................................................................................................................... 53
2.1.9: Competent E. coli Preparation Using Inoue Method ............................................................... 53
2.1.10: E. coli Transformation ........................................................................................................... 54
2.1.11: Glycerol Stock Preparation for Yeast and E. coli .................................................................. 55
2.1.12: Sequencing DNA from TCAG/BioBasic ............................................................................... 55
2.1.13: FM4-64 Staining for Fluorescence Microscopy ..................................................................... 55
2.2: Bait Generation ............................................................................................................................... 55
2.2.1: Primer Design for Baits ............................................................................................................ 55
2.2.2: Cutting AMBV and AMBV-YFP Plasmids with Restriction Enzymes ................................... 57
2.2.3: ORF Amplification, Gap Repair Homologous Recombination and ORF Sequencing ............ 57
2.3: Bait Validation: N G/N I Test ...................................................................................................... 59
ub ub
2.4: Bait Localization using Fluorescence Microscopy ......................................................................... 59
2.5: Testing Bait Function ...................................................................................................................... 60
2.6: Large Scale MYTH Transformation with cDNA and Genomic Libraries ...................................... 60
2.7: Bait Dependency Test ..................................................................................................................... 62
2.8: Prey Sequencing and Identification ................................................................................................ 63
2.9: Confirmation of Deletion Mutants .................................................................................................. 63
2.10: Estimating Free Zinc Ion Concentration in Zinc-Replete and Zinc-Limited Media ..................... 64
2.11: Zinc Shock Assay .......................................................................................................................... 64
2.12: qRT-PCR Experimental Protocol .................................................................................................. 65
2.12.1: RNA Isolation ........................................................................................................................ 65
2.12.2: cDNA Synthesis from RNA ................................................................................................... 66
2.12.3: qRT-PCR Primers, Program and Setup .................................................................................. 66
2.13: Localization of Zrc1p and Zrt1p ................................................................................................... 68
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2.13.1: Integration of MYTH Tag Downstream of ZRC1 and ZRT1 ORF ......................................... 68
2.13.2: Fluorescence Live Microscopy for Zrc1p, Zrt1p ................................................................... 69
2.14: Western Blot Protocol ................................................................................................................... 70
2.14.1: Protein Extraction................................................................................................................... 70
2.14.2: Measuring Protein Concentration Using Bradford Assay ...................................................... 71
2.14.3: 10% Resolving and 5% Stacking SDS-Polyacrylamide Gel .................................................. 72
2.14.4: Gel Electrophoresis, Transfer and Western Blot .................................................................... 72
2.14.5: Scanning Western Blot Films and Measuring Pixel Density with Image J Software ............ 73
Chapter 3: Results ........................................................................................................................ 75
3.1: MYTH Baits were Successfully Validated Using the N G/N I Control Test ............................... 76
ub ub
3.2: Proper Subcellular Localization of Nft1p, Pdr10p, and Vmr1p Validated Using Fluorescence
Microscopy ............................................................................................................................................. 77
3.3: Fusion of the MYTH Tag does not Affect ABC Transporter Function .......................................... 79
3.4: Protein-Protein Interactome of Nft1p, Pdr10p, Pdr18p and Vmr1p from MYTH .......................... 81
3.5: Studying Interactions of Zrt1p and Zrc1p with ABC Transporters ................................................. 92
3.5.1: Deletion of PDR15 and PDR18 Causes Sensitivity to Zinc Shock .......................................... 94
3.5.2: Deletion of PDR15 and PDR18 Causes Changes in ZRC1 and ZRT1 mRNA Levels ............. 96
3.5.3: Deletion of PDR18 Causes Changes in Zrc1p and Zrt1p Protein Levels................................. 98
3.5.4: Zrc1p and Zrt1p Properly Localize in pdr15∆ and pdr18∆ strains ........................................ 102
Chapter 4: Discussion ................................................................................................................ 110
4.1: Analysis of ABC Transporter Protein-Protein Interactome .......................................................... 111
4.1.1: Metabolism ............................................................................................................................. 112
4.1.2: Sphingolipid Biosynthesis ...................................................................................................... 114
4.1.3: Ergosterol Biosynthesis and Transport .................................................................................. 116
4.1.4: ABC Transporter Interactors Involved in Transport Function ............................................... 117
4.1.4.1: ABC Transporter Complexes .......................................................................................... 118
4.1.4.2: Possible Role of ABC Transporters In Osmoregulation ................................................. 119
4.1.5: Cell/Spore Wall Assembly and Maintenance ......................................................................... 121
4.2: Analysis of Zinc Shock Phenotype for pdr15∆ and pdr18∆ strains.............................................. 124
4.2.1: Alterations in Zinc Homeostasis in pdr15∆ strain ................................................................. 125
4.2.2: Alterations in Zinc Homeostasis in pdr18∆ strain ................................................................. 131
4.3: Future Directions ........................................................................................................................... 133
4.3.1: Confirmation of Protein-Protein Interactions and Mapping of Interaction Sites ................... 133
4.3.2: ABC Transporters and Zinc Homeostasis .............................................................................. 134
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4.4: Summary and Conclusion ............................................................................................................. 135
Appendix 1................................................................................................................................. 137
A1.1: Strains, Plasmids, Prey Libraries and Antibodies ...................................................................... 138
A1.2: Media Recipes ............................................................................................................................ 139
A1.2.1: 10X Amino Acid and Nucleotide Drop-out Solution (1 L) ................................................. 139
A1.2.2: LB Medium (Liquid or Solid, with or without Antibiotic) (1 L) ........................................ 140
A1.2.3: Synthetic Dropout (SD) Medium (Liquid or Solid, with or without Antibiotic) (1 L) ....... 140
A1.2.4: Synthetic Dropout (SD) + X-Gal Medium (Solid) (1 L) ..................................................... 141
A1.2.5: Terrific Broth (TB) Liquid Medium (with or without Antibiotic) (1 L) ............................ 141
A1.2.6: YPAD Medium (Liquid or Solid, with or without Antibiotic) (1 L) ................................... 141
A1.2.7: 2X YPAD (Liquid) (1 L) ..................................................................................................... 142
A1.2.8: Zinc-Limited Medium (500 mL) ......................................................................................... 142
A1.2.9: Zinc-Replete Medium (500 mL) ......................................................................................... 142
A1.3: Antibiotics .................................................................................................................................. 143
A1.3.1: 100 mg/mL Ampicillin 1000X Stock (10 mL) .................................................................... 143
A1.3.2: 200 mg/mL Geneticin (G418) 1000X Stock (10 mL) ......................................................... 143
A1.3.3: 50 mg/mL Kanamycin 1000X Stock (10 mL) ..................................................................... 143
A1.3.4: 100 mg/mL Nourseothricin 1000X Stock (10 mL) ............................................................. 143
A1.4: Chemical Solution Recipes ........................................................................................................ 143
A1.4.1: 10% Ammonium Persulfate (1 mL) .................................................................................... 143
A1.4.2: Cell Wall Disruption Solution for 96-well Yeast Miniprep ................................................ 143
A1.4.3: 1 M Dithiothreitol (DTT) (10 mL) ...................................................................................... 144
A1.4.4: 0.5 M Ethylenediaminetetraacetic Acid (EDTA), pH 8.0 ................................................... 144
A1.4.5: 6X Gel Loading Dye for Agarose Gels ............................................................................... 144
A1.4.6: 80% Glycerol (100 mL) ...................................................................................................... 144
A1.4.7: Inoue Buffer for Competent E. coli Preparation ................................................................. 145
A1.4.8: 1 M and 2 M Lithium Acetate for Yeast Transformation (1 L) .......................................... 145
A1.4.9: 5% Milk Blocking Solution for Western Blot (100 mL) ..................................................... 145
A1.4.10: Phosphate Solution for X-gal Medium (1 L) ..................................................................... 145
A1.4.11: 50% Polyethylene Glycol (PEG) 3350 for Yeast Transformation (50 mL) ...................... 146
A1.4.12: 0.1% Ponceau S for Western Blot (100 mL) ..................................................................... 146
A1.4.13: 2 mg/mL Salmon Sperm DNA (ssDNA) Solution for Yeast Transformation .................. 146
A1.4.14: 2X Sample Buffer for Proteins (50 mL) ............................................................................ 146
A1.4.15: 0.9% Sodium Chloride (100 mL) ...................................................................................... 147
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A1.4.16: 1 M Sodium Citrate, pH 4.2 (200 mL) .............................................................................. 147
A1.4.17: 10% Sodium Dodecyl Sulfate (SDS) ................................................................................ 147
A1.4.18: 1X and 10X SDS Gel Running Buffer (2 L) ..................................................................... 147
A1.4.19: 2 M Sodium Hydroxide (100 mL) ..................................................................................... 148
A1.4.20: 1 M Sorbitol (1 L) ............................................................................................................. 148
A1.4.21: 1X and 50X TAE Buffer for DNA-Agarose Electrophoresis (1 L) .................................. 148
A1.4.22: 10X TBS, pH 7.5 for Western Blot (1 L) .......................................................................... 148
A1.4.23: 1X TBST Solution for Western Blot (1 L) ........................................................................ 149
A1.4.24: 1X and 10X Transfer Buffer for Western Blot (2 L)......................................................... 149
A1.4.25: 50% Trichloroacetic Acid (100 mL) ................................................................................. 149
A1.4.26: 1.0 M Tris, pH 6.8, 7.5 or 8.0 (1 L) ................................................................................... 149
A1.4.27: 1.5 M Tris, pH 8.8 (1 L) .................................................................................................... 149
A1.4.28: 10X Tris-EDTA (TE) Solution for MYTH Large Scale Transformation (1 L) ................ 150
A1.4.29: 100 mg/mL X-gal Solution (1 mL) ................................................................................... 150
A1.4.30: 40 mM Zinc Chloride Stock Solution for Zinc Shock Assay (10 mL) ............................. 150
Appendix 2................................................................................................................................. 151
References.................................................................................................................................. 156

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List of Tables
Table Description Page
Number
Table 1: Primer Sequences for Amplification of ORF of Baits for Cloning into 56
AMBV and AMBV-YFP Plasmids
Table 2: Primer Sequences for Verifying ORF of Baits 58
Table 3: Primer Sequences for Confirmation of Deletion Mutants 64
Table 4: Primer Sequences for qRT-PCR 66
Table 5: Primer Sequences for Amplifying MYTH-YFP Tag for Integrating 68
Downstream of ZRC1 and ZRT1
Table 6: Primer Sequences for MYTH Tag Integration Confirmation Downstream 69
of ZRC1 and ZRT1 ORF
Table 7: Description and Localization from the Saccharomyces cerevisiae Database 81
(SGD) for the 43 Interactors from the MYTH Screening of Nft1p, Pdr10p, Pdr18p
and Vmr1p
Table 8: Human Homologs of Preys According to the InParanoid Database, and 91
Associated Diseases from OMIM Database
Table 9: Summary of qRT-PCR, Western Blot and Fluorescence Microscopy 108
Results for Zrc1p and Zrt1p in the pdr15∆, pdr18∆ and Wild-type (Y7092) Strains
Table 10: List of Yeast Strains Used in the Experiments 138
Table 11: Amino Acids and Nucleotides in 10X Drop-out Solution 139
Table 12: List of Ykr103wp Interactors from the BioGRID Database 152
Table 13: List of Ykr104wp Interactors from the BioGRID Database 152
Table 14: List of Vmr1p Interactors from the BioGRID Database 153
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153
Table 15: List of Pdr10p Interactors from the BioGRID Database
Table 16: List of Pdr15p Interactors from the BioGRID Database 154
Table 17: List of Pdr18p Interactors from the BioGRID Database 154

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