A Novel Role for the ARL2 GTPase in Cofactor-mediated Tubulin Folding Öffentlichkeit

Abstract

Microtubules are dynamic, filamentous polymers composed of αβ-tubulin and are essential cytoskeletal components of all eukaryotic cells. Microtubules are required for a variety of essential cellular functions, including cell morphogenesis, motility, mitosis, intracellular traffic, and formation of both primary cilia and flagella. Highlighting their cellular importance, microtubules are also the primary target for many cancer therapeutics. In addition to post-translational modifications and selective expression of tubulin isotypes, the functions of microtubules are strongly regulated by the dynamics of the individual microtubule polymers. Dynamic instability is a term that describes the process by which microtubules stochastically alternate between polymerization and depolymerization, which is largely controlled by the binding and hydrolysis of tubulin-bound guanine nucleotide. Despite our understanding of the many levels of microtubule regulation, one fundamental aspect of microtubule biology remains poorly understood: the biogenesis and assembly of the αβ-tubulin heterodimer. Tubulin is composed of α- and β-subunits, whose folding and assembly require a set of tubulin-specific chaperones, or cofactors, that form a series of folding intermediates in the eventual production of native αβ-tubulin dimer. Described herein is a novel role for the small, regulatory GTPase ARL2 in the cofactor-mediated tubulin folding and heterodimer assembly pathway. ARL2 is shown to strongly interact with the tubulin-specific chaperone D (TBCD), forming a complex that I argue is integral to the tubulin folding pathway. In contrast to previous models, I also propose that the cycling of guanine nucleotides on ARL2 is a key regulatory mechanism that is required for the folding and formation of native αβ-tubulin heterodimers.

Table of Contents

CHAPTER 1: INTRODUCTION p.1
MICROTUBULES p.2
TUBULIN FOLDING p.4
Figure 1-1 p.5
TUBULIN-SPECIFIC CHAPERONE D p.7
ARF-LIKE 2 p.8
TBCS IN DISEASE p.12
FOCUS OF THIS DISSERTATION p.13
REFERENCES p.14
CHAPTER 2: A TRIMER CONSISTING OF THE TUBULIN-SPECIFIC CHAPERONE TBCD, REGULATORY GTPASE ARL2, AND β-TUBULIN IS REQUIRED FOR MAINTAINING THE MICROTUBULE NETWORK p.25
ABSTRACT p.26
INTRODUCTION p.26
RESULTS p.29
TBCD and ARL2 are present in animal cells and tissues in a complex with an apparent molecular weight of ~200 kDa p.29
Figure 2-1 p.30
Figure 2-2 p.33
Recombinant GST-TBCD co-purifies with αβ-tubulin p.34
Figure 2-3 p.36
Co-expression of GST-TBCD and ARL2 results in the formation and ability to purify a TBCD/ARL2/β-tubulin trimer p.37
Figure 2-4 p.38
TBCE can bind to the TBCD/ARL2/β-tubulin trimer or to TBCB/α-tubulin p.40
Figure 2-5 p.42
ARL2 mutants that lose TBCD binding have reduced ability to alter microtubule densities p.44
Figure 2-6 p.46
Figure 2-7 p.48
DISCUSSION p.50
EXPERIMENTAL PROCEDURES p.57
REFERENCES p.64
CHAPTER 3: NUCLEOTIDE BINDING TO ARL2 IN THE TBCD/ARL2/β-TUBULIN COMPLEX DRIVES CONFORMATIONAL CHANGES IN β-TUBULIN p.69
ABSTRACT p.70
INTRODUCTION p.70
RESULTS p.75
The TBCD/ARL2/β-tubulin complex binds both GDP and GTP p.75
Figure 3-1 p.77
Figure 3-2 p.79
Figure 3-3 p.81
Molecular dynamics of TBCD in the TBCD/ARL2/β-tubulin complex are only minimally affected by the binding of guanine nucleotides p.83
Figure 3-4 p.85
Addition of guanine nucleotides to the TBCD/ARL2/β-tubulin complex results in conformational changes in β-tubulin that do not map onto regions involved in guanine-nucleotide binding p.86
Figure 3-5 p.87
HDX-MS analysis of ARL2 monomer reveals large changes, particularly at consensus
GTP-binding motifs p.89
Figure 3-6 p.91
Formation of the TBCD/ARL2/β-tubulin complex results in distinct changes in the molecular dynamics of ARL2 p.93
Figure 3-7 p.94
ARL2 binds guanine nucleotide in the TBCD/ARL2/β-tubulin complex p.96
Figure 3-8 p.97
DISCUSSION p.99
EXPERIMENTAL PROCEDURES p.105
REFERENCES p.111
CHAPTER 4: DISCUSSION p.119
SUMMARY p.120
CHALLENGING THE STATUS QUO p.121
Figure 4-1 p.123
FUTURE DIRECTIONS p.126
Figure 4-2 p.127
CONCLUDING REMARKS p.129
REFERENCES p.131

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Cell
Stichwort	ARF-like 2 (ARL2) Tubulin-specific Chaperone D (TBCD) small GTPase protein purification tubulin folding
Committee Chair / Thesis Advisor	Kahn, Richard A, Emory University
Committee Members	Hepler, John R, Emory University Sale, Winfield S, Emory University Ortlund, Eric, Emory University Conn, Graeme L, Emory University

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