The ELMO Domain Containing (ELMOD) Proteins: Phylogenetic, Structural, and Functional Characterization Restricted; Files Only

East, Michael (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/7w62f892j?locale=en
Published

Abstract

The ELMO domain containing proteins (ELMODs) are a novel group of GTPase activating proteins (GAPs) that act on members of the Arf family of regulatory GTPases, though they lack the previously characterized, conserved Arf GAP domain. Very little is known about the functions of the ELMODs but the human family members have been implicated in non-syndromic familial deafness or idiopathic pulmonary fibrosis. The ELMODs belong to the ELMO family of proteins which includes three ELMODs and three ELMOs in humans and is defined by the presence of the ELMO domain in each of its members. The ELMOs function as key regulators of cell migration and actin dynamics as unconventional guanine nucleotide exchange factors for the Rho family GTPase Rac1. Unlike the ELMODs, the ELMOs lack any detectable GAP activity for Arf family GTPases. In this dissertation, I show that ELMODs and ELMOs are two phylogenetically distinct groups of proteins despite being members of the same gene family. The ELMODs represent the more ancient form of the protein and were likely present in the last eukaryotic common ancestor, suggesting some ancient function(s) of the ELMODs important or even essential for eukaryotic life. I mapped the GAP motif of the ELMODs to a region within the ELMO domain and provided initial characterization of this motif by identifying an arginine residue essential for efficient GAP activity. I also provide the first functional information for the ELMODs and present models for the cellular roles of ELMOD1 and ELMOD3 as a regulator of Arf signaling at the Golgi and as an activator of RhoA signaling at the trailing edge of migrating cells, respectively. However, additional cellular localizations of each of the ELMODs and putative binding partners identified in this dissertation are suggestive of other, novel roles for the ELMODs in cells. Together these data represent the majority of the available information on the ELMODs and provide the foundation for our later studies to provide a better understanding of the biology of the ELMODs and their roles in cell biology and human diseases.

Table of Contents

General Introduction

1

Figure 1: The GTPase cycle.

3

Figure 2: Domain architecture of the ELMO family of proteins.

5

ELMO domains: evolutionary and functional characterization of a novel GTPase activating protein (GAP) domain for Arf family GTPases

14

Summary

16

Introduction

17

Experimental Procedures

20

Results

24

Figure 1: The six human ELMO domain-containing family members are equally divided into three ELMO and three ELMOD proteins

26

Figure 2: ELMOs cluster into a distinct phylogenetic sub-family

29

Figure 3: The ELMO sub-family is further divided into three distinct phylogenetic clades

30

Figure 4: Sequence alignment of the ELMODs reveals a highly conserved motif that includes a central arginine residue

32

Figure 5: The putative catalytic arginine and some of the other residues within the putative GAP domain of the ELMODs (top) are not conserved in members of the ELMO sub-family (bottom)

33

Figure 6: Mutation of the putative catalytic arginine residue reduces GAP activity of ELMOD1-myc/his and ELMOD2-myc/his in in vitro and cell-based assays

36

Figure 7: Over-expression of ELMOD1-HA alters Golgi morphology but this phenotype is absent for ELMOD1(R174K)-HA

40

Figure 8: Cellular localization of endogenous ELMOD1 and exogenous ELMOD1-HA or ELMOD2-HA

45

Discussion

47

References

55

Acknowledgments

61

Footnotes

61

Table S1: List of taxa and accession numbers for ELMO domain containing proteins used in our analyses

62

ELMOD3 is a novel regulator of the actin cytoskeleton through a RhoA dependent mechanism

65

Abstract

66

Introduction

66

Materials and Methods

69

Results

71

Figure 1: ELMOD3 migrates at a higher than expected apparent molecular weight on SDS gels

72

Figure 2: Endogenous ELMOD3 localizes to the trailing edge of migrating mouse embryonic fibroblasts (MEFs)

74

Figure 3: Endogenous ELMOD3 has a punctate staining pattern with organization along actin fibers in non-polarized mouse embryonic fibroblasts (MEFs)

75

Figure 4: ELMOD3 protein levels are unchanged after inhibiting protein synthesis for 16 hours

77

Figure 5: Endogenous ELMOD3 associates with the actin cytoskeleton

79

Figure 6: GFP-ELMOD3 causes dynamic plasma membrane blebbing

80

Figure 7: ELMOD3-HA causes changes in the actin cytoskeleton in a Rho-dependent fashion

82

Figure 8: Expression of dominant negative RhoA (T19N) inhibits blebbing and changes ELMOD3-HA localization

84

Figure 9: ELMOD3-HA induced plasma membrane blebbing is dependent on the RhoA signaling pathway

85

Discussion

86

References

91

Putative binding partners of ELMODs suggest novel functions and mechanisms

96

Introduction

97

Materials and Methods

99

Results and Discussion

103

Figure 1: Schematic of the SILAC co-IP methodology

105

Figure 2: Bovine testis enhances GST-ELMOD3 GAP activity

111

Figure 3: GST-ELMOD3 binds phosphoinositides

113

Figure 4: Staining of ELMOD3-HA and of PI(4,5)P2 show extensive overlap at the cell periphery

115

Figure 5: PI(4,5)P2 and PI(3,4,5)P3 have an asymmetrical distribution at the plasma membrane in migrating cells

117

References

118

Table 1: Putative binding partners of ELMOD1-HA

124

Table 2: Putative binding partners of ELMOD2-HA

126

Table 3: Putative binding partners of ELMOD3-HA

128

Discussion

130

ELMODs as GAPs

131

ELMOs and the ELMO domain

133

Figure 1: Model for the function of the ELMO domain in ELMOs and ELMODs

135

Models for the cellular functions of ELMODs

138

Figure 2: Schematic of confirmed and suspected subcellular localization of the ELMODs throughout the cell

139

Figure 3: The model for the function of ELMOD3 closely parallels its paralog ELMO1

146

General comments regarding models for the functions of ELMODs

151

Clinical aspects of the ELMODs

152

Concluding remarks

153

References

154

About this Dissertation

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research field
Keyword
Committee Chair / Thesis Advisor
Committee Members
Last modified No preview

Primary PDF

Supplemental Files