Characterization the role of kinectin in assembly of translation elongation complex to endoplasmic reticulum and its involvement in organelle motility

THE ROLE OF KINECTIN IN ASSEMBLY OF
TRANSLATION ELONGATION COMPLEX TO
ENDOPLASMIC RETICULUM AND ITS INVOLVEMENT
IN ORGANELLE MOTILITY












ONG LEE LEE
(B. Applied Sci, QUT, Australia)












A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY
NATIONAL UNIVERSITY MEDICAL INSTITUTES
NATIONAL UNIVERSITY OF SINGAPORE
2005

i
Acknowledgements

First of all, I would like to express my thanks to A/Prof Hanry Yu for giving me the
opportunity to pursue my postgraduate studies in his laboratory. I am grateful for his
support, guidance and invaluable advice throughout his supervision.

I would like to acknowledge past and present members of this laboratory for their help
and companionship during my stay here. I would like to extend my appreciation to
Connie, Adeline and Pao Chun whom we shared time troubleshooting problems and
hypothesizing new ideas together.

Many thanks to my friends, Aiwei, Veronica, Chris, Bee Leng, Bee Ling, Yuan Ming,
May and Andrea for their help and endless encouragement throughout my studies.

Last, but not least, my deepest thank and appreciation to my family members for their
continuous support and understanding which allows me to concentrate whole-
heartedly in my research project.

ii
Table of Contents
Acknowledgements i
Table of contents
ii
Summary viii
List of Tables x
List of Figures xi
List of Abbreviations xiv
List of Publications
xvi

Chapter 1: Introduction 1

Chapter 2: Background
2.1 Cytoskeleton 7
2.1.1 Actin microfilament 8
2.1.2 Intermediate filament 9
2.1.3 Microtubules 10
2.2 Microtubule based organelle transport 11

2.3 Molecular Motors 12

2.3.1 Dynein 13
2.3.2 Conventional Kinesin 14
2.3.3 Kinesin superfamily 15
2.4 Establishment and maintenance of organelle position in cells 17
2.4.1 Endoplasmic reticulum 18
2.4.2 Mitochondria 19
2.4.3 Activation of kinesin 21
2.5 Motor protein receptors 22
2.5.1 Coat Proteins 22
2.5.2 Scaffold Proteins 23
2.5.3 Small GTPase 23
2.5.4 Transmembrane protein 24
2.5.4.1 Amyloid precusor protein (APP) 24
2.5.4.2 Sunday Driver (SYD) 24
2.6 Kinectin 25

iii
2.6.1 Discovery 25
2.6.2 Distribution of kinectin 26
2.6.3 Structural properties of kinectin 27
2.6.4 160 kDa and 120 kDa kinectin 28
2.6.5 Kinectin splice variants 29
2.6.6 Kinectin’s role in organelle motility 30
2.6.7 Kinectin interaction with GTPases 32
2.6.8 Clinical implications of kinectin 34
2.7 Molecular mechanisms of eukaryotic translation 35
2.7.1 Translation initiation 36
2.7.2 Translation elongation 38

2.7.3 Translation termination 39
2.8 Elongation complex 40
2.8.1 Alpha subunit 41
2.8.2 Beta subunit 43
2.8.3 Delta subunit 44
2.8.4 Gamma subunit 46
2.8.5 Valysl-tRNA synthetase 48
2.8.6 Assembly of EF-1 subunits 48
2.8.7 Regulation of peptide elongation 52

Chapter 3: Materials and Methods
3.1 Isolation of native human kinectin isoforms 54
3.1.1 Polymerase chain reaction 54
3.1.2 Agarose gel electrophoresis 55
3.1.3 Extraction of DNA from agarose gel 55
3.1.4 Ligation I 56
3.1.5 Ligation II 56
3.1.6 Preparation of competent cells 56
3.1.7 Transformation 57
3.1.8 Small-scale plasmid preparation 57
3.1.9 Large-scale plasmid preparation 58
3.1.10 Quantitation of DNA 58
3.1.11 Restriction endonuclease digestion 59

iv
3.1.12 Automated sequencing 59
3.2 Construction of kinectin baits for yeast two-hybrid screening 60
3.2.1 Cloning of Baits A, B and D into BD vector 60
3.2.2 Small-scale transformation of baits into yeast 60
3.2.3 Preparation of yeast cultures for protein extraction 61

3.2.4 Preparation of protein extract from yeast 61
3.2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis 62
3.2.6 Coomassie Brilliant Blue staining 63
3.2.7 Western Transfer 63
3.2.8 Western Blot 63
3.3 Amplification of human fetal brain library 64
3.3.1 Library titering 64
3.3.2 Plasmid library amplification 65
3.4 Gal 4 Yeast two-hybrid screening 65
3.4.1 Library-scale yeast transformation 65
3.4.2 Activation of HIS3 reporter gene 65
3.4.3 Activation of Lac Z reporter gene 66
3.4.4 Plasmid isolation from yeast 66
3.4.5 Clones segregation 67
3.4.6 Clones identification 67
3.5 Protein Purification using affinity chromatography method 67
3.5.1 Protein expression in E. coli 67
3.5.2 Affinity purification of GST fusion proteins 68
3.5.3 Affinity purification of His
6
fusion proteins 69
3.5.4 Dialysis of purified proteins 69
3.6 Circular dichroism measurement for the baits 69
3.6.1 Bait proteins preparation 69
3.6.2 Circular dichroism measurement 70
3.7 In vitro GST pull-down assay 70
3.7.1 GST-tagged Bait proteins ligand preparation 70
3.7.2 GST pull-down assay 70
3.8 Mammalian cell culture 71
3.8.1 Subculturing of mammalian cells 71

3.8.2 Cryopreservation of cells 71

v
3.8.3 Thawing of cells 72
3.9 Co-immunoprecipitation assay 72
3.9.2 Transfection 72
3.9.3 Immunoprecipitation 72
3.10 Isolation of EF-1α, β, and γ subunit cDNA 73
3.11 Production of polyclonal antibody in rabbit 73
3.11.1 Purification of immunogen 73
3.11.2 Rabbit immunization 74
3.11.3 Serum collection 74
3.11.4 Purification of antibodies 74
3.11.5 In vitro phopshorylation of EF-1
δ
by cdc2 kinase 75
3.12 GST pull-down of native proteins 75
3.13 Real-time biomolecular interaction analysis 76
3.14 Identification of interacting domains 77
3.14.1 Identification of EF-1
δ
binding domain on Bait D 77
3.14.2 Identification of kinectin and EF-1γ binding domains 78
on EF-1δ
3.15 In vitro protein translation assay 79
3.16 Co-localization studies 80
3.16.1 Immunostaining 80
3.16.2 Confocal microscopy 80
3.17 Overexpression studies 81
3.17.1 Plasmid construction and verification 81

3.17.2 Overexpression of baits 81
3.18 Transient knockdown of kinectin using Morpholinos 81
3.18.1 Morpholinos designs 81
3.18.2 Special delivery into cells 82
3.18.3 RNA analysis 82
3.18.4 Immunoflorescence staining 83
3.18.5 Luciferase assay 83
3.19 Transient knockdown of kinectin using DNA-enzyme 84
3.19.1 Design of the DNA-enzyme 84
3.19.2 Transient transfection 85

vi
3.19.3 Total RNA isolation 85
3.19.4 Semi-quantitative RT-PCR analysis

86

3.19.5 Semi-quantitative western blot analysis 86
3.20 Transient knockdown of kinectin using siRNA 86
3.20.1 Design of siRNA 86
3.20.2 Transient transfection 87
3.21 Transient knockdown of kinectin using pSUPER vector 87
3.21.1 Design of pSUPER construct 87
3.21.2 Cloning strategy 87
3.21.3 Transient transfection 87
3.22 Stable kinectin knockdown cell line using pSilencer vector 89
3.22.1 Design of pSilencer/KNT construct 89
3.22.2 Establishment of stable cell line 89
3.22.3 Immunoflorescence staining 89

Chapter 4: Results and Discussion - Kinectin anchors EF-1δ to the ER
4.1 Isolation of native human kinectin spliced isoforms 91
4.2 Kinectin baits construction for yeast two-hybrid screening 96
4.3 Circular dichroism analysis of kinectin baits 101
4.4 Yeast two-hybrid library screening 102
4.5 Verification of positive clones using GST pull-down assay 109
4.6 Kinectin interacts with EF-1δ in mammalian cells 118
4.7 Characterization of anti-EF-1δ polyclonal antibody 118
4.8 Interaction analysis with endogenous proteins 120
4.9 Real-time bio-molecular interaction analysis 123
4.10 Characterization of the EF-1δ binding domain on kinectin 128
4.11 Excess kinectin fragments affect the in vitro protein synthesis 130
4.12 Intracellular localization of kinectin and EF-1
δ
134
4.13 Kinectin anchors EF-1δ to the ER 136
4.14 Discussion 140

Chapter 5: Results & discussion - Kinectin anchors EF-1 complex to the ER
5.1 Isolation of EF-1 subunits 147

vii
5.2 Interaction analysis of kinectin with EF-1 subunits using yeast 148
two-hybrid method
5.3 Verification the interactions using in vitro binding assay 151
5.4 Characterization of anti-EF-1β and EF-1γ polyclonal antibody 157
5.5 Association of kinectin with EF-1 subunits in intact cells 161
5.6 Characterization of kinectin and EF-1
γ
binding domain on EF-1

δ
161
5.7 Kinectin anchors the EF-1 subunits to the ER 166
5.8 Aberrant splicing of EF-1
δ
binding domain on kinectin mRNA 171
using morpholinos
5.9 Kinectin involvement in EF-1 complex anchorage to the ER is 180
confirmed by morpholino studies
5.10 Microtubule and ER network not affected by morpholino 188
knockdown
5.11 Kinectin plays a role in protein synthesis 188
5.12 Discussion 197

Chapter 6: Results & discussion - Kinectin is involved in the intracellular
dynamics of ER and mitochondria
6.1 DNA enzyme cleaves transcribed kinectin mRNA 209
6.2 DNA enzyme reduces the endogenous kinectin protein 210
6.3 No kinectin knockdown in cells using siRNA 213
6.4 No kinectin knockdown using pSUPER vector 214
6.5 Kinectin knockdown in cells stably transfected using pSilencer vector 216
6.6 Kinectin knockdown affect the localization of EF-1
β
and EF-1
γ
220
6.7 Kinectin is involved in ER membrane dynamics 222
6.8 Kinectin is also involved in mitochondria motility 225
6.9 Microtubule network is not affected by kinectin siRNA 227
6.10 Discussion 227

Conclusion and future prospectives
235
References 239
Appendices 269


viii
Summary

Kinectin has been proposed to be a membrane anchor for kinesin on
intracellular organelles. A family of nine human kinectin isoforms was isolated from
four different cDNA libraries. A kinectin isoform that lacks a major portion of the
kinesin-binding domain does not bind kinesin but interacts with another resident of
the endoplasmic reticulum, the translation elongation factor-1 delta (EF-1δ). This was
shown by yeast two-hybrid analysis and a number of in vitro and in vivo assays. EF-
1
δ
provides the guanine nucleotide exchange activities on EF-1
α
during the
elongation step of protein synthesis. The minimal EF-1δ-binding domain on kinectin
resides within a conserved region present in all the kinectin isoforms. Over-expression
of the kinectin fragments in vivo disrupted the intracellular localization of EF-1
δ

proteins. This report provides evidence to kinectin’s alternative function as the
membrane anchor for EF-1δ on the endoplasmic reticulum.
Since elongation factors exist as a quaternary complex consisting of EF-1αβγδ
subunits, we next characterized the assembly of the whole complex to ER via kinectin

by proposing two models. Our results from a series of in vitro and in vivo assays are
in favour of the first model which suggests that the anchorage of the EF-1βγδ
complex to ER is via kinectin instead of the second model whereby kinectin anchors
the EF-1
δ
onto specific regions of the ER membrane while the EF-1
βγ
complex
interacts with other regions of the ER membrane in kinectin- and EF-1
δ
- independent
manners. We have also demonstrated that the interaction of kinectin with EF-1
complex is physiologically important. In cells with EF-1δ-binding domain on kinectin
spliced out by morpholinos, we observed a down regulation of membraneous protein
synthesis in contrast to an upregulation of cytosolic protein synthesis. This could be
the case when at least a part of the translation factors is compartmentated in the cell

ix
(Richter and Smith, 1981). It has been suggested that some components of the protein
synthetic apparatus is limiting. The redistribution of such proteins can regulate the
rate of different reactions in protein biosynthesis (Richter and Smith, 1981; Ryazanov
et al., 1987). Thus, translation efficiency of mRNAs could be enhanced by kinectin’s
ability to localize elongation factors, synthetases and ribosomes into an aggregated
structure.
Besides characterizing the role of kinectin in protein synthesis, we attempted
to resolve the controversial issues on whether kinectin is indeed the kinesin receptor.
We have successfully demonstrated in vivo by RNA interference assay that kinectin is
involved in the intracellular dynamics of ER and mitochondria. Our current results,
together with well documented role of kinectin-kinesin interaction in intracellular
transport of ER and lysosomes, truly confirm the role of kinectin in organelle motility

(Kumar et al., 1995; Ong et al., 2000).
In this work, we have characterized the involvement of kinectin in two major
intracellular processes, namely, the protein synthesis and organelle motility. These
findings serve as a platform for more detailed mechanistic understanding on how
kinectin could interplay the two major functions together.


x
List of Tables
Table 1: Primers used for PCR amplification of kinectin domains

Table 2: Coding regions for truncated kinectin domains

Table 3: Primers used for PCR amplification of EF-1δ domains

Table 4: Coding regions for truncated EF-1δ domains

Table 5: Spectra properties of fluorochromes

Table 6. Distribution of human kinectin isoforms.

Table 7. Interaction of kinectin isoforms with Kif 5.

Table 8. Assay for autonomous activation of reporter genes.

Table 9. Putative positive interacting partners with Bait D from yeast two-hybrid
screening.

Table 10. Summary of in vitro interaction of positive clones from yeast two-hybrid

screening with kinectin baits.

Table 11. Interaction of kinectin fragments.


Table 12. Summary of in vitro interaction of kinectin bait D.

Table 13. Summary of kinectin and EF-1
γ
binding domain on EF-1
δ.



xi
List of Figures

Fig. 1 Translation initiation in eukaryotic cells

Fig. 2 Translation elongation in eukaryotic cells.

Fig. 3. Five proposed models for the assembly of elongation complex

Fig. 4. Kinectin isoforms from human fetal brain cDNA library

Fig. 5. Human kinectin isoforms.

Fig. 6. Baits expression in yeast.

Fig. 7. Circular dichroism analysis of Bait A, B and D

Fig. 8. Activation of HIS3 and Lac Z reporter gene in yeast two-hybrid screening.

Fig. 9. In vitro interaction of positive clones from yeast two-hybrid screening with
kinectin baits.

Fig. 10. Co-immunoprecipitation of kinectin bait D with EF-1
δ
.

Fig. 11. Specificity of anti-EF-1
δ
polyclonal antibody.

Fig. 12. Interaction of endogenous kinectin with EF-1δ.

Fig. 13. Analyzing EF-1δ binding to kinectin bait D by surface plasmon resonance.

Fig. 14. EF-1δ binding domain on kinectin using yeast two-hybrid method.

Fig. 15. In vitro interaction of EF-1δ with its binding domain on kinectin.

Fig. 16. Excess kinectin fragments affect the in vitro protein translation.

Fig. 17. Co-localization of endogenous kinectin and EF-1δ in CV1 cells.

Fig. 18. Distribution of EF-1
δ
proteins disrupted by kinectin over-expression.

Fig. 19. Two proposed models on how EF-1 complex anchors to ER.

Fig. 20. PCR amplification of EF-1α, β and γ subunits.

Fig. 21. In vitro interaction of kinectin bait D, EF-1β, EF-1γ and EF-1δ subunits.

Fig. 22. Specificity of anti-EF-1β and γ polyclonal antibody.

Fig. 23. Immunoprecipitation of endogenous kinectin, EF-1γ and EF-1β with EF-1δ.

xii
Fig. 24. Kinectin and EF-1γ binding domain on EF-1δ.

Fig. 25. Distribution of EF-1β proteins disrupted by kinectin over-expression.

Fig. 26. Distribution of EF-1γ proteins disrupted by kinectin over-expression.

Fig. 27. Splicing of kinectin pre-mRNA exon 36 in HeLa cells in the presence of HS1
morpholino.


Fig. 28. Splicing of kinectin pre-mRNA exon 37 in HeLa cells in the presence of HS2
morpholinos.

Fig. 29. Splicing of kinectin pre-mRNA exon 36 & 37 in HeLa cells in the presence of
HS1&2 morpholinos.

Fig. 30. Kinectin morpholinos affect the distribution of EF-1δ.

Fig. 31. Kinectin morpholinos affect the distribution of EF-1γ.

Fig. 32. Kinectin morpholinos affect the distribution of EF-1β.

Fig. 33. Kinectin morpholinos do not affect the microtubule network.

Fig. 34. Kinectin morpholinos do not affect the ER network.

Fig. 35. Membraneous and cytosolic luciferase constructs

Fig. 36. Kinectin morpholinos reduce membraneous luciferase protein synthesis

Fig. 37. Kinectin morpholinos increase cytosolic luciferase protein synthesis

Fig. 38. Effect of DNA-enzyme on endogenous kinectin mRNA and protein.

Fig. 39. Effect of RNAi on endogenous kinectin mRNA and protein.


Fig. 40. Effect of pSUPER/KNT on endogenous kinectin mRNA and protein.

Fig. 41. Effects of stable expression of pSilencer/KNT on endogenous kinectin
mRNA and protein.

Fig. 42. Effects of stable expression of pSilencer/KNT on distribution of kinectin.

Fig. 43. Effects of stable expression of pSilencer/KNT on distribution of EF-1β

Fig. 44. Effects of stable expression of pSilencer/KNT on distribution of EF-1γ.

Fig. 45. Effects of stable expression of pSilencer/KNT on distribution of endoplasmic
reticulum.


xiii
Fig. 46. Effects of stable expression of pSilencer/KNT on distribution of
mitochondria.

Fig. 47. Effects of stable expression of pSilencer/KNT on distribution of microtubule.

xiv
List of Abbreviations

A adenine
AA Aplastic anemia
Ab antibody
AD activation domain
APP amyloid precursor protein
ATP adenine triphosphate
BD binding domain
bp base pair
BSA bovine serum albumin
C cytosine
CD circular dichroism
cDNA complementary DNA
C. elegans Caenorhabditis elegans
Da dalton
DIG digoxigenin
DMEM Dulbecco’s modified Eagle’s medium

DMSO dimethylsulfoxide
dNTP deoxynucleotide triphosphate
DNA deoxyribonucleic acid
E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid
EF-1 translation elongation factor-1
EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid
ER endoplasmic reticulum
FCS foetal calf serum
FITC fluorescein isothiocyanate
G guanine
g g-force
GAPDH glyceraldehdyde-3-phosphate dehydrogenase
GFP green flouresent protein
GST glutathione S-transferase
GTP guanine triphosphate
h hour
HCl hydrochloric acid
HIS3 histidine
His
6
6xHis
Ig immunoglobulin
IPTG isopropyl β-D-thiogalactosidase
KAP kinesin associated protein
KHC kinesin heavy chain
Kifs kinesin isoforms
KLC kinesin light chain
KRP kinesin related protein
LB Luria Bertani

M molar
min minute
MT microtubule
mRNA messenger RNA

xv
MTOC microtubule organizing center
OD optical density
Oligos oligonucleotides
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
PEG polyethylene glycol
PMSF phenylmethylsulfonyl fluoride
RNA ribonucleic acid
RNC ribosome-nascent polypeptide complex
RT-PCR reverse transcription polymerase chain reaction
rpm revolution per minute
RU response unit
SDS sodium dodecyl sulfate
sec second
siRNA small interfering RNA
SPR surface plasmon resonance
SRP signal recognition particle
T thymidine
TBS Tris buffered saline
TEMED N, N, N’, N’-tetramethylethylenediamine
Tris hydroxymethyl-aminomethane
TRITC tetramethyrhodamine isothiocyanate
vd variable domain

UV ultraviolet
V volts
v volume
ValRS valyl-tRNA synthetase

xvi


Publications


1. Lee-Lee Ong, Angeline P.C. Lim, Connie P.N. Er, Sergei A. Kuznetsov and
Hanry Yu (2000) Kinectin-kinesin Binding Domains and Their Effects on
Organelle Motility, The Journal of Biological Chemistry, 275(42): 32854-
32860.

2. Lee-Lee Ong, Connie P.N. Er, Andrea Ho, May T. Aung and Hanry Yu
(2003) Kinectin Anchors the Translation Elongation Factor-1δ to the
Endoplasmic Reticulum, The Journal of Biological Chemistry, 278(34):
32115-32123.

3. Stephen C.H. Wong, Lee-Lee Ong, Connie P.N. Er, Shujun Gao, Hanry Yu
and Jimmy B.Y, So (2003) Cloning of rat telomerase catalytic subunit
functional domains, reconstitution of telomerase activity and enzymatic profile
of pig and chicken tissues, Life Sciences, 73: 2749-2760.

4. Niovi Santama, Connie P.N. Er, Lee-Lee Ong and Hanry Yu (2004)
Distribution and Functions of Kinectin Isoforms, Journal of Cell Science,
117:4537-4549.

5. Lee-Lee Ong, Pao-Chun Lin and Hanry Yu (2005) Regulation of protein
synthesis by the assembly of translation elongation factors on kinectin.
Manuscript in preparation.



Conference Poster Presentation

1.
Lee-Lee Ong
and Hanry Yu (2000) Identification and Characterization of
Kinectin-associated proteins, The Dynamics of the Cytoskeleton, Keystone
Symposia, Keystone, Colorado, U.S.A.

2. Lee-Lee Ong and Hanry Yu (2003) Assembly of Translation Elongation
Factor-1 Complex on Kinectin, The 4
th
Sino-Singapore Conference on
Biotechnology, 11-13 Nov, Singapore.

3. Lee-Lee Ong and Hanry Yu (2003) Assembly of Translation Elongation
Factor-1 Complex on Kinectin, The American Society For Cell Biology, 43
rd

Annual Meeting, Dec 7-13, San Francisco, U.S.A

1
Chapter 1: Introduction

Intracellular organelle transport is fundamental to cellular functions such as

endocytosis (Bomsel et al., 1990), secretion (Stearns and Wang, 1991; Yokota, 1989),
phagocytosis (Blocker et al., 1997) in macrophages, fast axonal transport in neurons
and antigen presentation in lymphocytes. All living cells transport materials between
various cellular compartments to maintain their metabolic functions and to interact
with the extracellular environment. Abnormalities of these cellular phenomena have
been correlated with various diseases such as motor neuron diseases (Kihira et al.,
1995), autoimmune glomerular diseases (Ott et al., 1989), neurodegeneration in
Alzheimer’s disease (De Strooper and Annaert, 2000; Selkoe, 1999), Behcet’s disease
and human hepatocellular carcinoma (Lu et al., 2003; Wang et al., 2004). Therefore,
the knowledge on the molecular mechanism of the transport process not only will help
us understand these diseases at the mechanistic level but will also eventually allow us
to design sophisticated drug delivery methods so that drugs can be delivered precisely
and activated only in specific compartments inside the cells.
Two types of intracellular organelle transport have been discovered (Langford,
1995). One is microtubule (MT)-based, which is defined as the movement of
membrane organelles along linear microtubule arrays. The other type of organelle
transport is actin-based which is defined as the movement of organelles along web-
like networks of actin filaments (Kuznetsov et al., 1992). No organelles have yet been
found to move on intermediate filaments.
Previous efforts to understand organelle transport have led to the partial
identification of some components of the organelle machinery, namely, microtubules
as tracks on which organelles are transported; motor proteins (such as kinesin, dynein
and myosins) which drive the organelles; activators that regulate the motor protein

2
activities; and motor receptors on the organelle membrane that anchor motor proteins
to regulate the directions of organelle transport.
In the past decade, numerous studies have revealed the identity of motors that
are involved in each of the membrane trafficking pathways (Goldstein and
Gunawardena, 2000; Kamal and Goldstein, 2000). However, one of the most poorly

understood aspects of microtubule-dependent trafficking is the identity of the
membranous cargo each motor carries and the nature of the motor-cargo interactions
(Kamal and Goldstein, 2002).
One important discovery was the identification of kinectin, which had initially
been proposed to be a membrane anchor for kinesin on intracellular organelles
(Kumar et al., 1995; Toyoshima et al., 1992). Kinectin consists of a 120 kDa
polypeptide and a 160 kDa polypeptide interacting through an
α
-helical coiled-coil
domain to form a heterodimer (Kumar et al., 1998a; Kumar et al., 1998b). The 120
kDa polypeptide is the truncated version of the 160 kDa polypeptide, lacking the first
232 amino acids, in the NH
3
-terminus (Kumar et al., 1998a). The NH
3
-terminus of the
160 kDa polypeptide consists of a trans-membrane domain that anchors kinectin to
organelle membranes, potentially with the help of the 7 myristylation sites throughout
the molecule (Kumar et al., 1998a; Yu et al., 1995). The COOH-terminus of kinectin
consists of two functional domains. The kinesin-binding domain can interact with the
cargo-binding site of the conventional kinesin and enhance the kinesin’s microtubule-
stimulated ATPase activity (Ong et al., 2000). A separate domain interacts with small
G-proteins such as Rho A, Rac 1 (Hotta et al., 1996; Neudauer et al., 2001) and plays
a key role in mediating the microtubule-dependent Rho G activity (Vignal et al.,
2001). Kinectin’s involvement in organelle motility is further supported by the
antibody inhibition whereby anti-kinectin monoclonal antibody blocks motor-

3
membrane binding and dramatically reduces both kinesin- and dynein-mediated
vesicle motility (Kumar et al., 1995). Our previous work has characterized the sites of

interaction between human kinectin and conventional kinesin (Kifs 5) using in vitro
and in vivo assays (Ong et al., 2000). The kinesin-binding domain on kinectin can
enhance the microtubule-stimulated ATPase activity and inhibit the kinesin-dependent
organelle motility in vivo (Ong et al., 2000).
However, the role of kinectin as a universal membrane anchor for kinesin has
been questioned when kinectin’s restricted intra-cellular and phylogenetic
distributions were discovered. Kinectin is not detected in axons of cultured neurons
where kinesin is the major motor responsible for fast anterograde transport
(Toyoshima and Sheetz, 1996). Furthermore, the kinectin gene is not found in
Caenorhabditis elegans or Drosophila genomes, where conserved conventional
kinesin heavy chain gene is present (Goldstein and Gunawardena, 2000). These
findings suggest that additional or alternative membrane anchors for kinesin must
exist for organelle motility. Recent yeast two-hybrid and biochemical studies have
identified a few new kinesin-interacting partners on different organelles. For example,
Sunday Driver interacts with the tricopeptide repeats of the kinesin light-chain subunit
of kinesin-1 and mediates the axonal transport of post-Golgi vesicles (Bowman et al.,
2000). Kinesin-1 was proposed to link to a class of transport vesicles via the JIP-1 and
JIP-2 scaffolding proteins that bind to members of the low-density lipoprotein
receptor family (Verhey et al., 2001). Another scaffold protein, 14-3-3 proteins, may
act as a membrane anchor for KIF1C (Dorner et al., 1998). The trans-membrane
amyloid precursor protein is another potential membrane anchor for kinesin-1, which
directly binds the tricopeptide repeats of the kinesin light-chain (Kamal et al., 2000).
The AP-1 clathrin-associated adaptor complex, which mediates the transport of

4
clathrin-coated vesicles from the trans-Golgi network to plasma membrane, binds
KIF13A (Nakagawa et al., 2000). There are studies revealing a potential interaction
between KIF3 and fodrin (brain spectrin) on neuronal vesicles (Takeda et al., 2000).
The recently identified dendrite-specific kinesin KIF17 can also interact directly with
the PDZ domain of mLin-10 (Setou et al., 2000). Therefore, a paradigm has emerged

that motor proteins utilize different membrane anchors and the same motor protein,
such as kinesin-1, can bind to different membrane anchors on different organelles
(Kamal and Goldstein, 2002). Since kinectin is primarily localized to the ER
(Toyoshima et al., 1992; Yu et al., 1995), sparingly on the lysosome (Vignal et al.,
2001) and mitochondria (Santama et al., 2004) but not Golgi apparatus

(Santama et
al., 2004), its primary functions may be restricted to these organelle compartments.
Different kinectin isoforms with combinations of variable domains (vd) have
been reported in human, mouse and fox genomes (Leung et al., 1996; Print et al.,
1994; Santama et al., 2004; Xu et al., 2002). There are at least five small (23-33
amino acid residues) variable domains scattered throughout the COOH-terminus of
kinectin (Leung et al., 1996). Two variable domains (vd3: amino acid residues 1177-
1200 and vd4: amino acid residues 1229-1256) overlap the kinesin-binding domain on
kinectin (Ong et al., 2000). It is interesting that the mapped down domain on kinectin
resides in the region where variable domain 3 and 4 reside (Ong et al., 2000).
This implies that the kinectin isoforms lacking either vd3 or vd4 cannot serve
as the membrane anchors for kinesin. Such isoforms without vd3 or vd4 have indeed
been identified in cells (Fig. 2 and Table 1) (Leung et al., 1996; Santama et al., 2004).
In this study, we are interested to investigate the function of one such kinectin isoform
lacking vd4 in ER. This work will contribute to a more unified understanding of the
functions of kinectin intracellularly.

5
Specific Aim 1: Characterization of kinectin-associated proteins
1.1 To identify the human kinectin isoforms.
1.2 To identify the protein interacts with one of the kinectin isoforms by yeast two-
hybrid screening.
1.3 In vitro characterization of kinectin and putative associated proteins.

Specific Aim 2: Mechanistic understanding of the role of kinectin with
Elongation Factor-1δ
2.1 To verify kinectin and Elongation Factor-1
δ
interactions by immunoprecipitation
assay.
2.2 Real-time binding kinetics of kinectin with Elongation Factor-1δ
2.3 To characterize the EF-1δ interacting domain on kinectin.
2.4 To investigate the effect of kinectin in the in vitro translation assay.
2.5 To investigate the co-localization of kinectin with EF-1δ by immunofluorescence
confocal microscopy.
2.6 To investigate the distribution of EF-1δ upon overxpression of kinectin.

Specific Aim 3: To investigate the mechanism of assembly of the entire
Elongation Factor complex onto ER membrane.

3.1 To characterize by yeast two-hybrid analysis the interacting pairs among EF-1α,
β, δ and γ subunits and kinectin.
3.2 To verify the interactions by in vitro binding assays.
3.3 To investigate the distribution of EF-1
β
and EF-1
γ
upon overxpression

of
kinectin.

6
3.4 To determine the distribution of EF-1δ, EF-1β and EF-1γ in cells with EF-1δ-

binding domain on kinectin being knocked down by morpholinos.
3.5 To assess the role of kinectin in protein synthesis.

Specific Aim 4: Biological relevance of kinectin in cells
4.1 To establish kinectin knockdown cells using DNA enzyme and small interference
RNA approaches.
4.2 To investigate the role of kinectin in intracellular dynamic of mitochondria and
endoplasmic reticulum in kinectin knockdown cells.

















7
Chapter 2: Background
2.1 Cytoskeleton

The cytoskeleton is a dynamic three-dimensional structure that fills the entire

cytoplasm. Their importance has been appreciated since the earliest days of cell
biology. The cytoarchitecture is responsible for the diversity in shapes and sizes of
cells and also contributed greatly to the multifaceted functions of each cell type
(Fuchs and Karakeisoglou, 2001). The cytoskeleton pulls the chromosomes aparts at
mitosis and then splits the dividing cells into two. Its drives and guides the
intracellular traffic of organelles, ferrying materials from one part of the cell to
another. Its supports the fragile plasma membrane and provide mechanical linkages
that let the cells bear stresses and strains without being ripped apart as the
environment shifts and changes. It enables some cells, such as sperm, to swim, and
others, such as machinery in the muscle cell for contraction and in the neuron to
extend an axon and dendrites. Its guides the growth of the plant cell wall and controls
the amazing diversity of cell shapes (Alberts et al., 2002a). The eukaryotic
cytoskeleton is composed primarily of three different types of fibrous structures,
namely, actin microfilaments, intermediate filaments and microtubules.
Each of these three principal types of protein filaments has a different
arrangement in the cell and a distinct function. By themselves, they can neither
provide shape nor strength to the cell. Different proteins regulate the cytoskeletal
network and thereby act together in determining cell shape and enabling motility
(Bear et al., 2001; Pan et al., 1993; Small et al., 2002). Their functions greatly depend
on a large retinue of accessory proteins, such as desmoplakin and plectins, which have
the capacity to physically link two or more cytoskeletal networks. Accessory proteins
are also essential for the controlled assembly of protein filament in particular

8
locations, and they provide the motors that either move organelles along the filaments
or move the filaments themselves (Fuchs and Yang, 1999).

2.1.1 Actin microfilament
Actin microfilaments are fine, thread-like protein fibers, 6 nm in diameter.
They are composed of monomeric cytoplasmic actin, a 43-45 kDa globular protein,

which is the most abundant cellular protein. Globular actin associates end-to-end to
form a protofilament in which polarity is maintained (Hamm-Alvarez, 1998). To form
the filament, two protofilaments of the same polarity wrap in a helix (375 angstrom
repeat) to form a microfilament. Assembly of actin can occur at either end, but with
different dynamics. One end of the microfilament is designated the barbed end and the
other is the pointed end. Actin filament assembly and function are known extensively
regulated by numerous capping proteins (Schafer and Cooper, 1995) and actin
monomer binding proteins (Sun et al., 1995) and its distribution is also sensitive to
intracellular signaling (Zigmond, 1996).
Although actin networks are dispersed throughout the cell, they are most
highly concentrated beneath the plasma membrane (Alberts et al., 2002a). The actin
filaments, depending on their orientation, can cause changes in shape of the cell
surface either as spikes, generated by discreet bundles, or ridges known as
lamellipodia produced by a flattened web of actin filaments. In addition, the
contraction of bundles of actin fibres in association with myosin is responsible for
muscle contraction. Microfilaments can also carry out cellular movements including
gliding, contraction, and cytokinesis (Alberts et al., 2002a; Small et al., 1999).