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

2.6.1 Discovery 25 2.6.3 Structural properties of kinectin 27 2.6.6 Kinectin’s role in organelle motility 30 2.6.7 Kinectin interaction with GTPases 32 2.6.8 Clinical implications of kin

THE ROLE OF KINECTIN IN ASSEMBLY OF

TRANSLATION ELONGATION COMPLEX TO

ENDOPLASMIC RETICULUM AND ITS INVOLVEMENT

NATIONAL UNIVERSITY OF SINGAPORE

2005

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

2.5.4.1 Amyloid precusor protein (APP) 24

2.6.1 Discovery 25

2.6.3 Structural properties of kinectin 27

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

2.8.7 Regulation of peptide elongation 52

Chapter 3: Materials and Methods

3.1 Isolation of native human kinectin isoforms 54

3.1.3 Extraction of DNA from agarose gel 55

3.1.6 Preparation of competent cells 56

3.1.11 Restriction endonuclease digestion 59

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

3.3 Amplification of human fetal brain library 64

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

3.5 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 His6 fusion proteins 69

3.6 Circular dichroism measurement for the baits 69

3.7.1 GST-tagged Bait proteins ligand preparation 70

3.11.5 In vitro phopshorylation of EF-1δ by cdc2 kinase 75

3.13 Real-time biomolecular interaction analysis 76

3.14 Identification of interacting domains 77

3.14.1 Identification of EF-1δ binding domain on Bait D 77

3.14.2 Identification of kinectin and EF-1γ binding domains 78

3.19 Transient knockdown of kinectin using DNA-enzyme 84

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

3.21 Transient knockdown of kinectin using pSUPER vector 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

Chapter 4: Results and Discussion - Kinectin anchors EF-1 δ 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

4.5 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-1δ 134

Chapter 5: Results & discussion - Kinectin anchors EF-1 complex to the ER

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-1γ binding domain on EF-1δ 161 5.7 Kinectin anchors the EF-1 subunits to the ER 166 5.8 Aberrant splicing of EF-1δ 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

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-1β and EF-1γ 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

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-1δ provides the guanine nucleotide exchange activities on EF-1α 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-1δ

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-1δ onto specific regions of the ER membrane while the EF-1βγ complex interacts with other regions of the ER membrane in kinectin- and EF-1δ- 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

(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

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-1γ binding domain on EF-1δ

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-1δ

Fig 11 Specificity of anti-EF-1δ 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-1δ 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

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

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

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

APP amyloid precursor protein

ATP adenine triphosphate

cDNA complementary DNA

C elegans Caenorhabditis elegans

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

IPTG isopropyl β-D-thiogalactosidase

KAP kinesin associated protein

KHC kinesin heavy chain

Kifs kinesin isoforms

KLC kinesin light chain

KRP kinesin related protein

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

siRNA small interfering RNA

SPR surface plasmon resonance

SRP signal recognition particle

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 4th 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, 43rdAnnual Meeting, Dec 7-13, San Francisco, U.S.A

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

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 NH3-terminus (Kumar et al., 1998a) The NH3-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-

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

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

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-1 δ

2.1 To verify kinectin and Elongation Factor-1δ 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-1β and EF-1γ upon overxpression of kinectin

3.4 To determine the distribution of EF-1δ, EF-1β and EF-1γ in cells with binding domain on kinectin being knocked down by morpholinos

EF-1δ-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

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

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)

2.1.2 Intermediate filament

Intermediate filamenents are ropelike fibers which with average 10 nm in diameter, and thus are "intermediate" in size between actin filaments (6 nm) and microtubules (25 nm) (Alberts et al., 2002a) There are at least 67 human genes encoding functional intermediate-filament proteins (Hermann et al., 2003; Zimek et al., 2003) The individual polypeptides of intermediate filaments are elongated molecules with an extended central α-helical rod domain of about 310 amino acids that forms a parallel coiled-coil with another monomer A pair of parallel dimers then associates in an anti-parallel fashion to form a staggered tetramer The tetramers pack together laterally to form the filament with no structural polarity (Alberts et al., 2002a; Chang and Goldman, 2004)

In most vertebrate cells, intermediate filaments form extensive network within the cytoplasm These networks extend radially in all directions to form cage-like structures that surround the nucleus to the cell surface (Chang and Goldman, 2004) Intermediate filaments impart intracellular mechanical strength and are consequently especially abundant in tissues such as epidermis and muscle that undergo substantial physical stress (Fuchs and Karakeisoglou, 2001) There are increasing number of intermediate filament-microtubule, intermediate filament-actin and intermediate filament-intermdiate filament crossbridge molecules, which range from molecular motors to multidomain proteins such as plectin, revealing a central role for intermediate filaments in coordinating cytoskeletal interactions (Helfand et al., 2002; Hollenbeck et al., 1989; Prahlad et al., 1998; Yabe et al., 2000) Furthermore, many kinases and cofactors bind to and phosphorylate intermediate filaments, placing them

in a favourable position for mediating the signaling events that seem to be related to

many aspects of the molecular crosstalk among the cytosketal systems (Meyer and Feldman, 2002; Sin et al., 1998; Tzivion et al., 2000)

2.1.3 Microtubules

A microtubule is a polymer of globular tubulin arranged into tubular and hollow filamentous tube with constant diameter of 20-25 nm (Alberts et al., 2002a) Tubulin exists predominantly as heterodimer of two subunits, α and β of about 55 and

50 kDa respectively, tightly bound together by noncovalent bonds The microtubule is

a 13-protofilaments tube, each protofilament composed of alternating α-tubulin and β-tubulin molecules (Gelfand and Bershadsky, 1991; Mandelkow and Mandelkow, 1995) The alignment of tubulin dimers within the microtubules leaves α-tubulins exposed to one end and β-tubulins exposed at the other, which gives microtubules intrinsic structural polarity (Nogales et al., 1999) The GTP bound to α-tubulin monomer is physically trapped at the dimer interface and is never hydrolyzed or exchanged In contrast, the nucleotide bound to the β-tubulin may be in either GTP or GDP form As a consequence of their structural polarity, microtubules also display kinetic polarity in that the rate of polymerization is different at the two ends; the faster growing end is the plus end (β-tubulins exposed), and the slower growing end is the minus end (α-tubulin exposed)

Microtubules are markedly reorganized throughout the life of the cell to support cellular functions such as modulation of cell shape, cell motility and cell division (Cole and Lippincott-Schartz, 1995; Garrett and Kapoor, 2003; Sato and Toda, 2004; Scliwa and Honer, 1993; Skoufias and Scholey, 1993; Stukenberg, 2003) One such major reorganization takes place during mitosis where spindle microtubules function in the dynamics of chromosome alignment and segregation (Morris et al.,

1998) The polarized arrays of microtubules provide tracks for the transport of organelles Microtubules determine the positions of the membrane-enclosed organelles and direct intracellular transport In most cells, microtubules grow outwards from an organizing center (MTOC) located adjacent to the nucleus, and extend throughout the cytoplasm towards the cell periphery (Lane and Allan, 1998) γ-tubulin, a protein with high homology to α/β-tubulin, is responsible for the nucleation of microtubules at the MTOC (Meads and Schroer, 1995; Vinh et al., 2002) In such cells, the slow-growing ends of microtubules (denoted minus ends) are all close to the MTOC, meaning that any organelles or vesicles moving towards the minus ends of microtubules will accumulate at the cell center, whilst organelles moving towards microtubule plus ends will translocate towards the cell periphery (Lane and Allan, 1998) In nerve axons, the microtubules are arranged longitudinally with the plus end pointing away from the cell body, whereas in epithelial cells microtubules are organized with plus end pointing towards the basement membrane (Hirokawa, 1998)

2.2 Microtubule based organelle transport

Intracellular transport is essential for morphogenesis and functioning of the cells After synthesis, proteins and lipids are sorted and transported to specific destinations within the cell (Miki et al., 2001) The trafficking of vesicles and organelles often occurs over long distances For example, membrane receptors destined for synapses in neuronal cells need to be transported from the cell body down axons that can reach a meter in length Diffusion would be prohibitively slow and cells have therefore evolved molecular motors that transport vesicle cargoes along microtubule or actin tracks (Verhey and Rapoport, 2001) The average rate for fast

axonal transport along microtubules is 0.5-5 µm/sec (Hirokawa, 1998) Small cells and local transport appear to rely on actin tracks (Atkinson et al., 1992), which serve

as a substrate for a large superfamily of myosin motors (Mermall et al., 1998) Large cells, long distances, and nuclear division appear to rely primarily on microtubule tracks, along which members of the kinesin and dynein superfamilies are involved (Goldstein and Philp, 1999)

2.3 Molecular Motors

Molecular motors bind to a polarized cytoskeletal filament and transduce chemical energy derived from ATP hydrolysis into mechanical work used for cellular motility (Howard and Hyman, 2003) Dozens of different motor proteins coexist in every eukaryotic cell They differ in the type of filament they bind to (either actin or microtubules), the direction in which they move along the filament, and the ‘cargo’ they carry Many motor proteins carry membrane-enclosed organelles such as mitochondria, Golgi stacks or secretory vesicles to their appropriate locations in the cell (Cole and Lippincott-Schartz, 1995) Other motor proteins cause cytoskeletal filaments to slide against each other, generating the force that drives such phenomena

as muscle contraction, ciliary beating and cell division (Alberts et al., 2002a)

The cytoskeletal motor proteins associate with their filament tracks through a

‘head’ region, or motor domain, that binds and hydrolyzes ATP Coordinated with their cycle of nucleotide hydrolysis and conformational change, the proteins cycle between states in which they are bound strongly to their filament tracks and states in which they are unbound Through a mechanochemical cycle of filament binding, conformational change, filament release, conformational relaxation, and filament

rebinding, the motor protein and its associated cargo move one step at a time along the filament (typically a distance of a few nanometers) (Alberts et al., 2002a)

2.3.1 Dynein

Organelles are able to move in both directions along microtubules Plus directed motility is driven by conventional kinesin and a number of KRPs By contrast, transport in the opposite direction along microtubule is driven predominantly

end-by cytoplasmic dynein (Lane and Allan, 1998)

Dynein was originally identified as a force-generating ATPase in

Tetrahymena cilia (Gibbons and Rowe, 1965), and a cytoplasmic dynein was later

discovered to power minus-end-directed motion in nonciliated cells (Paschal et al., 1987) Dynein is a multimeric complex (1-2 MDa) consisting of two heavy chains (>500 kDa) complexed with three 74 kDa intermediate chains; four light intermediate chains of 60-60 kDa; and one or two light chain of 8-11 kDa (Lane and Allan, 1998) The central and COOH terminal region of dynein form a globular domain interacting with microtubules and having motor activity, and the amino terminal region is though

to be the site of binding of cargoes (Koonce et al., 1992; Mikami et al., 1993; Zhang

et al., 1993) The rate of dynein driven microtubule motility can reach 3-5 µm/s from

in vitro assays (Kagami et al., 1990)

The best characterized binding partner for dynein is dynactin, a multisubunit complex essential for most dynein activities (Gill et al., 1991) Although it is well established that dynactin is required for dynein attachment to membranes, dynactin itself cannot bind to those membranes, so there must be another component serving as

a membrane anchor for dynactin (Gill et al., 1991; Karcher et al., 2002) Dynactin contains 11 subunits: p150Glued, p62, dynamitin (p50), actin-related protein 1 (Arp1),

Arp 11, β-actin, CapZα/β, p27, p25 and p24 (Echeverri et al., 1996; Holzbaur et al., 1991; Koonce et al., 1992; Paschal et al., 1993; Schafer et al., 1994; Schroer, 2004; Vaughan and Vallee, 1995) These play an important regulatory role in the binding ability of dynein to the microtubule

The coordinated action of flagellar dynein generates specific flagellar waveform and thus provides the motive force necessary to move the cell through the solution (reviewed in (Mitchell, 1994; Witman et al., 1994)) In contrast, cytoplasmic dyneins are involved in a variety of intracellular motile processes including maintenance of Golgi apparatus and trafficking of membraneous vesicles and other intracellular particles (Holzbaur et al., 1991; Lin and Collins, 1992; Vaisberg et al., 1996) Cytoplasmic dynein is indispensable for spindle pole assembly and stabilization (Gaglio et al., 1997; Merdes et al., 1996) Inhibition of either dynein or dynactin function leads to an embryonic lethal phenotype in both invertebrates and mice (Gepner et al., 1996; Harada et al., 1998) The direct heavy chain phosphorylation regulates the functional activity of cytoplasmic dynein (Dillman and Pfister, 1994)

2.3.2 Conventional Kinesin

Conventional kinesin was discovered in squid axoplasm as an active, microtubule-dependent organelle transporter (Brady, 1985; Vale et al., 1985a) It is found in many animal species, although not in budding yeast (Moore and Endow, 1996)

It is composed of two 120 kDa heavy chains (KHC) and two 64 kDa light chain (KLC) (Hirokawa et al., 1989) When observed by the low-angle rotary-shadowing electron microscopy, it has a rodlike structure composed of two globular

heads (10 nm in diameter), a stalk and a fanlike end, with a total length of 80 nm (Hirokawa et al., 1989) The detailed analyses of structure, sequence and expression strongly suggest that the region of KHC that generates force and movement (globular head motor domain) is composed of two regions, a 330 amino acid catalytic domain that hydrolyzes ATP and interacts with the microtubule track (Vale and Fletterick, 1997) and a short 40 amino acid neck domain important for processive movement and control of direction (Case et al., 1997; Romberg et al., 1998) The amino terminal motor domain of KHC is attached to an α-helical coiled coil stalk domain and a globular tail domain This motor protein moves at about 0.5 µm/ sec in moility assay

Conventional kinesin has been localized using immunofluorescence in a punctate distribution in mammalian cells, sea urchin cells and squid axoplasm, associated with a variety of organelles (Brady et al., 1990; Henson et al., 1992; Lippincott-Schwartz et al., 1995; Pfister et al., 1989; Schmitz et al., 1994; Wright et al., 1991) Antibody inhibition of KHC and KLC in extruded axoplasm models of axonal transport blocks both plus and minus end directed movement, the latter inhibition is thought to be a results of steric problems in the experiments (Brady et al.,

1990) Genetic analyses of conventional kinesin function in C elegans and Drosophila melanogaster have shown that kinesin mutations lead to neuronal defect

phenotypes of varying severity, including paralysis and death (Gho et al., 1992; Hurtley and Helenius, 1989)

2.3.3 Kinesin Superfamily

Initially, it was thought that all of these movements could be provided by conventional kinesin alone It is now clear that there are several subfamilies of kinesin related proteins A systematic moleclular biological search of genes coding for

proteins containing adenosine triphosphate (ATP)-binding and microtubule binding consensus sequences led to the discovery if the kinesin superfamily proteins (Kifs) that participate in axonal transport (Aizawa et al., 1992) Today, by combining molecular biological approaches with Basic Alignment Search Tool (BLAST) search

of proteins in public and private genome databases, a total of 45 KIFS have been identified in mouse and human genomes (Miki et al., 2001) A new kinesin tree includes 155 proteins from 11 species was reported recently (Dagenbach and Endow, 2004)

The KIFS have been classified based on the position of the motor domain, namely, N-kinesin with motor domain at amino terminal; M-kinesin with motor domain in the middle and C-kinesin with motor domain at the COOH terminal (Miki

et al., 2001) N-kinesin can be subdivided into 11 classes, namely, Kif1, Kif3, Kif4, Kif5, Kif13, Kif17 as the major members M-kinesin consists of only Kif2 family C-kinesin is composed of KifC1 and KifC2/C3 families Kifs of other species could be classified under these 14 classes (Kirokawa and Takemura, 2004)

Based on the in vitro motility assays, it appears that N-kinesins move towards

the plus ends of microtubules, while C-kinesins move towards the minus-ends of microtubules M-kinesin has plus-end-directed motility and a unique microtubule-depolymerizing activity They are move approximately at 0.1-1.5 µm/ sec as

determined by in vitro motility assay (Kirokawa and Takemura, 2004)

The KIFS take various molecular shapes Kif1A, Kif1Bα and Kif1Bβ are monomeric; many Kifs are homodimers, conventional kinesin or Kif5 form heterotetramer, consisting of two KHC and KLC KIF3 forms heterotrimeric complex, which composed of Kif3A, Kif3B and kinesin associated protein 3 (KAP3)

Although the motor domain is highly conserved among different Kifs, other regions of the molecules are quite divergent and these regions serve as cargo-binding domain The sequence diversity of the cargo-binding domains explains why Kifs transport a wide variety of cargoes, including membraneous organelles, macromolecular complexes and mRNAs However, each cargo appears to be selectively recognized and transported to a precise destination (Aizawa et al., 1992; Okada et al., 1995; Yonekawa et al., 1998; Zhao et al., 2001) Knockout mice of KIF1A or KIF1Bβ show motor and sensory nerve defects and have a reduced number

of synaptic vesicles at the synaptic terminals (Yonekawa et al., 1998; Zhao et al., 2001) In Alzheimer’s disease patients brain, vesicles containing amyloid precursor protein (APP) are transported in the axon by KIF5 (Kamal et al., 2001; Kamal et al., 2000) Other organelles such as mitochondria, lysosomes and tubulin oligomers are also transported by Kif5 (Nakata and Hirokawa, 1995; Tanaka et al., 1998; Terada et al., 2000) Kif3 complex transport vesicles associated with fodrin (Takeda et al., 2000) In the dendrites, vesicles containing N-methyl-D-aspartate (NMDA)-type glutamate receptors are transported by Kif17 from the cell body to the postsynaptic sites (Gundersen et al., 1998; Setou et al., 2000)

2.4 Establishment and maintenance of organelle position in cells

In cells, one of the important function membrane molecular proteins is the maintenance of organization of organelles The endoplasmic reticulum (ER) (Terasaki

et al., 1986), early endosomes (Hopkins et al., 1990), lysosomes (Swanson et al., 1987) and Golgi complex (Cooper et al., 1988) have been shown to have the capacity

to form extended tubular processes that move along microtubules Golgi membranes are normally localized at the minus ends of microtubules in the perinuclear region of

non-polarized cells In the presence of brefeldin A, extensive Golgi tubules form and extend along microtubules towards their plus ends (Lippincott-Schwartz et al., 1991) The cytosolic acidification in various cells types results in the redistribution of late endosomes (Parton et al., 1991) and lysosomes (Heuser, 1989) towards the plus ends

of microtubules, whereas alkalinization causes a shift in distribution towards their minus ends

Most organelles have the capacity to bind to both plus and minus end directed

motors, and the in vitro and organelle-motor competition binding studies support the

concept that a regulated motor complex which contains both plus and minus end directed motor activities and shared activator molecules may control the direction of organelle movement (Schroer and Sheetz, 1991; Yu et al., 1992) Both microtubules and motor proteins are responsible for the position of organelles at particular locations within cells and also maintenance of spatial distribution among the organelles (Lane

and Allan, 1998)

2.4.1 Endoplasmic reticulum

The ER is an extensive network of interconnected membranes that is comprised of nuclear envelope, rough ER, smooth ER and intermediate compartment which extends to occupy the whole cell (Krijnse-Locker et al., 1995) It was thought that such distribution maximizes the surface area of the ER, which will facilitate various roles such as calcium regulation, lipid synthesis and translocation of newly synthesized proteins into the ER membrane and lumen

The dynamics of the ER network depend on microtubules When microtubules are deploymerized, the ER retracts towards the cell centre The ER is then capable of extending outwards once again along newly formed microtubules (Lee et al., 1989)

In electron microscopy studies, a close relationship of ER cisternae and microtubules has been observed in a variety of animal cells (Masurosky et al., 1981; Tokunaga et al., 1983) Three possible mechanisms for microtubule-dependent construction and motility of ER networks were revealed from the time-lapse microscopy (Baumann and Walz, 2001) The ER tubule is stably attached to a microtubule, and the microtubule is translocated along the substrate or cellular structure; new tubules may

be dragged along by the tips of polymerizing microtubules; or an tubule slides along a stationary microtubule by the activity of ER-bound motor proteins such as kinesin or cytoplasmic dynein

The evidences supporting the role of kinesin in the plus-end directed extension

of the ER come from a variety of studies Immunofluoresence results using kinesin antibodies generally show punctate staining patterns which are often closely associated with ER membrane (Hollenbeck, 1989) Kinesin has been identified on ER vesicles in squid axoplasm by immunogold labeling (Tabb et al., 1998) The antibodies to kinectin, an abundant kinesin-binding protein gives a reticular staining pattern characteristic of ER in several cell types (Toyoshima et al., 1992) The

anti-inhibition of ER motility by anti-kinesin or anti-kinectin antibodies in in vitro assay

(Kumar et al., 1995; Lane and Allan, 1999) The suppression of kinesin heavy chain expression in cultured rat hippocampal neurons by anti-sense oligonucleotides resulted in the retraction of ER network from the periphery into the center of the cells, without affecting the distribution of microtubules (Feiguin et al., 1994)

2.4.2 Mitochondria

Mitochondria are vital determinants of both the life and death of cells (Newmeyer and Ferguson-Miller, 2003) Mitochondria appear to adopt a variety of

different shapes in living cells, ranging from multiple small compartments to tubular networks (Collins et al., 2002; Legros et al., 2002) This suggests an important role for the cytoskeleton in maintaining their intracellular distribution (Allan and Schroer, 1999)

Ultrastructural examination in neurons have shown intimate associations and even cross-bridges between mitochondria and microtubules (Hirokawa, 1982) and between mitochondria and actin filaments (LeBeuz and Willemot, 1975) In neurons, mitochondria are distributed through a combination of transport and stopping events (Allen et al., 1982) Disruption of axonal transport leads to nonuniform distribution of organelles along axon Isolated mitochondria display both plus and minus end directed movement along microtubules (Morris and Hollenbeck, 1995)

Three members of the kinesin superfamily have been implicated in transport

of mitochondria, namely, conventional kinesin Kif5B (Tanaka et al., 1998), KLP67A (Pereira et al., 1997) and Kif 1B (Nangaku et al., 1994) Conventional kinesin, Kif5b,

is localized to mitochondria in vivo and the null mutants in mouse exhibits a clear

defect in mitochondrial dispersion in extra-embryonic cells (Tanaka et al., 1998) In L929 cells, inhibition of conventional kinesin by SUK4 antibody resulted in perinuclear clustering of mitochondria (De Vos et al., 2000) Kif1B was found concentrated in the mitochondrial fraction and purified Kif1B could transport mitochondria along microtubule in vitro (Nangaku et al., 1994) Kinesin light chain are thought to form the cargo-binding domain of motor protein with mitochondria in various cultured cells (Khodjakov et al., 1998) Treatment with tumour necrosis factor induces hyperphosphorylation of kinesin light chain and inhibits kinesin-mediated transport of mitochondria (De Vos et al., 2000)

It is unclear whether cytoplasmic dynein is the mitochondrial minus directed motor, since the overexpression of p50/ dynamitin had no effect on mitochondrial position (Burkhardt et al., 1997) Recently, the expression of dominant negative mutant of Drp1 (dynamin-related protein) results in the formation of highly interconnected, fused mitochondria (Smirnova et al., 1998) Tctx1, a dynein light chain that is capable of supporting dynein-mediated transport, has shown to interact with voltage-dependent anion selective channel (VDAC) in yeast two hybrid screening (Schwarzer et al., 2002) The latter is the major channel for the movement

end-of adenosine nucleotides for the mitochondria

2.4.3 Activation of kinesin

The organelle distribution is likely to be achieved by regulation of kinesin activity The majority of kinesin in cells is associated with neither microtubules nor vesicles It is apparently kept in an inactive state when not transporting cargo, preventing its futile movement along microtubules Kinesin is in a folded conformation such that the KHC globular tail domain interacts with and inhibits the KHC motor domain The extreme COOH terminus of kinesin tail domain is necessary for this inhibition, partially via interaction with the motor’s neck domain Cargo binding by the carboxy-terminal tail may release the motor domain from this inactive conformation, allowing them to interact with microtubules The role of motor’s light chain in this regulation is not clear, but may serve to stabilize the inactive form (Coy

et al., 1999; Friedman and Vale, 1999; Hackney and Stock, 2000; Verhey et al., 1998)

In other models, the binding of the motor to its cargo might not automatically lead to activation A kinase or phosphatase could modify kinesin bound to a vesicle

and activate its motility There are plenty of evidence that both KHC and KLC can be phosphorylated but the regulatory role in vivo remains to be established (Verhey and Rapoport, 2001)

2.5 Motor protein receptors

In the past decade, major emphasis has been placed on identifying different cargos and motor proteins and the identification of motor receptors has been slow in coming The followings are the kinesin-associated proteins identified so far

2.5.1 Coat Proteins

Transport of cargo proteins between membrane-bound organelles is generally thought to occur via carrier vesicle The first step is self-assembly of coat proteins (such as clathrin and coatomer proteins) on the cytoplasmic side of the membrane, and they are used to shuttle cargo between membranes in distinct steps of the secretory pathway The AP-1 clathrin-associated adaptor complex helps to mediate the transport of clathrin-coated vesicles from the trans-Golgi network to plasma membrane, was found to bind to the Kif13A (Nakagawa et al., 2000)

Spectrin is another type of coat protein that has been implicated in membrane organization Several isoforms of spectrin have been discovered to interact with membraneous organelles (De Matteis and Morrow, 2000) Spectrin binds to the multi-protein dynactin complex, an adaptor thought to link dynein to its cargoes A neuronal isoform of spectrin, fodrin, was found to interact with KAP3, the accessory subunit of Kif3 that has been suspected to be involved in organelle binding (Takeda et al., 2000)

The high degree of heterogeneity of spectrin, owing to multiple genes and alternative mRNA splicing, may be important for recruiting particular motors or combinations of motors to organelles (Klopfenstein et al., 2000)

2.5.2 Scaffold Proteins

Sequence analyses have revealed that the tail domains of several motors contain protein interaction modules such as SH2, SH3, pleckstrin homology, tetratricopeptide and PDZ domains Kif17 has been found to associate with a large PDZ-containing complex that forms on neuronal membranes (Setou et al., 2000) A two-hybrid screening identified another scaffold protein, four isoforms of the 14-3-3 family, interact with Kif1C (Dorner et al., 1999) Co-immunoprecipitation demonstrated that the two proteins interact only when a serine in the motor’s COOH terminal domain was phosphorylated by casein kinase II This finding suggest that components of signaling cascades are recruited to scaffold assemblages along with downstream target motors in order to drive the regulated movement of attached cargo (Klopfenstein et al., 2000) There is work demonstrating that kinesin light chain binds

to JIP-1 and JIP-2 proteins, which are thought to serve as scaffolding proteins for the c-Jun N-terminal kinase (JNK) signaling pathway (Verhey et al., 2001) Additional data suggest that interactions of kinesin and JIP proteins may be essential for proper localization of JIPS to nerve terminal

2.5.3 Small GTPases

Small G proteins participate in a wide variety of regulation and signaling events The GTP-binding proteins (also called GTPases) constitute a large family of proteins that all contain variations on the same GTP-binding globular domain of about