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 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 wholeheartedly in my research project. i 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 Chapter 2: Background 2.1 Cytoskeleton 2.1.1 Actin microfilament 2.1.2 Intermediate filament 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 2.6 Kinectin 2.5.4.1 Amyloid precusor protein (APP) 24 2.5.4.2 Sunday Driver (SYD) 24 25 ii 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 iii 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 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 His6 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 iv 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-1δ 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-1δ 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 v 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-1δ 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 vi 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 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-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 6.10 Discussion 227 Conclusion and future prospectives 235 References 239 Appendices 269 vii 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. EF1δ 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 viii (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. 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Bacterial culture media LB broth Reagent Amount Source Tryptone 10 g/L Difco, USA Yeast extract g/L Difco, USA Sodium chloride g/L Sigma, USA Water top up to L - Adjust to pH with 5N NaOH - Autoclave at 121oC for 15 - Store at 4oC LB/Amp agar - 18 g of agar added to L of LB broth - Autoclave at 121oC for 15 - Add ampicillin to 50 µg/ml - Pour plates and store at 4oC SOC broth Reagents Amount Source Sodium chloride g/L Sigma, USA Tryptone 10 g/L Sigma, USA Yeast extract 0.5 g/L Sigma, USA Potassium chloride 250 mM Sigma, USA Magnesium chloride 10 mM Sigma, USA Water top up to L - Adjust to pH with NaOH - Autoclave at 121oC for 15 269 - Add dextrose to final concentration of 20 mM - Store at 4oC FSB broth Reagents Amount Source Potassium acetate (pH 7.5) 10 mM Sigma, USA Manganese chloride 45 mM Sigma, USA Calcium chloride 10 mM Sigma, USA Potassium chloride 100 mM Sigma, USA Hexamminecobalt chloride mM Sigma, USA Glycerol 10% Sigma, USA - Dissolve all components in water - Ajust to pH 6.4 with 0.1N HCl - Filter the medium through 0.22 µm membrane filter - Store at 4oC Antibiotic stock solution Antibiotic Stock concentration Source Ampicillin 100 mg/L in water Sigma, USA Chloramphenicol 20 mg/L in ethanol Sigma, USA Kanamycin 50 mg/ml in water Sigma, USA - stock solution stored at –20oC 270 II. Yeast Two-hybrid media and stock solution YPD broth Reagents Amount Source Bacto peptone 20 g/L Difco, USA Yeast extract 10 g/L Difco, USA Water top up to L - Adjust to pH 5.8 with HCl - Autoclave at 121oC for 15 - Add dextrose to final concentration of 2% - Store at 4oC YPD agar - 20g of agar added to L of YPD broth - Autoclave at 121oC for 15 - Pour plates and store at 4oC SD medium Reagents Amount Source Yeast nitrogen base 6.7 g/L Difco, USA without amino acids Appropriate 10X dropout 100 ml supplements Water top up to 1L - Adjust to pH 5.8 with NaOH - Autoclave at 121oC for 15 - Add dextrose to final concentration of 2% - Store at 4oC 271 SD agar - 20g of agar added to L of SD medium - Autoclave at 121oC for 15 - Add dextrose to final concentration of 2% - Pour plates and store at 4oC 10X Dropout supplements Reagents Amount Source L-Isoleucine 300 mg/L Sigma, USA L-Valine 1500 mg/L Sigma, USA L-Adenine hemisulfate 200 mg/L Sigma, USA L-Arginine HCl 200 mg/L Sigma, USA L-Histidine HCl 200 mg/L Sigma, USA L-Leucine 1000 mg/L Sigma, USA L-Lysine HCl 300 mg/L Sigma, USA L-Methionine 200 mg/L Sigma, USA L-Phenylalanine 500 mg/L Sigma, USA L-Threonine 2000 mg/L Sigma, USA L-Tryptophan 200 mg/L Sigma, USA L-Tyrosine 300 mg/L Sigma, USA L-Uracil 200 mg/L Sigma, USA - Dropout supplements prepared lacking one or more components. - A combination of minimal SD base and dropout supplement will produce synthetic, defined minimal medium lacking one or more specific nutrients. - Autoclave at 121oC for 15 - Store at 4oC 272 X-gal substrate solution Prepare 20 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside in N,Ndimethylformamide. Store in dark at –20oC. III. Cell culture media and reagents Dulbecco’s Modified Eagle Medium (DMEM), pH 7.2-7.4 Reagents Amount Source DMEM powder satchet Gibco, New Zealand Fetal calf serum 100 ml/L Gibco, New Zealand Sodium bicarbonate 4.5 g/L Sigma, USA Penicillin/Streptomycin 10 ml/L Sigma, USA (100X) Water Top up to L - Filter the medium through 0.22 µm membrane filter - Store in dark at 4oC Pencillin and Streptomycin Solution (100X) Reagents Amount Source Penicillin G sodium x 106 units/100 ml Sigma, USA Stretomycin sulfate g/ 100 ml Sigma, USA Water Top up to 100 ml - Filter the solution through 0.22 µm membrane filter - Store at -20oC 273 Phosphate Buffered Saline (PBS) Reagents Amount Source g/L Merck, USA Potassium chloride 0.2 g/L Merck, USA Sodium phosphate 1.44 g/L Merck, USA Potassium phosphate 0.24 g/L Merck, USA Sodium chloride Water Top up to 1000 ml Trypsin-EDTA Solution Reagents Amount Source Trypsin 0.5 g/L Sigma, USA EDTA 0.2 g/L Sigma, USA PBS Top up to 1000 ml - This solution was then sterilized through membrane filtration. Cryo-preservation medium Reagents Amount Source Dimethylsulphoxide ml Sigma, USA Foetal calf serum ml Gibco, New Zealand Growth medium ml As above 274 IV. SDS-polyacrylamide gel electrophoresis reagent solution Gel loading buffer (6X) Reagents Amount Source Bromophenol blue 0.9 g/L Sigma, USA Xylene cyanol FF 0.9 g/L Sigma, USA 600 ml/L Sigma, USA 60 mM Sigma, USA Reagents Amount Source Tris base 181.5 g/L Sigma, USA Reagents Amount Source Tris base 60 g/L Sigma, USA Reagents Amount Source Tris base 15.1 g/L Sigma, USA Glycine 72 g/L Sigma, USA SDS g/L Sigma, USA Glycerol EDTA Separating gel buffer (4X) - adjusted to pH 8.8 Stacking gel buffer (4X) - adjusted to pH 6.8 Electrophoresis buffer (5X) 275 Coomassie destaining solution Reagents Amount Source Methanol 5% Merck, Germany Acetic acid 10% Merck, Germany Reagents Amount Source Methanol 50% Merck, Germany Acetic acid 10% Merck, Germany 0.1% (w/v) Sigma, USA Reagents Amount Source Tris Base 2.4 g/L Sigma, USA 11.26 g/L Sigma, USA 200 ml Merck, Germany Composition Source Coomassie Staining solution Coomassie Brillant Blue R250 Western Blot Transfer Buffer Glycine Methanol Western blot buffers Buffer TBS buffer TBST buffer Blocking buffer 10 mM Tris-HCl, pH 7.4 Sigma, USA 150 mM NaCl Sigma, USA 20 mM Tris-HCl, pH 7.4 Sigma, USA 500 mM NaCl Sigma, USA 0.05% (v/v) Tween-20 Sigma, USA 0.2% (v/V) Triton X-100 Sigma, USA 5% skim milk in TBS Diploma, Australia buffer 276 V. DNA cloning reagents TBE buffer (1X) Reagents Amount Source Tris base 10.8 g/L Sigma, USA Boric acid 5.5 g/L Sigma, USA EDTA 20 mM Sigma, USA Reagents Amount Source Tris-HCl, pH 20 mM Sigma, USA EDTA 10 mM Sigma, USA 100 ug/ml Sigma, USA Reagents Amount Source Sodium hydroxide 200 mM Sigma, USA SDS 1% (v/v) Sigma, USA Amount Source 3M Sigma, USA Reagents Amount Source Sodium chloride 750 mM Sigma, USA MOPS, pH 50 mM Sigma, USA Triton X-100 0.15% (v/v) Sigma, USA Buffer P1 RNase A Buffer P2 Buffer P3 Reagent Potassium acetate, pH 5.5 Buffer QBT 277 Buffer QC Reagents Amount Source 1M Sigma, USA MOPS, pH 50 mM Sigma, USA Isopropanol 15% (v/v) Sigma, USA Reagents Amount Source Sodium chloride 1.25 M Sigma, USA Tris-HCl, pH 8.5 50 mM Sigma, USA 15% (v/v) Sigma, USA Reagents Amount Source Tris-HCl, pH 10 mM Sigma, USA EDTA mM Sigma, USA Sodium chloride Buffer QF Isopropanol Buffer TE VI. Fixative 3.7% Paraformaldehyde - Dissolve 3.7g of paraformaldehyde in 100 ml of PBS - Warm the solution at 37oC until fully dissolved - Store in aliquots at –20oC. 278 [...]... 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-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. .. 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 5 3.4 To determine the distribution of EF-1δ, EF-1β and EF-1γ in cells with EF-1δbinding domain on kinectin being knocked down by morpholinos 3.5 To assess the role of kinectin in protein synthesis... 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 4 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 twohybrid screening 1.3 In vitro... 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... 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 6 Chapter 2: Background 2.1 Cytoskeleton The cytoskeleton is a dynamic three-dimensional structure that fills the entire cytoplasm Their importance... 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... 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 microtubulestimulated... 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 Ckinesin is composed of KifC1 and KifC2/C3 families Kifs of other... 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. .. 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 . 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. 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. 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