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J. Biol. Chem., Vol. 278, Issue 34, 32115-32123, August 22, 2003
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to the Endoplasmic Reticulum*
From the
National University Medical Institutes
and ¶Department of Physiology, Faculty of
Medicine, National University of Singapore, Singapore 117597
Received for publication, October 25, 2002 , and in revised form, May 27, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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). This was shown by yeast two-hybrid analysis and a number
of in vitro and in vivo assays. EF-1 provides the
guanine nucleotide exchange activities on EF-1
during elongation step
of protein synthesis. The minimal EF-1-binding domain on kinectin
resides within a conserved region present in all the kinectin isoforms.
Overexpression of the kinectin fragments in vivo disrupted the
intracellular localization of EF-1 proteins. This report provides
evidence of an alternative kinectin function as the membrane anchor for
EF-1 on the endoplasmic reticulum and provides clues to the EF-1
complex assembly and anchorage on the endoplasmic reticulum. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helical coiled-coil domain to form a heterodimer
(4). The 120-kDa polypeptide is
the truncated version of the 160-kDa polypeptide, lacking the first 232 amino
acids, in the N terminus (4).
The N terminus of the 160-kDa polypeptide consists of a transmembrane domain
that anchors kinectin to organelle membranes, potentially with the help of the
7 myristylation sites throughout the molecule
(3,
4). The C 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
(5). A separate domain
interacts with small G-proteins such as Rho A and Rac 1
(6,
7) and plays a key role in
mediating the microtubule-dependent Rho G activity
(8). The role of kinectin in
organelle motility is further supported by antibody inhibition and
kinectin-domain overexpression studies, in which the organelle motility is
adversely affected both in vitro and in vivo
(2,
5).
The role of kinectin as a universal membrane anchor for kinesin has been questioned when kinectin's restricted intracellular 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 (9). Furthermore, the kinectin gene is not found in Caenorhabditis elegans or Drosophila genomes, where conserved conventional kinesin heavy chain gene is present (10). 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 examples, 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 (11). 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 (12). Another scaffold protein, 14-3-3 proteins, may act as a membrane anchor for KIF1C (13). The transmembrane amyloid precursor protein is another potential membrane anchor for kinesin-1, which directly binds the tricopeptide repeats of the kinesin light chain (14). 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 (15). There are studies revealing a potential interaction between KIF3 and fodrin (brain spectrin) on neuronal vesicles (16). The recently identified dendrite-specific kinesin KIF17 can also interact directly with the PDZ domain of mLin-10 (17). 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 (18). Since kinectin is primarily localized to the ER (1, 3), sparingly on the lysosome (8) and mitochondria2 but not Golgi apparatus, 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 (19–21).2 There are at least five small (23–33 amino acid residues) variable domains scattered throughout the C terminus of kinectin. Two variable domains (vd3: amino acid residues 1177–1200 and vd4: amino acid residues 1229–1256) overlap the kinesin-binding domain on kinectin (5). 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. 1A) (20).2 We have investigated the function of one such kinectin isoform lacking vd4 in ER.
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We have isolated from a human fetal brain cDNA library a naturally occurring kinectin isoform that contains variable domains vd1, vd2, vd3, vd5, and vd6 but lacks vd4 (Fig. 1A). This kinectin isoform did not interact with the conventional kinesin heavy chain in a yeast two-hybrid analysis (Fig. 1A). However, it interacted with another resident of the ER, the translation elongation factor-1 delta, in a series of in vitro and in vivo experiments.
EF-1 is a subunit of the protein complex that participates in the
elongation step during the translation of mRNA. The EF-1 complex consists of
four subunits, namely, , 
, , and 
. The GTP binding
protein, EF-1, is responsible for the transfer of aminoacyl-tRNA to
ribosome with the hydrolysis of GTP. The EF-1 and EF-1 subunits
are responsible for the nucleotide exchange activity, which converts the
EF-1•GDP to the active EF-1•GTP
(22–24).
Both and sequences appear unrelated other than the C-terminal
domain, which is responsible for the nucleotide exchange activity
(25). The exact function of
EF-1 is not yet known, although there are studies indicating that
EF-1 can enhance the guanine nucleotide exchange activity of
EF-1, and its association with tubulin suggests that EF-1 may
help to anchor EF-1 to cell structures like microtubules and membranes
(26,
27).
We report here that kinectin interacts with EF-1 to anchor it to ER.
Disruption of the kinectin-EF-1 interaction interferes with the
EF-1 distribution in ER. The results provide us with a better
understanding of the kinectin function in the ER.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Construction of the Kinectin Bait D—A pair of oligonucleotides flanking 2539–4143 base pair (bp) of the human kinectin gene (GenBankTM accession number Z22551 [GenBank] ) was designed to isolate different kinectin isoforms. A standard polymerase chain reaction (PCR) (28) was carried out using the human fetal brain, fetal liver, adult brain, and prostate libraries (Clontech). The amplified PCR products were subcloned into pTOPO vector (Invitrogen) by TA cloning. The different kinectin isoforms were grouped by restriction mapping, and their sequences were verified by sequencing. The region between 3142 and 4139 bp (amino acid residues 1024–1356) of an isoform was subcloned in-frame to the GAL4 DNA-binding domain (BD) of the pAS2–1 vector (Clontech) and renamed as bait D.
Yeast Two-hybrid Screening—Yeast two-hybrid screening (Clontech) was performed according to the supplied protocol. The expression of baits A, B, and D fusion proteins in yeast were determined by immunoblotting the yeast extract with GAL4 DNA-BD monoclonal antibody (Clontech).3 The kinectin bait D was used to screen a human fetal brain Matchmaker cDNA library fused to the GAL4 DNA-activation domain (AD) of the pACT2 vector (Clontech). The bait and the amplified library were sequentially transformed into yeast strain Y190 (Clontech) using the lithium acetate method (29). The transformants were assayed for their expression of histidine (HIS3) reporter gene by plating them onto the synthetic medium deficient in tryptophan, leucine, and histidine. 25 mM 3-amino-1,2,4-triazole was included to limit the number of false HIS3 positives (30). Colonies that grew successfully in the selective medium were further screened for their expression of the lacZ reporter gene by colony lift assay (31). The cDNAs in the AD vector of the persistent positive clones were isolated. False positives were further eliminated by testing whether the clones could activate both HIS3 and lacZ reporter genes with control baits (baits A, B, and BD vector alone).
The primary sequences of the positive clones were determined with an ABI PRISM Big DyeTM Terminator Cycle Sequencing Ready Kit using an ABI PRISMTM 377 DNA sequencer, according to the manufacturer's instructions. The results were analyzed with the LASER-GENE (DNASTAR) and the BLAST 2.0 (National Library of Medicine) software.
In Vitro GST Pull-down Assay—The glutathione
S-transferase (GST) fusion constructs with the kinectin baits A, B,
and D were made by subcloning the corresponding cDNA into the expression
vector pGEX4T-1 (Amersham Biosciences). A His6-tagged fusion
construct with EF-1 was made by subcloning the EF-1 cDNA into
pRSET expression vector (Invitrogen). The fusion proteins were expressed in
Escherichia coli strain BL21/pLysS (DE3) (generous gift from Niovi
Santama, Cyprus Institute of Neurology and Genetics, University of Cyprus).
Protein expression was induced by the addition of
isopropyl--D-thiogalactoside. Intact bacterial cells
containing the expressed fusion proteins were collected by centrifugation at
6000 x g for 10 min. Protein extracts were obtained by freezing
and thawing the cell pellet and resuspending the pellet in GST purification
buffer (1x phosphate-buffered saline (PBS), 50 mM Tris-HCl,
pH 8, 0.5 mM MgCl2, 0.1% (v/v) Triton X-100) plus 5
mM dithiothreitol and a mixture of protease inhibitors (1 µg/ml
each of aproptinin, leupeptin, pepstatin A and 1 mM
phenylmethylsulfonyl fluoride). Equal amounts of the GST fusion proteins with
baits A, B, and D were immobilized onto glutathione-agarose beads by
end-over-end mixing in a 1.5-ml microcentrifuge tube for 2 h at 4 °C. The
beads were washed three times with GST purification buffer to remove unbound
proteins. The expressed His6-EF-1 (36 kDa) fusion protein
extract was allowed to interact with the GST-bait fusion protein-coated beads
for 2 h at 4 °C with end-over-end mixing. Any unbound proteins were
removed through extensive washings. The proteins that remained bound to the
immobilized GST-baits were released by boiling in SDS gel sample buffer,
analyzed by SDS-polyacrylamide gel (12%) electrophoresis (PAGE), and
immunoblotting with mouse RGS-His antibody (Qiagen). Antibody binding
was detected with goat anti-mouse secondary antibody, coupled to horseradish
peroxidase according to the manufacturer's instructions (Sigma).
Co-immunoprecipitation of Kinectin with
EF-1—African green monkey kidney cells, COS7, were
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
(v/v) fetal calf serum (FCS), L-glutamine, penicillin, and
streptomycin. For transient transfection assays, GST-tagged kinectin bait D
(64 kDa) and FLAG-tagged EF-1 (36 kDa) were generated by subcloning the
corresponding cDNA into the pXJ-GST and pXJ-FLAG mammalian expression vectors,
respectively (32). Both
constructs were co-transfected into COS7 cells cultured in a 60-mm dish using
TransFastTM transfection reagent (Promega). The transfected cells were
washed with ice-cold 1x PBS at 48 h after transfection. The cells were
isolated into 1 ml of the ice-cold GST purification buffer containing protease
inhibitors (as described above). Cell lysate was prepared by passing the cells
through 25-gauge needle for 10 times. Intact cells and debris were removed by
centrifugation at 1000 x g for 10 min at 4 °C. 60 µl of
the equilibrated 50% glutathione-agarose bead suspension was added to the cell
lysate and incubated for 2 h at 4 °C. Immunoprecipitates were detected by
immunoblotting with a mixture of anti-GST (Santa Cruz Biotechnology) and
anti-FLAG M2 monoclonal antibodies. Antibody binding was detected as described
above.
Anti-EF-1 Polyclonal Antibody
Preparation—The EF-1 recombinant protein was expressed in
E. coli BL21 using pRSET vector (Qiagen). The fusion protein was
purified using the nickel-nitrilotriacetic acid agarose bead affinity column
(Qiagen). The purified EF-1 protein was injected into rabbit by
standard protocol (33). The
antiserum was affinity-purified using the first 100 amino acids of the
EF-1 protein coupled to cyanogen bromide-activated Sepharose 4B
(34).
In Vitro Phosphorylation of EF-1 by cdc2
Kinase—HeLa cell lysate was prepared using SF buffer (50
mM HEPES-NaOH pH 7.4, 150 mM NaCl, 10 mM
MgCl2, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1%
sodium orthovanadate, 25 mM -glycerophosphate) in the
presence of protease inhibitors (as described above). 1 µg of the HeLa cell
lysate was incubated with or without 10 units of cdc2 kinase in kinase
reaction buffer (50 mM Tris-HCl, 10 mM
MgCl2,2mM dithiothreitol, 1 mM EGTA, 0.01%
Brij 35, 100 µM ATP) at 30 °C for 30 min. The reaction
samples were separated by SDS-PAGE and the EF-1 was detected by
immunoblotting with the purified anti-EF-1 polyclonal antibody.
Antibody binding was detected with swine anti-rabbit secondary antibody
(Dako), coupled to horseradish peroxidase according to the manufacturer's
specifications.
In Vitro Binding of the Endogenous EF-1 (32 kDa) with
Kinectin Baits—Bacterially-expressed kinectin baits A, B, and D
were coupled onto glutathione-agarose beads as described above. HeLa cells
were cultured in DMEM supplemented with 10% FCS, L-glutamine,
penicillin, and streptomycin. The cells were harvested and re-suspended with
one volume of the GST purification buffer in the presence of protease
inhibitors. The cell lysate was prepared as described above and was added to
the beads coated with GST-bait fusion protein and incubated with end-over-end
mixing for 2 h at 4 °C. Immunoprecipitates were detected by immunoblotting
with the purified anti-EF-1 polyclonal antibody.
In Vitro Binding of the Endogenous Kinectin with
EF-1—Bacterially expressed EF-1 were coupled
onto glutathione-agarose beads. The HeLa cell lysate was prepared as described
above and was added to the beads coated with GST-EF-1 and incubated
with end-over-end mixing for 2 h at 4 °C. Immunoprecipitates were detected
by immunoblotting with CT-1 monoclonal antibody (generous gift from Dr. Martin
Krönke, University of Cologne, Germany). Antibody binding was detected as
described above.
Real-time Biomolecular Interaction Analysis—The GST-tagged
kinectin bait D (64 kDa) and His6-tagged EF-1 (52 kDa)
recombinant proteins were expressed in E. coli BL21 using pGEX4T-1
and pET32 (Novagen) vectors, respectively. The GST-tagged and
His6-tagged recombinant proteins were purified according to
manufacturers' protocols (Amersham Biosciences & Novagen). The
biomolecular interaction analysis was based on surface plasmon resonance (SPR)
technology, which was performed with BIACORE 3000 (BIAcore AB). HBS buffer (10
mM HEPES-NaOH pH 7.4, 150 mM NaCl, 3.4 mM
EDTA, 0.005% surfactant P20) was used as flow buffer, and the flow rate was
kept at 5 µl/min. 1 mg/ml of GST-only and GST-bait D proteins were coupled
to CM5 sensor chip surfaces to yield a signal of 4000 response unit (RU) using
standard amine coupling procedure
(35). A control reference flow
cell was included where no ligand bound. The surfaces were then inactivated
with 1 M ethanolamine. 50 µl of 1 µM of the
analyte was injected for 10 min and the binding kinectics was monitored for 16
min. After each interaction cycle, the surfaces were regenerated with 10
mM glycine, pH 2.5. The analytes were injected in triplicates in
random order. All interaction experiments were performed at 25 °C. All
binding curves were corrected for refractive index changes and nonspecific
binding by subtraction of the control reference flow cell. The association and
dissociation rate constants were deduced from the initial signal courses of
the association and dissociation phases, assuming 1:1 Langmuir binding and
employing the BIAevaluation 3.0 software (BIAcore AB).
Mapping of the EF-1 Binding Domain on Kinectin Bait
D—The EF-1 cDNA obtained from the yeast two-hybrid screening
was subcloned into the BD vector. A series of truncated clones of the kinectin
bait D (D1 to D18) were constructed (Fig.
5) by PCR. The amplified fragments were subcloned into the AD
vector, and their interaction with the EF-1 was assayed using the yeast
two-hybrid analysis as described above. A total of 50 yeast transformants were
picked for each truncated clone to assay for the activation of HIS3
and lacZ reporter genes. Clones were considered as positive only when
more than 90% of the transformants activated both HIS3 and
lacZ reporter genes.
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Immunofluorescence Assay—African green monkey kidney cells,
CV1 cells, were seeded on coverglass overnight and maintained in DMEM
supplemented with 10% FCS, L-glutamine, penicillin, and
streptomycin. The cells were fixed with 3.7% paraformaldehyde in PBS for 10
min at 37 °C and permeabilized with 0.01% saponin in PBS for 5 min at room
temperature. The fixed cells were blocked with 10% FCS in PBS for an hour
prior to incubation with the primary antibodies, CT-1 monoclonal antibody, and
anti-EF-1 polyclonal antibody overnight. The cells were washed three
times and followed by 1 h of incubation with the secondary antibodies,
DIG-conjugated sheep anti-mouse IgG and Biotin-conjugated swine anti-rabbit
IgG (Dako). The cells were incubated for another 1 h with the tertiary
antibodies, FITC-conjugated sheep anti-DIG IgG and TRITC-conjugated Extravidin
after the three washes with PBS. The coverglasses were washed thoroughly,
mounted in FluorSaveTM (Calbiochem) and imaged using a Carl Zeiss LSM510
laser-scanning confocal microscope.
Overexpression Studies—The EF-1 binding domains on
kinectin, baits D and D12, were subcloned into the pEGFP-C vector (Clontech)
for optimal expression in mammalian cells. The pEGFP-C vector alone was used
as control. CV1 cells were cultured and transfected as described above. 48-h
post-transfection, the cells were treated with 1 µg/ml of nocodazole at 37
°C for 2 h and followed by another 3 h in the absence of nocodazole. Cells
were subsequently fixed and stained with anti-EF-1 polyclonal antibody
prepared as described above. Transfected cells were identified by green
fluorescence protein expression and scored either with distinct or disrupted
EF-1 distribution. A total of 30 cells were observed for each clone
during each experiment. The data tabulated were based on three independent
experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-helical coiled-coil. Baits A, B, and D showed no
interaction with the conventional kinesin heavy chain in yeast two-hybrid
analysis (Fig. 1A).
This is consistent with our previous findings that the vd3 and vd4 of kinectin
are required for kinesin binding
(5).
In order to identify interacting partners of the kinectin isoforms that
lack the kinesin-binding domain, we performed a yeast two-hybrid screening of
the human fetal brain cDNA library. A total of 17 unique positive-clones were
identified from screening 
2.35 x 107 transformants.
Among them, one of the positive clones has sequence identical to the published
human translation elongation factor-1 delta sequence (EF-1,
GenBankTM accession number NM001960). The plasmid obtained has an insert
of 1200 bp in length and encodes the entire open reading frame of the
EF-1. EF-1 interacts specifically only with bait D, but not
baits A or B in the yeast two-hybrid analysis
(Fig. 1A). Baits A and
B proteins were produced with the predicted molecular mass in
yeast.3
GST-Kinectin Bait D Specifically Interacts with EF-1 in
Vitro—To verify that kinectin bait D binds to the full-length
EF-1 in vitro, we first performed a GST pull-down analysis.
GST fusion constructs with the kinectin baits A, B, and D were made and
expressed in E. coli. Equal amounts of the individual fusion protein
extracts were immobilized onto glutathione-agarose beads. The beads were
washed extensively to remove unbound proteins such that only the kinectin
baits remained bound to the beads. The His6-tagged EF-1
construct was expressed in E. coli, and the cell extract was allowed
to interact with the immobilized GST-bait fusion proteins. The proteins
remained bound to the immobilized GST-baits after extensive washings were
analyzed by immunoblotting with anti-His antibody
(Fig. 1B). Association
of the EF-1 in vitro with the kinectin bait D, but not with
baits A or B, is consistent with the interactions observed in the yeast
two-hybrid analysis.
Kinectin Interacts with EF-1 in Mammalian
Cells—To assess whether the interaction of EF-1 and kinectin
occurs in mammalian cells, GST-tagged bait D and FLAG-tagged EF-1 were
co-transfected into COS7 cells. Cell lysate was prepared 48 h after
transfection and precipitated with glutathione-agarose beads. The protein
complexes pulled down by the beads were analyzed by immunoblotting with
anti-GST and anti-FLAG antibodies. The FLAG-tagged EF-1 indeed
co-precipitated with the GST-bait D and not with GST-only (control)
(Fig. 2), indicating that
kinectin bait D interacts specifically with EF-1 in COS7 cells.
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Interaction Analysis with Endogenous Proteins—To further
confirm their interaction, we investigated whether the ectopically expressed
kinectin would interact with the endogenous EF-1 in HeLa cells.
Polyclonal anti-EF-1 antibody was produced using purified
His6-tagged protein containing the first 100 amino acids of the
human EF-1 as antigen. The affinity-purified antibody recognizes two
polypeptide bands (32 kDa) on SDS-PAGE in HeLa cell lysate
(Fig. 3A). The N
terminus of the EF-1 protein has shown no significant homology with
other proteins (36). The
purified antibody is specific for EF-1 and has exhibited no
cross-reactivity with EF-1
(25)
(Fig. 3B). The two
bands that the purified antibody recognizes are the un-phosphorylated and
phosphorylated forms of EF-1
(37,
38). When phosphorylated
in vitro by cdc2 kinase, the un-phosphorylated EF-1 undergoes
a shift in electrophoretic mobility to the phosphorylated form
(34)
(Fig. 3C).
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The HeLa cell lysate was incubated with the immobilized GST-bait fusion
proteins. The endogenous proteins that remained bound to the baits were
analyzed by immunoblotting with the purified anti-EF-1 antibody. The
association of the endogenous EF-1 with the kinectin bait D, but not
with baits A or B, supports the observation of specific kinectin-EF-1
interaction (Fig. 3D)
in vivo. Similarly, association of the endogenous kinectin with
EF-1 was observed when the HeLa cell lysate was incubated with the
immobilized GST-EF-1 proteins (Fig.
3E).
Real-time Biomolecular Interaction Analysis—To evaluate the
kinectin-EF-1 interaction quantitatively, we used a biosensor
instrument, BIAcoreTM. BIAcore analysis is based on the surface plasmon
resonance (SPR) measuring changes in refractive index upon binding of soluble
analyte to an immobilized ligand
(39). The purified recombinant
proteins used for the analysis were shown in
Fig. 4A. GST-only and
GST-bait D were separately immobilized onto the sensor chip and 1 micromolar
of either His6-EF-1 or His6-only recombinant
proteins were injected over the sensor chip surface at a constant flow rate.
The SPR signal is expressed as a sensorgram, in RU plotted as a function of
time (40).
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We found no association of both His6 and
His6-EF-1 with the immobilized control GST-only ligand
(Fig. 4B); however,
His6-EF-1 showed a gradual association (association phase)
with the immobilized GST-bait D (Fig.
4C). When His6-EF-1 was replaced with
flow buffer, dissociation of the EF-1 from GST-bait D was seen
(dissociation phase). The association rate for the binding of EF-1 was
determined to be 6.55 x 103
M–1s–1 and the
dissociation rate to be 2.23 x 104
s–1. Kinetic analysis based on the sensorgram
yielded the apparent dissociation constant of 34 nM. No significant
binding was observed for His6 to the GST-bait D.
Characterization of the EF-1 Binding Domain on
Kinectin—In order to identify the minimal sufficient domain on
kinectin that interacts with EF-1, we have constructed a series of 18
truncated fragments of kinectin by both N- and C-terminal deletions
(Fig. 5). Their interactions
with EF-1 were examined with the yeast two-hybrid analysis. Among the
18 clones, 12 were positive as indicated by the activation of both
HIS3 and lacZ reporter genes. The proteins synthesized from
clone D1, D4, D5, D6, D7, D8, D9, D11, D12, D14, D15, and D16 could interact
with the EF-1-bait, whereas D2, D3, D10, D13, D17, and D18 could
not.
All the positive clones were confirmed experimentally for their interaction
with EF-1 in the GST pull-down assay. The proteins synthesized from
clone D1, D4, D5, D6, D7, D8, D9, D11, D12 but not D14, D15, and D16 could
interact with EF-1. The minimal sufficient domain for
EF-1-binding on kinectin is the clone D12 (amino acid residues
1116–1175), which encodes a 60-amino acid fragment in the conserved
region of the kinectin sequence (Fig.
6). The binding affinity of D14, D15, and D16 to the EF-1
could be weak or transient and therefore was only detected in the yeast
two-hybrid analysis but not in the GST pull-down assay. Therefore, D12 was
used in further experiments to confirm its functional significance.
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Intracellular Localization of Kinectin and
EF-1—For kinectin to interact with EF-1
in vivo, they need to encounter each other (i.e. co-localize
in cells). We used confocal immunofluorescence to study the intracellular
localization of kinectin and EF-1 proteins. Indirect double
immunofluorescence staining was performed using monoclonal anti-kinectin
antibody (41) and
affinity-purified polyclonal anti-EF-1 antibody, on CV1 cells.
Endogenous kinectin displayed an ER-like staining pattern, which overlapped
significantly with that of the EF-1
(Fig. 7). Thus, the similar
subcellular distributions of kinectin and EF-1 in the ER-like network
suggest that the two proteins can interact with each other in vivo,
with the possible function of kinectin anchoring EF-1 to the ER.
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Kinectin Anchors EF-1 to the ER—To test the
hypothesis that kinectin might anchor EF-1 to the ER, we examined the
effect of the kinectin fragments (D and D12) overexpression on the EF-1
distribution in vivo. The kinectin baits D and D12 were cloned into a
pEGFP-C vector and introduced into CV1 cells. 48 h post-transfection, the
cells were treated with nocodazole for 2 h to perturb the microtubules and
allowed the ER network (42)
and EF-1 to retract toward the cell center.3 The EF-1
distribution in the cell was imaged by confocal immunofluorescence microscopy
with anti-EF-1 antibody. The control cells (untransfected cells or
cells transfected with pEGFP-C vector alone) exhibited the typical ER-like
staining pattern in CV1 cells (Fig.
8). 89% of the pEGFP-C vector transfected control cells and all
the untransfected control cells exhibited the ER-like EF-1 network
(Table I). However, only 26 and
30% of cells transfected with baits D and D12, respectively exhibited the
ER-like EF-1 network. It is possible that the overexpressed kinectin
fragments disrupted the ER, or EF-1 anchors to ER. Nevertheless,
kinectin seems to be important for maintaining the distinct EF-1
distribution in ER-like network manner.
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We further investigated if the overexpression of the kinectin fragments
disrupted the ER. The cells, transfected with baits D and D12, were stained
with an antibody against calnexin, which is a marker protein for ER. All the
transfected cells showed similar staining pattern as the untransfected control
cells (Fig. 8C),
indicating that the ER network remained intact when kinectin fragments were
overexpressed. In another word, the disruption of the EF-1 network is
likely due to the detachment of EF-1 from the intact ER network when
the overexpressed kinectin fragments disrupted the kinectin-EF-1
interaction. Therefore, kinectin is important for anchoring EF-1 to
ER.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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as a candidate binding partner of kinectin. The results
from subsequent GST pulldown, co-immunoprecipitation, and BIAcore real-time
binding analysis, have confirmed that EF-1 is indeed a binding partner
of kinectin. The observed interaction of kinectin bait D with EF-1 is
unlikely to be due to nonspecific interaction, commonly seen between
coiled-coil domains spanning leucine zipper motifs as the kinectin bait D does
not contain such motifs. Our circular dichroism analysis of the kinectin baits
A, B, and D indicated that all three baits exhibit the typical -helical
structures as previously predicted
(3,
4,
41), suggesting that the
differences in the ability of the kinectin baits to interact with EF-1
are unlikely due to gross misfolding of the baits, even though we cannot rule
out the possibility of minor misfolding.3
We have constructed a series of deletion mutants of kinectin to
characterize the minimal sufficient domain for the kinectin-EF-1
interaction. The EF-1-binding domain on kinectin resides near the C
terminus and is in close proximity to the kinesin-binding domain
(5). The resolution of the fine
mapping of the EF-1-binding domain on kinectin was restricted by the
qualitative nature of the yeast two-hybrid assay. Despite >90% of the D14,
D15, and D16 transfected yeast cells can activate both HIS3 and
lacZ reporter genes, a larger fragment D12 was deduced to be the
minimal binding domain. D12 is the minimal domain on kinectin that can
interact with the EF-1 protein in both yeast two-hybrid and GST
pull-down assay. The minimal EF-1-binding domain on kinectin is located
within the conserved region, between vd2 and vd3, indicating that EF-1
can interact with all kinectin isoforms. Unlike EF-1, the conventional
kinesin heavy chain can only interact with the kinectin isoforms containing
both vd3 and vd4 (5). Our
findings suggest that all kinectin isoforms are capable of interacting with
EF-1 and only a subset of isoforms, which contains vd3 and vd4, is
likely to play an additional role in organelle motility
(5). In preliminary in
vitro binding studies, we have not observed an interference of the
kinesin-kinectin interaction by the addition of excess recombinant
EF-1.3 Further studies are needed to elucidate the
relationship between the two adjacent binding domains on kinectin.
Both, transient expression of the full-length kinectin and
immunolocalization of the endogenous kinectin in cells, display an ER-like
staining pattern (3,
41). Previous studies have
also demonstrated that EF-1 clearly co-localizes with the
protein-disulfide isomerase, which is one of the most abundant ER-resident
proteins (34). We have shown
here that kinectin and EF-1 co-localize significantly in CV1 cells, and
display an ER-like staining pattern. Such a co-localization of kinectin with
EF-1 is consistent with the hypothesis that kinectin anchors
EF-1 to the ER.
The hypothesis has been confirmed in the kinectin fragment overexpression
study, where a disruption of the EF-1 network was observed. The
overexpression of the kinectin fragments containing the EF-1-binding
domain but lacking the transmembrane N terminus competed with the EF-1
binding to the native kinectin on ER. ER network remained intact in this
overexpression study suggesting that the observed disruption of the
EF-1 network was likely due to the disruption of the EF-1
anchorage to the intact ER network via the kinectin residing on ER. The
anchorage of EF-1 and EF-1 complex by kinectin on ER might be important
for coupling protein synthesis and membrane translocation machineries on
ER.
EF-1 plays an important role in the regulation of protein synthesis
(43). EF-1 is a
component of the EF-1 complex responsible for GDP/GTP
exchange on EF-1 (22,
24,
36,
44).
The only known kinectin-interacting protein in the EF-1 complexes
identified from yeast two-hybrid screening is EF-1. In addition, no
direct interaction of kinectin with EF-1, , or subunit
observed from one-to-one yeast two-hybrid analysis.3 The exact
mechanism of how kinectin may affect protein synthesis will need to be address
in the future. The current findings indicate that kinectin seems to play an
important role in supporting protein synthesis by anchoring the EF-1 complex
via EF-1.
The precise mode of the interaction of the EF-1 complex
with ER is unknown (34). Our
current findings suggest two possible models on how EF-1 complexes are
anchored to the ER membrane. The first model suggests that the anchorage of
the EF-1 complex to ER is via kinectin. In
Artemia, EF-1 was found to co-elute with the heterodimer
EF-1 during purification
(45). It has also been
reported that EF-1 contains 2 internal repeats
(VFGEXNXS) at amino acid residues 35–42 and
355–362. It is conceivable that the region of these two octapeptides of
the EF-1 could be part of binding motif for EF-1 and EF-1,
respectively, hence suggesting that EF-1 resides between the EF-1
and EF-1 in the complex
(44,
46). Since kinectin is an
integral membrane protein on ER, it can interact with EF-1 and anchor
the entire EF-1 complex onto ER. The C-terminal part of
EF-1 interacts with EF-1 to support the guanine nucleotide
exchange activity of the EF-1 in protein synthesis
(46). The restricted
localization of kinectin to ER could be related to its primary role as the
membrane anchor for the translation elongation factors. The concentration of
the translation elongation factors to ER should allow efficient protein
synthesis by preventing the intermittent diffusion of the translation
elongation factors into the cytoplasm
(47).
An alternative model suggests that kinectin anchors the EF-1 onto
specific regions of the ER membrane while the EF-1 complex
interacts with other regions of the ER membrane in kinectin-and
EF-1-independent manners. EF-1 was found to have specific
affinity for membranes and tubulin
(45,
48). EF-1 was also found
to be an actin-binding protein associating the protein synthesis apparatus
with the cytoskeleton (49).
This model can explain the presence of two different guanine-nucleotide
exchange proteins in the EF-1 complex in cells. The C termini of both
EF-1 and EF-1 contain domains that are responsible for the
guanine nucleotide-exchange activities on EF-1 but their N termini
exhibit no significant sequence homology
(25,
36). It is possible that the
EF-1 complex and the EF-1-kinectin complex serve similar
functions on different regions of the ER membrane. Even though the first model
seems better supported by our and other reported findings, further
investigations are needed to clarify the detailed mechanism of the anchorage
of EF-1 complex to ER.
In conclusion, our findings reveal a new role of kinectin in cells in
addition to its involvement in organelle motility. This report provides
evidences suggesting that kinectin acts as a membrane anchor of EF-1
onto ER.
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FOOTNOTES |
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Research scholar of the National University of Singapore.
|| To whom correspondence should be addressed: Block MD9, 03-03, 2 Medical Dr., Singapore 117597. Tel.: 65-6874-1644; Fax: 65-6778-8161; E-mail: nmiyuh{at}nus.edu.sg.
1 The abbreviations used are: ER, endoplasmic reticulum; EF-1, translation
elongation factor-1; vd, variable domain; BD, binding domain; AD, activation
domain; HIS3, histidine; GST, glutathione S-transferase;
PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; FCS,
fetal calf serum; SPR, surface plasmon resonance; RU, response unit; DIG,
digoxigenin; GFP, green fluorescent protein.
2 N. Santama, C. P. N. Er, L. L. Ong, and H. Yu, manuscript in
preparation.
3 L. L. Ong, C. P. N. Er, A. Ho, M. T. Aung, and H. Yu, unpublished
observations.
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ACKNOWLEDGMENTS |
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