From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030-3305
Received for publication, December 19, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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The Tctex1/Tctex2 family of dynein light chains
associates with the intermediate chains at the base of the soluble
dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further
address the role of these light chains in dynein activity, the
structural and biochemical properties of several members of this
polypeptide class were examined. Gel filtration chromatography and
native gel electrophoresis indicate that recombinant
Chlamydomonas flagellar Tctex1 exists as a dimer in
solution. Furthermore, yeast two-hybrid analysis suggests that this
association also occurs in vivo. In contrast, both murine
and Chlamydomonas Tctex2 are monomeric. To investigate
protein-protein interactions involving these light chains, outer arm
dynein from Chlamydomonas flagella was cross-linked using
dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is
closely related to the highly conserved LC8 protein found in many
enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.
Dyneins are molecular motors that translocate toward the minus-end
of microtubules. These enzymes provide the motive force to power
eukaryotic cilia and flagella and occur in the cytoplasm where they are
involved in a wide variety of motile activities that are of fundamental
importance to the cell. For these molecular motors to generate useful
work, both microtubule motor and cargo-binding activities must be
subject to precise temporal and spatial regulation. For flagellar
dyneins, the ultimate cargo is another axonemal microtubule, and thus
control for this process can be built directly into the flagellar- or
ciliary-specific enzyme. The situation for cytoplasmic dynein is more
complex, because there are many different cargoes that must be
transported within a single cell. Indeed, the mechanisms by which
individual cellular cargoes are attached to particular cytoplasmic
dynein motors and whether this attachment is an integral property of
specific enzyme subsets are of major importance for understanding
motor-driven transport.
In general, dyneins are constructed around ~520 kDa heavy chains that
consist of multiple AAA1
(ATPases associated with cellular
activities) domains (see Ref. 1 for recent review). AAA
proteins represent an ancient and diverse family of ATPases ranging
from bacterial metal chelatases to eukaryotic microtubule-severing
proteins. The dynein HCs exhibit both ATPase and microtubule motor
activities. These proteins comprise the globular head and
microtubule-binding stalk domains of the complex (2, 3). In addition,
the N-terminal ~160 kDa of each HC forms a stem that interacts both
with additional HCs and an intermediate chain/light chain complex to
form the basal cargo-binding domain of the enzyme (4-7). Each IC
consists of a C-terminal region comprised of multiple WD-repeats (a
motif of ~40 residues containing an invariant Trp-Asp dipeptide) and
a gene-specific N-terminal region (8-10). In flagellar dyneins, the
ICs show little homology with each other in the gene-specific regions
and likely play very different roles in the function of this enzyme.
For example, in the Chlamydomonas outer arm, IC1 (previously
termed IC78) has been shown to interact with Cytoplasmic dynein contains two light intermediate chains that are
phosphorylated in a cell cycle-dependent manner (16). One
of these polypeptides mediates the interaction of dynein with pericentrin (17). Thus, light intermediate chains also are involved in
cargo-binding activities. Two general classes of LCs (<~25 kDa) have
been identified in dynein (18). The first comprises a diverse series of
proteins that associate directly with individual HCs. These
polypeptides are thought to be involved in the control of motor
functions in response to various regulatory inputs. Thus far, they have
been identified only in flagellar dyneins (see Ref. 18 for review).
The second LC class includes three distinct protein families
(designated LC8, LC7/roadblock and Tctex1/Tctex2) that associate with the ICs at the base of the soluble dynein particle. Members of
these protein families are found in both cytoplasmic and flagellar dyneins. The LC8 protein is highly conserved from
Chlamydomonas to humans (19); it is not dynein-specific and
is found in many other multimeric enzymes such as neuronal nitric oxide
synthase (20) and myosin V (21). This protein exists as a dimer (22) and exposes two identical surfaces that appear to bind a variety of
proteins (23), including dynein ICs and specific cellular cargoes such
as the proapoptotic factor Bim (24) and Drosophila Swallow
(25). In multicellular organisms, complete loss of LC8 function results
in embryonic lethality (26). However, in Chlamydomonas, LC8-null mutants grow normally but are unable to make flagella due to
disruption of intraflagellar transport (27). Multiple LC8 variants have
been identified in a variety of organisms, including Drosophila, schistosomes (28), and mammals. In
Chlamydomonas, flagellar outer arm dynein contains both LC8
and a homologue (LC6) that share ~40% sequence identity (19).
The second LC family (LC7/roadblock) is essential for both flagellar
and cytoplasmic dynein function (29). Although the precise role played
by these proteins remains obscure, expression of at least one member of
this class is down-regulated in response to light within rat visual
cortex (30).
The Tctex1 and Tctex2 proteins were originally identified in mouse
testis as candidates for involvement in the non-Mendelian transmission
of variant forms of mouse chromosome 17 known as the t
haplotypes (31, 32). Subsequently, a Tctex2 homologue was found within
the Chlamydomonas outer dynein arm (33), and Tctex1 was
identified in both cytoplasmic dynein and flagellar inner arm I1 (34,
35). Several independent studies have implicated Tctex1 in the
attachment of specific cargoes to the dynein motor (36-39). For
example, in the vertebrate photoreceptor Tctex1 binds directly to the
C-terminal tail of rhodopsin whereas the related LC rp3 does not (36).
Disruption of this interaction due to mutations in rhodopsin leads to
retinitis pigmentosa, because rhodopsin-bearing vesicles can no longer
be transported to the base of the connecting cilium for insertion into
the membrane stacks. In humans, the Tctex1 gene maps at or close to the
retinal cone dystrophy-1 locus (40). Intriguingly, several reports
suggest that Tctex1 and its close homologue rp3 (41) are differentially expressed in fetal and adult brain (38, 41). Thus, regulation of
cellular LC content might provide one mechanism to control dynein-cargo interactions.
To further understand the role played by the Tctex1/Tctex2 family
proteins in dynein function, we have investigated the properties of
these LCs both in mammals and Chlamydomonas, because the two systems provide complementary information. In this report, we describe
the identification of additional members of the Tctex1/Tctex2 LC family
and detail an analysis of LC expression patterns in various human
tissues during development. Using specific antibodies, we demonstrate
that Tctex2, like Tctex1, is present in both flagellar and cytoplasmic
dyneins. We also show that Tctex1, but not Tctex2, is dimeric in
solution and furthermore, that members of this LC class interact
directly with LC8 family proteins in the Chlamydomonas outer arm.
Purification of Axonemes and Dynein--
Wild-type
Chlamydomonas were deflagellated using dibucaine and
isolated by standard methods (42). Membranes were removed with 1%
Nonidet P-40. Dynein was extracted from the resulting axonemes by
treatment with 0.6 M NaCl. Subsequently, the
Cytoplasmic dynein was obtained from rat brain, liver, kidney, spleen,
and testis homogenates by immunoprecipitation using monoclonal antibody
74-1, which reacts specifically with IC74 (44). These samples were
kindly provided by Drs. M. Salata and K. Pfister (University of Virginia).
Cross-linking with Dimethylpimelimidate--
Protein·protein
interactions within Chlamydomonas axonemes and purified
dynein were detected by cross-linking with dimethylpimelimidate (DMP)
(45). Samples were exchanged into 100 mM triethanolamine, pH 8.2, and treated with 0-10 mM DMP for 60 min at room
temperature. The cross-linker was dissolved in methanol and added
directly to the sample to achieve a final solvent concentration of 10% (v/v). Reactions were terminated by addition of gel sample buffer.
Preparation of Recombinant Proteins and Antibodies--
The
entire coding region for murine Tctex2 was cloned into the pMal-c2
vector, expressed as a C-terminal fusion with maltose-binding protein
(MBP) and purified by amylose affinity chromatography. The LC and
fusion partner were separated by digestion with Factor Xa. The entire
fusion protein was used as the immunogen for preparation of rabbit
polyclonal antibody R7714. The serum was blot-purified against the
isolated LC (46), and the resulting antibody preparation was used at a
dilution of 1/50 for immunoblot analysis. The Chlamydomonas LC6 fusion protein and corresponding rabbit antibody R4928 were prepared in a similar manner. Purification of rp3, Tctex1, and Chlamydomonas LC2 fusion proteins and the preparation of
antibodies R5205 and R5391 that recognize Tctex1 and LC2, respectively,
have been described previously (33, 34, 47).
Northern Analysis of Light Chain Expression--
A dot blot
arrayed with poly(A)+ RNA from 76 distinct human adult and
fetal tissues was obtained from CLONTECH
Laboratories Inc. (Palo Alto, CA). The blot was probed sequentially
with cDNAs encoding human rp3 (specific activity = 2.7 × 108 cpm.µg Native and Denaturing Polyacrylamide Gel
Electrophoresis--
Denatured proteins were separated using 8 and
12.5% acrylamide slab gels and 5-15% acrylamide gradient gels
containing SDS. Gels were stained with Coomassie Blue or were blotted
to nitrocellulose in 10 mM NaHCO3, 3 mM Na2CO3, 0.01% SDS, 20%
methanol. Blots were probed with affinity-purified antibodies, and
reactivity was assessed using a peroxidase-conjugated secondary
antibody and a chemiluminescent detection system (ECL, Amersham
Pharmacia Biotech).
Native gel electrophoresis was used to determine the solution molecular
weight of recombinant polypeptides (22, 48). Proteins were
electrophoresed in gels of different acrylamide concentration and the
negative slope of 100 (log [RF × 100]) used to determine the retardation coefficient (KR). Standard proteins used were jack bean urease (545-kDa hexamer and 272-kDa trimer), bovine serum albumin (132-kDa dimer and 66-kDa monomer), ovalbumin (45 kDa), bovine carbonic anhydrase (29 kDa), and
Gel Filtration Chromatography--
The native molecular weight
of recombinant Tctex1 also was assessed by gel filtration
chromatography in 20 mM Tris.Cl, pH 8.0, 1 mM
dithiothreitol, 0.5 mM EDTA, 150 mM KCl on a
Superose 6 column (Amersham Pharmacia Biotech) using a Bio-Rad
Biologics chromatography workstation. Molecular mass standards
used were: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa),
chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa).
Circular Dichroism Spectroscopy--
Recombinant
Chlamydomonas Tctex1 was prepared at 47.8 µM.
The circular dichroism spectrum was measured in the far UV range between 190 and 280 nm using a Jasco J-715 spectropolarimeter. The
signal at 222 nm was converted to mean residue ellipticity [ Yeast Two-hybrid Screen--
The full-length murine Tctex1
cDNA was cloned into the pAS2-1 vector resulting in the in-frame
fusion of Tctex1 with the DNA-binding domain of the yeast GAL4 protein.
A mouse brain cDNA library (9- to 12-week male BALB/c)
containing 3.5 × 106 independent clones
(CLONTECH Laboratories Inc.) was cloned into pACT2
to produce fusions between the encoded proteins and the DNA activation
domain of GAL4. The yeast strain (Y190) used for the screening assay
contained both HIS3 and lacZ reporter genes under
control of a GAL4-responsive upstream activation site. Lack of
autonomous activation by the Tctex1/DNA-binding domain fusion was
demonstrated by plating cells transformed with the bait plasmid alone
on media lacking histidine. For the assay, bait and library plasmids
were transformed simultaneously into yeast and positive interactions
initially identified by growth of His+ cells on media
lacking histidine. Putative positives were then further tested by
assaying colonies for Computational Methods--
Members of the Tctex1/Tctex2 protein
family were identified in the nonredundant and EST data bases at NCBI
using BLAST. Multiple sequence alignment was prepared using CLUSTALW,
and the output was processed using BOXSHADE. The phylogenetic analysis
was performed using the Phylip suite of programs. Specifically,
distances were calculated with PROTDIST and FITCH, and the unrooted
tree was drawn using DRAWTREE. Secondary structure was predicted using PHD.
Phylogeny of the Tctex1/Tctex2 LC Family--
Previously, we
identified several dynein components belonging to the Tctex1/Tctex2
light chain family. These included Tctex1 (in cytoplasmic dynein and
flagellar inner arm I1 (35)), its close homologue rp3 (in cytoplasmic
dynein (47)), and the more distantly related Tctex2 (in flagellar outer
arm dynein (33)). Recent analysis of the nonredundant and EST data
bases at NCBI using BLAST has allowed us to identify several additional
members of this class of dynein component of both mammalian and
nematode origin. A ClustalW sequence comparison of the currently
identified members of this family is shown in Fig.
1a. These proteins share most
identity in the C-terminal regions and have gene-specific N-terminal
sections that show varying degrees of similarity with each other. The
phylogenetic relationships within this class of polypeptide were
calculated using the Phylip suite of programs and are shown in Fig.
1b. This analysis demonstrates that the Tctex1 and Tctex2
light chains define two major subgroups of this dynein LC class and
reveals that Tctex2-related proteins derive from many sources other
than testis, suggesting that they may not be flagella-specific.
Structural and Solution Properties of Tctex1 and Tctex2--
To
further address the role played by these proteins in both flagellar and
cytoplasmic dynein function, the structural and solution properties of
the Tctex1/Tctex2 LC class were examined. A secondary structure
analysis of Chlamydomonas Tctex1 using PHD (50) indicates
that the N-terminal half of the protein is likely formed from two Tctex1 Is Dimeric in Solution--
Following denaturing
electrophoresis, most recombinant Tctex1 migrates with
Mr = 13,000, which is completely consistent with the calculated molecular mass of 12,805 Da (includes a single additional N-terminal His residue) (Fig.
2, right inset). However, an
extra band at Mr ~ 27,000 also is evident.
This band is recognized by antibody R5205 and therefore likely
represents a Tctex1 dimer. To test whether native Tctex1 is indeed
dimeric, the solution molecular weight of the recombinant protein was
determined by gel filtration chromatography (Fig. 2). The major Tctex1
peak has a mass of ~31.5 kDa, strongly suggesting that this protein is dimeric in solution. No peak corresponding to the Tctex1 monomer was
observed under native conditions even at high dilution, suggesting a
very high affinity between monomers.
To confirm that Tctex1 is dimeric, the molecular mass was also
determined by native gel electrophoresis using the method of Hedrick
and Smith (22, 48). This analysis yielded an estimate of 22 kDa,
supporting the dimeric nature of this protein (Table I). To assess whether other members of
this protein family exist as oligomers, the solution molecular weight
of MBP/Tctex2 and MBP/LC2 fusion proteins also were determined (Table
I). The native molecular weight of the control protein MBP/lacZ was
very close to the calculated value, indicating that MBP itself does not
dimerize (see also Ref. 22). Both MBP/Tctex2 and MBP/LC2 yielded
molecular weight estimates that strongly suggest these proteins are
monomeric.
Further evidence for the dimerization of Tctex1 was obtained from a
yeast two-hybrid screen of a mouse brain library using murine Tctex1 as
the bait. The overall goal of this screen was to identify potential
dynein-cargo interactions mediated by Tctex1. A number of novel
interactions between Tctex1 and other proteins were identified.
Intriguingly, ~10% of the confirmed clones recovered from the screen
were found to encode Tctex1 itself, suggesting that the Tctex1-Tctex1
interaction can occur in vivo. Clones encoding other members
of this dynein LC family were not obtained from this screen even though
at least one, rp3, is expressed at much higher levels in brain than
Tctex1 (see below).
Interaction of Tctex2 with an LC8-related Protein--
The
Tctex1 and Tctex2 LCs associate with the dynein ICs and several
additional LCs at the base of the soluble dynein particle (47). In
Chlamydomonas flagellar outer arm dynein, LC2 (Tctex2) is
essential for the assembly of the dynein particle into the axoneme
(51). To further investigate interactions involving this LC class,
Chlamydomonas axonemes were treated for 60 min with 0-10
mM DMP (Fig. 3a),
which cross-links primary amines with a final linker length of 9.2 Å.
Reactions were terminated by addition of gel sample buffer, and the
samples were electrophoresed. Following Coomassie Blue staining,
cross-linking was evident as a smearing of individual bands and an
increase in the background staining in samples treated with the highest
concentrations of DMP (Fig. 3b). To identify interactions
involving LC2, both purified dynein and isolated axoneme samples were
treated with DMP, blotted to nitrocellulose, and probed with antibodies
that react with individual dynein components. In both dynein and
axoneme samples, a single major cross-linked product of
Mr 32,000 was observed, suggesting that LC2
(Mr = 20,000 (52); actual mass = 15,883 Da)
had become cross-linked to a protein of ~14 kDa (Fig.
4, a and b).
Examination of identical samples with antibody R4928 revealed that this
band also contained LC6 (mass = 13,857 Da), an LC that is closely
related to the highly conserved LC8 protein found in many different
enzyme systems (19). Antibodies against other dynein LCs did not detect this band. These data suggest that LC2 and LC6 interact directly in situ. The minor band migrating slightly more slowly than
the LC2-LC6 complex recognized by antibodies R4928 and R5391 likely represents a variant of reduced charge generated by modification of
additional Lys residues. Similar charge modification products were
observed previously following treatment of LC8 with amine-selective cross-linking reagents (22).
In the purified dynein sample, an additional minor DMP product of
Mr ~ 100,000 containing both LC2 and LC6 was
observed. This band is of the appropriate size to represent attachment
of the LC2-LC6 complex to a dynein IC. The R4928 antibody against LC6 also detected one DMP-generated band of Mr
29,000 that did not contain either LC2 or any other dynein LC. Given
that there are two copies of LC6 per dynein particle (52)
and that the related LC8 protein is dimeric (22), this band most
probably derives from direct LC6-LC6 cross-linking. The calculated mass
of the LC6 dimer (27,714 Da) is consistent with this interpretation.
Inner arm I1 contains both Tctex1 and LC8 (35). However, in contrast to
LC2 (Tctex2) and LC6 in the outer arm, no products containing Tctex1
and LC8 were observed in DMP-cross-linked axoneme samples (data not shown).
Tctex2 Is a Component of Cytoplasmic Dynein--
Tctex2, unlike
Tctex1, has been described previously only in flagellar outer arm
dynein (33). To further examine whether Tctex2 is indeed
flagellar-specific, murine Tctex2 was expressed as a C-terminal fusion
with maltose-binding protein (Fig.
5a) and used as the immunogen
for antiserum production in rabbit R7714. Tctex2 was separated from the
fusion partner by digestion with Factor Xa (Fig. 5a), and
the purified light chain used to obtain a Tctex2-specific antibody
fraction from R7714 serum by blot affinity. The specificity of antibody
R7714 was assessed by immunoblot analysis against recombinant members
of the Tctex1/Tctex2 family, including the Chlamydomonas
Tctex2 homologue (LC2) and mammalian Tctex1, Tctex2, and rp3 (Fig.
5b). Immunoreactivity was observed only against mammalian
Tctex2, indicating that the antibody is indeed highly specific and can
readily distinguish between various members of this LC class. Tctex2 is
expressed at high levels in mammalian testis (32) (and see below).
Therefore, the specificity of the R7714 antibody was further assessed
by immunoblot analysis against a whole testis homogenate (Fig.
5c). Both R7714 and R5205 (versus Tctex1)
antibodies revealed single immunoreactive bands in testis; with R5205 a
very minor band at Mr = 61,000 also was
detected following very prolonged exposure.
To determine whether Tctex2 occurs in cytoplasmic as well as flagellar
dynein, the cytoplasmic isozyme was purified by immunoprecipitation from rat brain, kidney, liver, spleen, and testis extracts using monoclonal antibody 74-1 (44). Purified dynein from all these tissues
has previously been shown to contain both the Tctex1 and rp3 LCs (47).
In contrast, immunoblot analysis using the R7714 antibody against
Tctex2 detected immunoreactive bands only in cytoplasmic dynein samples
derived from kidney and spleen but not in those from brain, liver, and
testis (Fig. 5d). The lack of signal in the testis
cytoplasmic dynein sample suggests that the immunoprecipitates obtained
using the 74-1 monoclonal antibody contain only cytoplasmic dynein and
are not contaminated with detectable amounts of sperm flagellar
dyneins. These data suggest that Tctex2, like Tctex1, is present in
subsets of both flagellar and cytoplasmic dyneins. Although the R7714
antibody did not react with other members of this protein family on
immunoblots, it does remain possible that the protein detected in
cytoplasmic dynein samples by R7714 is closely related, but not
identical, to Tctex2.
Tissue-specific Differential Expression--
In addition to the
high level expression of Tctex2 in testis (32), previous studies have
suggested that Tctex1 and rp3 are differentially expressed during brain
development (38, 41). Because these LCs have different binding
affinities for at least one cytoplasmic dynein cargo (36), differential
LC expression may act as a mechanism to regulate the interaction of
specific cellular cargoes with the cytoplasmic dynein motor. Therefore, to further investigate the tissue-specific distribution of the Tctex1/Tctex2 family, poly(A)+ RNA from a wide variety of
adult and fetal human tissues was examined by Northern dot blot
analysis. The multiple tissue mRNA array was probed sequentially
with human clones encoding rp3, Tctex1, Tctex2, and the AI421187 (from
glioblastoma) and AI492091 (from B-lymphocyte, essentially identical to
the kidney-derived AW612564) ESTs (Fig.
6). All five probes revealed
tissue-specific alterations in expression level. For example, rp3 was
heavily expressed in adult brain, kidney, and liver but not in bone
marrow or ovary. In contrast, the close homologue Tctex1 was readily detected in many tissues, including bone marrow but was less evident in
adult brain. Tctex2 and related ESTs exhibited a more restricted distribution. Minor amounts of Tctex2 were detected in most samples with enhanced levels found only in testis, adult liver, and fetal thymus. All LCs showed differential expression between certain adult
and fetal tissues, suggesting that the levels of these proteins are
controlled in a developmentally regulated manner. Interestingly, not
all LCs were regulated in the same fashion. For instance in liver,
Tctex2, rp3, and AI421187 were significantly up-regulated in the adult
tissue whereas both Tctex1 and AI492091 showed little alteration. In
contrast, both Tctex2 and AI492091 were up-regulated in fetal thymus
whereas the other proteins were not.
Members of the Tctex1/Tctex2 class of dynein LCs associate with
the ICs and several additional LCs at the base of the soluble motor
particle. Genetic studies in Chlamydomonas have revealed that strains lacking the Tctex2 homologue LC2 fail to assemble outer
dynein arms and thus have compromised motility (53). Furthermore, there
is now considerable support for the hypothesis that these proteins
mediate specific cytoplasmic dynein-cargo interactions, including those
with rhodopsin (36), Fyn kinase (37, 38), and the vesicle-associated
protein Doc-2 (39). To gain further insight into the role of this LC
class in dynein function we report here on the structure and expression
of these polypeptides and on the protein-protein interactions in which
they are involved.
Models for protein-protein associations within the IC-LC complex of
both cytoplasmic dynein and the flagellar outer arm are shown in Fig.
7. Both sets of ICs interact with HCs,
the LC8 dimer, a member of the LC7/roadblock family and cargo
(
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-tubulin in
situ (11). This raised the possibility that ICs are responsible
for direct cargo binding, although it is now clear that, at least in
the flagellum, additional components are required to specify the
precise attachment site (12). In contrast, Chlamydomonas IC2
(previously IC69 or IC70) apparently mediates regulatory processes that
impinge on motor function (13). Cytoplasmic dynein has a single class of IC (IC74) that interacts directly with the dynactin activator of
dynein-based vesicular transport (14). In mammals there are several
IC74 genes, and evidence for both alternative splicing of IC74
transcripts and differential phosphorylation of the resulting proteins
has been obtained (10, 15). The clear similarities between flagellar
and cytoplasmic dyneins raised the possibility that the various IC74
isoforms bind specific intracellular cargoes. However, considering the
large number of different proteins, organelles, and other complexes
that must be transported at various times during the cell cycle, it
does not seem reasonable that IC74 isoforms alone can provide
sufficient specificity for cargo attachment.
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DISCUSSION
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subparticle of the outer arm was purified by centrifugation through a
5-20% sucrose density gradient (43).
1), Tctex1 (1.0 × 108 cpm.µg
1), and Tctex2 (7.7 × 108 cpm.µg
1), and then with ESTs encoding
two Tctex2 homologues derived from human glioblastoma (AI421187;
8.8 × 108 cpm.µg
1) and B-lymphocyte
(AI492091; 4.2 × 107 cpm.µg
1) libraries.
-lactalbumin (14.2 kDa). A plot of log KR
versus log Mr for these proteins
yielded a standard curve from which the native mass of the test samples
was determined.
]222 in mdeg.cm2.dmol
1
using [
]222 = 100 × CD signal/n
(number of residues) × l (path length = 0.1 cm) × C ([protein] in mM). The
approximate helical content of Tctex1 was determined using a value of
[
]222 =
32,600 mdeg.cm2.dmol
1 for a completely helical
protein (49).
-galactosidase activity. Following
confirmation of the specificity of the interaction, the Tctex1-binding
partners were identified by sequence analysis.
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Fig. 1.
Sequence analysis and phylogeny of the
Tctex1/Tctex2 family. a, alignment of the Tctex1/Tctex2 LC
family generated using CLUSTALW and shaded with BOXSHADE.
The aligned sequences are: human glioblastoma EST
(ai421187), Anthocidaris crassispina LC1
(suTctex2; BAA24185), murine Tctex2 (tctex2;
U21673), human testis Tctex2 EST (humtctex2; AA781436),
human kidney EST (aw612564), murine embryo EST
(w64276), murine Tctex1 (tctex1; A32995), human
Tctex1 (humtctex1; U56255), Chlamydomonas Tctex1
(chltctex1; AF039437), human rp3 (humanrp3;
U02556), Chlamydomonas LC2 (chlLC2; U89649),
Caenorhabditis elegans ORF (d1009-5), C. elegans EST (C48724), C. elegans ORF
(t05c12-5). b, following alignment using
ClustalW, distances were calculated with PROTDIST and FITCH from the
Phylip suite of programs, and the tree was constructed using DRAWTREE.
The unrooted tree reveals two major subclasses of this LC family
centered on the Tctex1 and Tctex2 proteins.
helices both of which are predicted with high reliability values. The
C-terminal section of Tctex1 appears to consist of several
strands,
although the prediction of the precise boundaries of these elements is
significantly less reliable.2
To assess the general validity of this structure prediction, the
circular dichroism spectrum of Tctex1 in the far UV was measured. The
spectrum shows clear evidence for a mixture of helix and sheet structures as predicted by the program PHD. From the signal at 222 nm, the mean residue ellipticity [
]222 was
calculated. Assuming [
]222 =
32,600
mdeg.cm2.dmol
1 for a completely helical
protein (49), 25.6% of residues within Tctex1 are in a helical
conformation. This value agrees very well with the secondary structure
prediction of 29.8%.
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Fig. 2.
Tctex1 is a dimer in solution. To
determine the native molecular weight and oligomerization status of
this class of LC, recombinant Chlamydomonas Tctex1 was
subjected to gel filtration chromatography on a Superose 6 column. A
single major peak with a molecular mass of 31.5 kDa was observed. Some
aggregated protein of very high molecular weight also is evident in the
chromatogram. The left inset shows the column calibration
using serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa) as standards. Right
inset, electrophoretic analysis on a 12.5% acrylamide gel of the
Tctex1 preparation prior to (left lane) and following
(right lane) concentration in a Centricon-10 unit. Equal
volumes of the two preparations were loaded. Some Tctex1 protein
migrates as a dimer even in the presence of SDS. These data suggest
that Tctex1 (monomer molecular mass = 12.8 kDa) exists as a dimer
in solution.
Oligomeric status of Tetex1 and Tetex2 proteins in solution
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Fig. 3.
Cross-linking of Chlamydomonas
axonemes with DMP. a, the structure of
dimethylpimelimidate (DMP) is shown. Under basic conditions, both
imidoester groups react with primary amines to yield a covalent linkage
with a length of 9.2 Å. b, Chlamydomonas
flagellar axonemes (~150 µg/lane) in 100 mM
triethanolamine, pH 8.2, were treated with 0-10 mM DMP for
60 min. The reactions were terminated by addition of gel sample buffer,
electrophoresed in a 5-15% acrylamide gradient gel, and stained with
Coomassie Blue.
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Fig. 4.
DMP cross-linking reveals the interaction of
the Tctex1/Tctex2 and LC8 protein families in outer arm
dynein. a, purified Chlamydomonas outer arm
dynein was treated with the indicated concentrations of DMP. Following
electrophoresis and blotting to nitrocellulose, the samples were probed
with antibodies R5391 and R4928 that are specific for LC2 (Tctex2) and
LC6 (a homologue of LC8), respectively. After treatment with DMP, both
antibodies detect the same major product of Mr
32,000, indicating that LC2 and LC6 have been cross-linked to each
other. The minor product at Mr ~ 100,000 likely derives from cross-linking of the LC2·LC6 complex to one
dynein IC. b, isolated Chlamydomonas flagellar
axonemes were treated with 10 mM DMP and probed with the
R5391 and R4928 antibodies. Both antibodies detect the
Mr 32,000 band observed in a,
indicating that the interaction between LC2 and LC6 also occurs
in situ.
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Fig. 5.
Tctex2 is present in mammalian cytoplasmic
dynein. a, murine Tctex2 was expressed as a C-terminal
fusion with maltose-binding protein and purified by amylose affinity
chromatography. The resulting fusion protein was used as the immunogen
for preparation of antiserum R7714. Following digestion with Factor Xa,
the Tctex2 light chain was separated from the fusion partner. In the
uncut sample, a band of Mr ~ 130,000 corresponding to MBP/Tctex2 dimer is evident. The intensity of this
band is diminished following Factor Xa digestion. b,
polyclonal rabbit antiserum R7714 was raised against murine Tctex2
expressed as a C-terminal fusion with maltose-binding protein.
Following blot purification versus recombinant Tctex2, the
specificity of the antibody was assessed by immunoblot analysis of
several recombinant dynein LCs, including human rp3,
Chlamydomonas LC2 (a Tctex2 homologue),
Chlamydomonas Tctex1, and murine Tctex2. c,
approximately 150 µg/lane testis homogenate were electrophoresed in a
5-15% acrylamide gradient gel. Total protein is shown in the
left lane (Coomassie Blue stain). Identical samples were
blotted to nitrocellulose and probed with blot-purified antibodies
R5205 and R7714 versus Tctex1 and Tctex2. Both antibodies
are highly specific and recognize single bands of the appropriate
Mr (14,000 and 22,000, respectively).
d, cytoplasmic dynein was immunoprecipitated from rat brain,
kidney, liver, spleen, and testis using monoclonal antibody 74-1 (44).
Following electrophoresis and immunoblotting using blot-purified R7714,
bands of Mr ~ 20,000 were detected in
cytoplasmic dynein samples from kidney and spleen but not in those from
brain, liver, and testis.
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Fig. 6.
Tissue-specific expression of Tctex1/Tctex2
family proteins in humans. Northern dot blot analysis of
poly(A)+ RNA from various human tissues. The array was
obtained from CLONTECH and sequentially probed with
the following cDNAs: rp3, Tctex1, Tctex2, AI421187, and AI492091.
The latter EST derived from B-lymphocytes and is essentially identical
to the kidney-derived EST AW612564 used to construct the phylogenetic
tree shown in Fig. 1b.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin or dynactin). The major difference is that the outer
arm ICs associate with monomeric Tctex2 and the LC6 dimer, whereas most
cytoplasmic dynein ICs bind the Tctex1 dimer as apparently is also the
case for inner arm I1. It will be of interest to determine whether a
single cytoplasmic dynein particle can directly bind both the Tctex1
dimer and a Tctex2 monomer, whether their association is mutually
exclusive or if interaction of Tctex2 requires accessory proteins such
as LC6.
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Fig. 7.
Model for protein-protein interactions within
dynein IC·LC complexes. These models depict known
protein-protein interactions within the IC·LC complexes from
Chlamydomonas outer arm dynein and mammalian cytoplasmic
dynein. Although certain similarities are evident, the models also
suggest that a major difference is in the binding of either a Tctex1
dimer or monomeric LC2 (Tctex2) combined with the presence of, and
interaction with, the LC8 homologue LC6. Although flagellar dyneins
contain either Tctex1 or Tctex2, it is unclear whether binding of
Tctex1 and Tctex2 to cytoplasmic dynein is mutually exclusive. The LC8
dimer structure derives from Protein Data Bank accession number 1B1W
(23).
Identification of additional mammalian Tctex2-related proteins is of considerable interest, because this LC subclass had previously been reported only in the flagellar outer arm (33). Although there is significant similarity between these proteins, Tctex2 and its close relatives include N-terminal extensions of varying lengths that may mediate gene-specific functions. Several of these novel homologues are derived from cells/tissues that do not contain cilia/flagella raising the possibility that members of this LC group also may occur in the cytoplasm. Examination of cytoplasmic dynein samples revealed the presence of Tctex2 in kidney and spleen but not in brain, liver, or testis. Because Tctex2 is heavily expressed in testis where it is a component of flagellar dynein, this suggests that the cytoplasmic dynein immunoprecipitates were not contaminated with detectable amounts of the flagellar enzyme. These data support the hypothesis that Tctex2 proteins, like Tctex1, are present in both flagellar and cytoplasmic dyneins, although their tissue distribution is considerably more restricted.
Both Tctex1 and Tctex2 were originally described as testis-specific proteins, although that interpretation has now been revised. Previous studies indicated that Tctex1 and its close homologue rp3 are differentially expressed during brain development (38, 41) with Tctex1 mRNA being more prominent in fetal than adult brain whereas rp3 followed the opposite pattern. The Northern analysis presented here confirms that rp3 is greatly up-regulated in the adult tissue whereas Tctex1 is present in much lower amounts. Although, intriguingly, both proteins may be readily detected in cytoplasmic dynein samples derived from adult rat brain (47). Tctex1 especially was present in high levels in many tissues, whereas rp3 expression was considerably more restricted. Tctex2 and its related homologues also were found in lesser amounts, and indeed Tctex2 and the glioblastoma-derived EST were present at high levels in only a few tissues, with clear differences between certain fetal and adult samples. These expression studies suggest that cytoplasmic dynein derived from various tissues differs in terms of LC content, supporting the notion that the resulting dynein subtypes may exhibit distinct cargo-binding activities.
Murine Tctex1 and Tctex2 are encoded within a region of chromosome 17 known as the t complex (31, 32). Variant forms of this chromosomal region (the t haplotypes) are transmitted in a non-Mendelian fashion by heterozygous males due to expression of mutant responder and distorter proteins that lead to defects in sperm bearing the wild-type version of the chromosome (reviewed in Ref. 54). Homozygosity for t haplotypes results in either embryonic lethality due to recessive lethal factors (for identical t haplotypes; tx/tx) or male sterility (with complementing t haplotypes; tx/ty) caused by a series of sterility factors. Tctex1 and Tctex2 are candidates for the proximal and central distorter/sterility factors based on gene location, testis-enriched expression and the presence of t haplotype-encoded mutations that might affect protein function (31, 32). The distorters and sterility factors were previously thought to be identical, because homozygosity for distorters results in infertility. However, recent high resolution mapping of the t complex region indicates that at least the proximal distorter and sterility factors are distinct genetic entities (55).
Tctex1 was initially found in cytoplasmic dynein (34) and subsequently in flagellar inner arm I1 (35). Analysis of Chlamydomonas flagella also revealed the presence of Tctex2 as an LC within the outer arm (33). This protein is encoded at the locus ODA12, and null mutants are unable to assemble outer arms and consequently swim slowly (53). These observations gave rise to the hypothesis that defects in flagellar dyneins provide the underlying biological basis for the transmission ratio distortion phenomenon (33, 35). This hypothesis has recently received considerable support from the observation that the strongest distorter maps at the same location as an axonemal dynein HC encoded at the Hybrid Sterility-6 locus (56). Furthermore, the responder has now been identified as a mutant form of a protein kinase required for sperm motility (57), suggesting that differential phosphorylation of dynein proteins may be involved in production of sperm with defective motility. Intriguingly, a Tctex2 homologue in both sea urchin and chum salmon sperm is phosphorylated during the activation of flagellar motility (58), although it is not yet clear whether this phosphorylation event is required for sperm activation or is a consequence of it.
In this report, we have identified additional members of the
Tctex1/Tctex2 family of dynein LCs and found that they are
differentially expressed in both a tissue-specific and developmentally
regulated manner. These observations lend further support to the
hypothesis that LC expression patterns might affect the cargo-binding
capabilities of cytoplasmic dynein.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Zheng-yu Peng (University of Connecticut Health Center) for his assistance with circular dichroism spectroscopy and Drs. Kevin Pfister and Mark Salata (University of Virginia) for the cytoplasmic dynein samples.
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FOOTNOTES |
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* This study was supported in part by Grant GM51293 from the National Institutes of Health and by the Heritage Affiliate of the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular Genetics, The Ohio State
University, Columbus, OH 43210.
§ An investigator of the Patrick and Catherine Weldon Donaghue Medical Research Foundation. To whom correspondence should be addressed: Dept. of Biochemistry, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3305. Tel: 860-679-3347; Fax: 860-679-3408; E-mail: steve@king2.uchc.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011456200
2 The proposed arrangement of secondary structural elements within Tctex1 is also supported by NMR spectroscopic studies (H. Wu, M. W. Maciejewski, S. E. Benashski, G. P. Mullen, and S. M. King, (2001) J. Biomol. NMR, in press.
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ABBREVIATIONS |
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The abbreviations used are: AAA, ATPases associated with cellular activities; DMP, dimethylpimelimidate; EST, expressed sequence tag; HC, heavy chain; IC, intermediate chain; LC, light chain; MBP, maltose-binding protein.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
King, S. M.
(2000)
J. Cell Sci.
113,
2521-2526 |
2. | Gee, M. A., Heuser, J. E., and Vallee, R. B. (1997) Nature 390, 636-639[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Koonce, M. P.
(1997)
J. Biol. Chem.
272,
19714-19718 |
4. | Sakakibara, H., Takada, S., King, S. M., Witman, G. B., and Kamiya, R. (1993) J. Cell Biol. 122, 653-661[Abstract] |
5. | Witman, G. B., King, S. M., Moss, A. G., and Wilkerson, C. G. (1991) in Comparative Spermatology 20 Years After (Baccetti, B., ed) , pp. 439-443, Raven Press, New York |
6. | Sale, W. S., Goodenough, U. W., and Heuser, J. E. (1985) J. Cell Biol. 101, 1400-1412[Abstract] |
7. |
King, S. M.,
and Witman, G. B.
(1990)
J. Biol. Chem.
265,
19807-19811 |
8. | Mitchell, D. R., and Kang, Y. (1991) J. Cell Biol. 113, 835-842[Abstract] |
9. | Wilkerson, C. G., King, S. M., Koutoulis, A., Pazour, G. J., and Witman, G. B. (1995) J. Cell Biol. 129, 169-178[Abstract] |
10. | Paschal, B. M., Mikami, A., Pfister, K. K., and Vallee, R. B. (1992) J. Cell Biol. 118, 1133-1143[Abstract] |
11. |
King, S. M.,
Wilkerson, C. G.,
and Witman, G. B.
(1991)
J. Biol. Chem.
266,
8401-8407 |
12. | Takada, S., and Kamiya, R. (1994) J. Cell Biol. 126, 737-745[Abstract] |
13. |
Mitchell, D. R.,
and Kang, Y.
(1993)
J. Cell Sci.
105,
1069-1078 |
14. |
Karki, S.,
and Holzbaur, E. L.
(1995)
J. Biol. Chem.
270,
28806-28811 |
15. |
Pfister, K. K.,
Salata, M. W.,
Dillman, J. F., 3rd,
Vaughan, K. T.,
Vallee, R. B.,
Torre, E.,
and Lye, R. J.
(1996)
J. Biol. Chem.
271,
1687-1694 |
16. | Niclas, J., Allan, V. J., and Vale, R. D. (1996) J. Cell Biol. 133, 585-593[Abstract] |
17. |
Tynan, S. H.,
Purohit, A.,
Doxsey, S. J.,
and Vallee, R. B.
(2000)
J. Biol. Chem.
275,
32763-32768 |
18. | King, S. M. (2000) Biochim. Biophys. Acta 1496, 60-75[Medline] [Order article via Infotrieve] |
19. |
King, S. M.,
and Patel-King, R. S.
(1995)
J. Biol. Chem.
270,
11445-11452 |
20. |
Jaffrey, S. R.,
and Snyder, S. H.
(1996)
Science
274,
774-777 |
21. | Espindola, F. S., Suter, D. M., Partata, L. B. E., Cao, T., Wolenski, J. S., Cheney, R. E., King, S. M., and Mooseker, M. S. (2000) Cell Motil. Cytoskelet. 47, 269-281[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Benashski, S. E.,
Harrison, A.,
Patel-King, R. S.,
and King, S. M.
(1997)
J. Biol. Chem.
272,
20929-20935 |
23. | Liang, J., Jaffrey, S. R., Guo, W., Snyder, S. H., and Clardy, J. (1999) Nat. Struct. Biol. 6, 735-740[CrossRef][Medline] [Order article via Infotrieve] |
24. | Puthalakath, H., Huang, D. C., O'Reilly, L. A., King, S. M., and Strasser, A. (1999) Mol. Cell 3, 287-296[Medline] [Order article via Infotrieve] |
25. | Schnorrer, F., Bohmann, K., and Nusslein-Volhard, C. (2000) Nat. Cell Biol. 2, 185-190[CrossRef][Medline] [Order article via Infotrieve] |
26. | Dick, T., Ray, K., Salz, H. K., and Chia, W. (1996) Mol. Cell. Biol. 16, 1966-1977[Abstract] |
27. |
Pazour, G. J.,
Wilkerson, C. G.,
and Witman, G. B.
(1998)
J. Cell Biol.
141,
979-992 |
28. | Yang, W., Jones, M. K., Fan, J., Hughes-Stamm, S. R., and McManus, D. P. (1999) Biochim. Biophys. Acta 1432, 13-26[Medline] [Order article via Infotrieve] |
29. |
Bowman, A. B.,
Patel-King, R. S.,
Benashski, S. E.,
McCaffery, J. M.,
Goldstein, L. S.,
and King, S. M.
(1999)
J. Cell Biol.
146,
165-180 |
30. |
Ye, F.,
Zangenehpour, S.,
and Chaudhuri, A.
(2000)
J. Biol. Chem.
275,
27172-27176 |
31. | Lader, E., Ha, H. S., O'Neill, M., Artzt, K., and Bennett, D. (1989) Cell 58, 969-979[CrossRef][Medline] [Order article via Infotrieve] |
32. | Huw, L. Y., Goldsborough, A. S., Willison, K., and Artzt, K. (1995) Dev. Biol. 170, 183-194[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Patel-King, R. S.,
Benashski, S. E.,
Harrison, A.,
and King, S. M.
(1997)
J. Cell Biol.
137,
1081-1090 |
34. |
King, S. M.,
Dillman, J. F., 3rd,
Benashski, S. E.,
Lye, R. J.,
Patel-King, R. S.,
and Pfister, K. K.
(1996)
J. Biol. Chem.
271,
32281-32287 |
35. |
Harrison, A.,
Olds-Clarke, P.,
and King, S. M.
(1998)
J. Cell Biol.
140,
1137-1147 |
36. | Tai, A. W., Chuang, J. Z., Bode, C., Wolfrum, U., and Sung, C. H. (1999) Cell 97, 877-887[Medline] [Order article via Infotrieve] |
37. | Mou, T., Kraas, J. R., Fung, E. T., and Swope, S. L. (1998) FEBS Lett. 435, 275-281[CrossRef][Medline] [Order article via Infotrieve] |
38. | Kai, N., Mishina, M., and Yagi, T. (1997) J. Neurosci. Res. 48, 407-424[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Nagano, F.,
Orita, S.,
Sasaki, T.,
Naito, A.,
Sakaguchi, G.,
Maeda, M.,
Watanabe, T.,
Kominami, E.,
Uchiyama, Y.,
and Takai, Y.
(1998)
J. Biol. Chem.
273,
30065-30068 |
40. | Watanabe, T. K., Fujiwara, T., Shimizu, F., Okuno, S., Suzuki, M., Takahashi, E., Nakamura, Y., and Hirai, Y. (1996) Cytogenet. Cell Genet. 73, 153-156[Medline] [Order article via Infotrieve] |
41. | Roux, A.-F., Rommens, J., McDowell, C., Anson-Cartwright, L., Bell, S., Schappert, K., Fishman, G. A., and Musarella, M. (1994) Hum. Mol. Genet. 3, 257-263[Abstract] |
42. | Witman, G. B. (1986) Methods Enzymol. 134, 280-290[Medline] [Order article via Infotrieve] |
43. | King, S. M., Otter, T., and Witman, G. B. (1986) Methods Enzymol. 134, 291-306[Medline] [Order article via Infotrieve] |
44. | Dillman, J. F., 3rd, and Pfister, K. K. (1994) J. Cell Biol. 127, 1671-1681[Abstract] |
45. | Benashski, S. E., and King, S. M. (2000) Methods 22, 365-371[CrossRef][Medline] [Order article via Infotrieve] |
46. | Olmsted, J. B. (1986) Methods Enzymol. 134, 467-472[Medline] [Order article via Infotrieve] |
47. | King, S. M., Barbarese, E., Dillman, J. F., 3rd, Benashski, S. E., Do, K. T., Patel-King, R. S., and Pfister, K. K. (1998) Biochemistry 37, 15033-15041[CrossRef][Medline] [Order article via Infotrieve] |
48. | Hedrick, J. L., and Smith, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164[Medline] [Order article via Infotrieve] |
49. | Chen, Y.-H., and Yang, J. T. (1971) Biochem. Biophys. Res. Commun. 44, 1285-1291[Medline] [Order article via Infotrieve] |
50. | Rost, B., and Sander, C. (1993) J. Mol. Biol. 232, 584-599[CrossRef][Medline] [Order article via Infotrieve] |
51. |
Pazour, G. J.,
Koutoulis, A.,
Benashski, S. E.,
Dickert, B. L.,
Sheng, H.,
Patel-King, R. S.,
King, S. M.,
and Witman, G. B.
(1999)
Mol. Biol. Cell
10,
3507-3520 |
52. | Pfister, K. K., Fay, R. B., and Witman, G. B. (1982) Cell Motil. 2, 525-547[Medline] [Order article via Infotrieve] |
53. |
Pazour, G. J.,
Dickert, B. L.,
and Witman, G. B.
(1999)
J. Cell Biol.
144,
473-481 |
54. |
Olds-Clarke, P.
(1997)
Rev. Reprod.
2,
157-164 |
55. |
Planchart, A.,
You, Y.,
and Schimenti, J. C.
(2000)
Genetics
155,
803-812 |
56. | Fossella, J., Samant, S. A., Silver, L. M., King, S. M., Vaughan, K. T., Olds-Clarke, P., Johnson, K. A., Mikami, A., Vallee, R. B., and Pilder, S. H. (2000) Mamm. Genome 11, 8-15[CrossRef][Medline] [Order article via Infotrieve] |
57. | Herrmann, B. G., Koschorz, B., Wertz, K., McLaughlin, J., and Kispert, A. (1999) Nature 402, 141-146[CrossRef][Medline] [Order article via Infotrieve] |
58. | Inaba, K., Kagami, O., and Ogawa, K. (1999) Biochem. Biophys. Res. Commun. 256, 177-183[CrossRef][Medline] [Order article via Infotrieve] |