Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06032-3305
Molecular analysis of a 19,000-Mr protein from the Chlamydomonas flagellum reveals that it is homologous to the t complex-encoded protein Tctex-2, which is a candidate for one of the distorter products that cause the extreme transmission ratio distortion (meiotic drive) of the murine t complex. The 19,000-Mr protein is extracted from the axoneme with 0.6 M NaCl and comigrates with the outer dynein arm in sucrose density gradients. This protein also is specifically missing in axonemes prepared from a mutant that does not assemble the outer arm. These data raise the possibility that Tctex-2 is a sperm flagellar dynein component. Combined with the recent identification of Tctex-1 (another distorter candidate) as a light chain of cytoplasmic dynein, these results lead to a biochemical model for how differential defects in spermiogenesis that result in the phenomenon of meiotic drive might be generated in wild-type vs t-bearing sperm.
The murine t complex (haplotype) is an alternate
form of the proximal 30-40 Mb of chromosome 17. The t form of this chromosome contains a series of
inversions that serve to suppress recombination with wild
type and, therefore, the t complex is normally inherited intact. The most striking property of this famous genetic unit is that heterozygous (+/t) males pass the t-bearing copy of
the chromosome to >95% of their progeny. This phenomenon is referred to as transmission ratio distortion or meiotic drive (see Silver [1985, 1993] for detailed discussion).
Genetic studies have revealed that, in mice, ratio distortion derives from the combined effects of four to five gene
products: a series of "distorters" (Tcd-1 to Tcd-4) that interact with a "responder" protein (Tcr) which is expressed
late in spermiogenesis after the completion of meiosis
(Lyon, 1984 Although much is known about the t complex in genetic
terms, the molecular mechanisms involved in these disparate effects on spermiogenesis and neural tube development have remained obscure. A number of studies have
identified excellent candidates for both the distorters and
the responder. These assignments have been based on genetic mapping of the various loci using partial t haplotypes derived from rare recombinants, combined with aberrant
levels of mRNA expression in the testis, the presence of
t-specific mutations, and/or phenotypic effects on sperm
morphology. Examples include Tctex-1 (t-complex testis
expressed) (Lader et al., 1989 Recently, we identified the t complex-encoded protein
Tctex-1 as a novel light chain (LC)1 of cytoplasmic dynein
that is differentially expressed in various tissues (King et
al., 1996a Detailed biochemical analysis of mammalian flagellar
dyneins is difficult because of several features inherent in
the design of mammalian spermatozoa (discussed in Gatti
et al., 1989 In this report we describe the molecular cloning and
analysis of a 19,000-Mr LC from the Chlamydomonas outer
dynein arm. This molecule, which is tightly associated with
the Axoneme Isolation and Dynein Purification
Flagella were prepared from Chlamydomonas reinhardtii using standard
methods and demembranated with NP-40 (King, 1995 For most experiments and for peptide sequencing, the wild-type strain
1132D Peptide Sequencing
Concentrated dynein components were separated by electrophoresis in a
5-15% acrylamide gradient gel and blotted to polyvinylidene difluoride
membrane (Immobilon Psq; Millipore Corp., Woburn, MA) in 10 mM
NaHCO3, 3 mM Na2CO3, 0.01% SDS, and 20% methanol. The 19,000-Mr
LC band was identified by staining with amido black, excised, and incubated with trypsin. Peptides eluting from the membrane were purified by
reverse phase chromatography on a C8 column. Two peptides were sequenced using an Applied Biosystems 492A sequencer (Foster City, CA) in
the Protein Chemistry Facility at the Worcester Foundation for Biomedical Research (Shrewsbury, MA).
Molecular Cloning
To amplify a region of the 19,000-Mr LC for use as a probe, we used a 3 Amplification products were sequenced, and one judged to encode part
of the 19,000-Mr LC was used to screen the Computational Methods
Sequence assembly was performed using the GCG suite of software (Devereux et al., 1984 Fusion Protein Expression and Antibody Preparation
The coding region for the 19,000-Mr LC was obtained by the PCR using
primers that resulted in a blunt 5 Electrophoresis and Immunoblotting
All samples were separated in 5-15% acrylamide gradient gels and either
stained with Coomassie blue or blotted to nitrocellulose using the buffer
system described above and probed as described in King et al. (1996b) The Chlamydomonas outer dynein arm contains eight LCs
(Pfister et al., 1982 A gene-specific primer based on the sequence (K)GLYYE
and an oligo(dT) adaptor primer were used in the PCR
and yielded a product of ~460 bp. Upon sequencing, this
product was found to encode the stop codon TAA immediately after the primer. As the peptide upon which this
primer was based had been predicted to be at the COOH
terminus, this product potentially encoded the 19,000-Mr LC and therefore was used to screen a The longest clone is 1,047 bp in length (Fig. 1) and contains a single open reading frame of 408 bp that encodes a
protein of 136 residues with a predicted molecular weight
of 15,825 daltons and an isoelectric point (pI) of 7.11. The
reading frame is preceded by a 144-bp 5
Southern blot analysis of Chlamydomonas genomic
DNA revealed single bands in both BamHI and SmaI digests (Fig. 2 a), suggesting that a single gene for this protein exists within Chlamydomonas. On Northern blots of
total RNA derived from nondeflagellated cells and from
cells actively undergoing flagellar regeneration, one message of ~1.6 kb was found (Fig. 2 b). The amount of this
RNA was greatly upregulated in regenerating cells, as expected for an integral axonemal component.
The secondary structure of the 19,000-Mr LC was predicted using PHD (Fig. 3 a; Rost and Sander, 1993
Examination of the GenbankTM and Expressed Sequence Tag databases using BLAST (Altshul et al., 1990 Further examination of the databases using the 19,000- Mr LC and Tctex-2 sequences revealed several additional
matches of high statistical significance. These include another t complex-encoded protein Tctex-1 that is a candidate for the Tcd-1t distorter and that we have recently
identified as an LC of cytoplasmic dynein that is differentially expressed in various tissues (King et al., 1996b
Phylogenetic analysis (Fig. 4 b) indicates that the six
known members of the Tctex-1 family fall into two main
groups. (There are at least two additional members of this
protein family exemplified by partial sequences in the Expressed Sequence Tag database.) Both known cytoplasmic
dynein LCs and one of the Caenorhabditis elegans proteins
(T05C12-5) comprise one group, whereas the second C. elegans protein (D1009-5), Tctex-2, and the Chlamydomonas outer arm light chain form the second. This indicates that mouse Tctex-2 is much more closely related to
the Chlamydomonas flagellar protein than it is to mouse
Tctex-1.
Even though it lacks a signal sequence, murine Tctex-2
has previously been described as a peripheral sperm membrane protein (Huw et al., 1995
The blot-purified R5391 antibody was then used to follow immunoreactive proteins during flagellar fractionation
(Fig. 5 c). Importantly, two proteins are recognized within
intact flagella: the original 19,000-Mr polypeptide and a
second molecule of Mr ~15,000. This is completely consistent with the previous sequence analysis of murine Tctex-2
that revealed two alternatively spliced variants within the
testis that encode proteins of similar Mr to those observed
in Chlamydomonas (see Huw et al., 1995 To determine whether all of the extracted 19,000-Mr protein was associated with the outer arm, a high salt axonemal extract was sedimented through a sucrose density gradient, and the fractions were probed for the presence of
both the 19,000-Mr protein (antibody R5391) and IC78 using mAb 1878A (King et al., 1986
To gain further insight into the axonemal location of the
smaller immunoreactive species, axonemes were prepared
from mutants lacking the outer (oda9) or inner (ida1, ida2,
ida4) arms, the radial spokes (pf14), and the central pair
microtubule complex (pf18) and probed with the R5391
antibody. The 19,000-Mr band was absent from axonemes
prepared from oda9 as expected for an outer arm component (Fig. 6 b). However, the lower band was present in all
mutants examined, suggesting that this protein is not a component of either row of dynein arms nor is it associated with the radial spokes or central pair microtubule
complex (not shown).
Here we identify a Chlamydomonas flagellar outer arm dynein LC as a homologue of the murine t complex-encoded
protein Tctex-2. Tctex-2 is of interest as it is a candidate
for one of the three to four distorter products that interact
with a cell-specific responder and lead to the extreme meiotic drive exhibited by the t complex. The data presented
here strongly suggest that Tctex-2, which is known to be a
sperm tail-specific protein (Huw et al., 1995 Transmission Ratio Distortion of the t Complex: A
Biochemical Hypothesis
The most puzzling aspect of the meiotic drive of the t complex is that only those sperm bearing the wild-type chromosome 17 are poisoned. Analysis of chimeric mice containing both +/t and +/+ spermatocytes indicates that t
sperm only show distortion over wild-type sperm that derived from the +/t, and not those from the +/+, spermatocyte (Seitz and Bennett, 1985 If Tctex-2 dysfunction itself results in nonmotile sperm
and thus represents one end of the pathway, the question
then becomes how can the ratio of wild-type to t-encoded
Tctex-2 protein be distorted during spermiogenesis? Here
our previous observation that Tctex-1 (a candidate for the
Tcd-1t distorter) is an LC of cytoplasmic dynein (King et al.,
1996a To obtain ratio distortion, the responder must be retained in close proximity to the nucleus that encoded it. As
spermatids form a syncitium after meiosis, it has been difficult to envisage how this might be achieved. However,
identification of axonemal and cytoplasmic dynein LCs as
candidate distorters provides a potential clue. Axoneme
assembly occurs at a specific perinuclear site that is defined by the basal body. This structure and its many associated proteins are not freely diffusible, being retained in
close association with each sperm nucleus. Therefore, we
suggest that the responder is a protein (possibly basal
body associated) that is involved in directing the assembly
of the sperm axoneme; i.e., it acts as a gatekeeper to determine what can and cannot enter the growing flagellum. A
general scheme for how distortion might then arise is outlined in Fig. 7 a.
Ratio distortion would be achieved through a combination of (i) the ability of Tcrt to interact only with wild-type
distorters and thus to incorporate only wild-type flagellar
dynein into the axonemes of t spermatozoa, which are thus
protected from the deleterious effects of the t-encoded distorters; and (ii) poisoning of wild-type sperm through the axonemal incorporation of t mutant flagellar dynein allowed by Tcr+. Assuming wild-type and t mutant distorters
are present in equimolar amounts within the syncitium,
wild-type axonemes would be predicted to incorporate
~50% of the t mutant flagellar dynein, which could be
more than sufficient to cause motility defects. Distorter allele-specific effects could be readily understood in the following terms: (i) alterations in the relative affinities of distorters for each other and/or for Tcr+ and Tcrt could lead
to subtle alterations in the amount and potency of t mutant
flagellar dynein incorporated into a given sperm flagellum; and (ii) direct consequences for dynein function. The presence of Tctex-2 in sperm from t/t mice (Huw et al., 1995 Possible Roles for Cytoplasmic Dynein
Newly synthesized flagellar components need to be directed to the site of flagellar assembly at the perinuclear
basal body. Assuming that cytoplasmic microtubules within
spermatids adopt the standard orientation with plus ends
toward the cell periphery, this activity would presumably
require a minus end-directed motor such as cytoplasmic
dynein of which the putative distorter Tctex-1 is an integral component. There are several possible scenarios for
the role that cytoplasmic dynein containing t-specific mutations in the Tctex-1 LC might play in ratio distortion.
These are illustrated in Fig. 7 b.
(i) Cytoplasmic dynein is involved in the interaction
with Tcr to determine whether wild-type or mutant flagellar dyneins are incorporated. In this case, to achieve distortion, cytoplasmic dynein containing t mutant Tctex-1
would have to transport only flagellar dynein containing t
mutant Tctex-2 to the site of axoneme assembly. Likewise,
wild-type cytoplasmic dynein would transport wild-type
flagellar dynein. Which cargo(s) are allowed to dock and
enter the growing flagellum would be determined by the
interaction of the responder with the cytoplasmic dynein
motor; i.e., cytoplasmic dynein containing t mutant Tctex-1
would cause distortion because it would transport flagellar
dynein containing only t mutant Tctex-2 that would be incorporated by the wild-type responder but not by the t mutant responder. Note that the interaction between cytoplasmic and flagellar dyneins need not be direct here, and
indeed this coupling could provide a site of action for one
or more additional distorter products.
(ii) Although all Tctex-1 within mammalian brain is cytoplasmic dynein associated (King et al., 1996), immunological analysis has suggested that sperm tails also contain
Tctex-1 (or a related homologue) (O'Neill and Artzt, 1995 (iii) This cytoplasmic motor complex might play no direct part in ratio distortion; i.e., both wild-type and t mutant cytoplasmic dynein either are not involved at all or
they both transport flagellar components (+ and t mutant)
to the site of axoneme assembly, but have no role in the interaction with Tcr.
Predictions of the Model
The model presented above makes a number of predictions, at least some of which are readily testable. For example, (i) flagellar axonemes from motile (t-bearing) sperm
should contain only wild-type Tctex-2 (and perhaps also
Tctex-1), whereas those from immotile (+-bearing) sperm
should have both the wild-type and mutant proteins. (ii)
For the model to be correct, defective Tctex-2 must lead to
aberrant motility. Therefore, Chlamydomonas strains lacking the 19,000-Mr Tctex-2 homologue should exhibit a detectable swimming defect. (A Chlamydomonas mutant
lacking the 19,000-Mr protein exhibits an outer arm assembly defect; consequently, such cells swim more slowly than
wild-type; Pazour, G., A. Koutoulis, H. Sheng, R.S. PatelKing, S.M. King, and G.B. Witman, unpublished results.)
The same should also be true for Tctex-1 if that protein's distorter phenotype derives from its function within the
flagellum rather than the cytoplasm. (iii) The responder
(Tcr) (for which there is a good candidate; Cebra-Thomas
et al., 1991 Intraflagellar Associations of Tctex-2
The characterization of Tctex-2 as a putative t complex
distorter is based on gene location (mapping of rare partial
haplotypes places it near Tcd-3), testis-specific expression,
and the presence of several t-specific mutations that are
predicted to cause significant alteration in protein structure (see Huw et al., 1995 As noted by Huw et al. (1995) An Extended Family of Dynein LCs
Detailed sequence analysis revealed that this Chlamydomonas LC and Tctex-2 are both related to recently identified LCs (Tctex-1 and rp3) within the mammalian cytoplasmic dynein complex (see King et al., 1996a Within the Chlamydomonas flagellum, the 19,000-Mr
LC is tightly associated with the Immunoblot analysis of Chlamydomonas flagella revealed two protein bands recognized by the R5391 antibody. All of the upper band was associated with the outer
arm. However, the smaller protein was tightly associated
with the flagellar axoneme remnant after high salt extraction and was not missing in mutants lacking the outer or
inner arms, radial spokes, or central pair complex. Thus, the axonemal location of this protein remains unknown
but, given the highly specific nature of the antibody used,
it likely represents an additional member of this protein
family. In mice, an alternatively spliced cDNA that encoded a smaller variant of Tctex-2 also was identified (Huw
et al., 1995 In conclusion, we demonstrate here that a Chlamydomonas homologue of the putative murine t complex distorter Tctex-2 is an LC of outer arm dynein. Our results
suggest that the t-encoded mutations in Tctex-2 lead to
dysfunction of the flagellar dynein molecular motor. This
in turn may contribute to non-Mendelian chromosome
segregation by disrupting normal flagellar motility of
+-bearing sperm. Further detailed functional analysis of
this LC in both Chlamydomonas and mice will provide additional clues as to the important role it plays in dynein activity and to the mechanisms responsible for meiotic drive.
). All of the genes encoding these proteins are
located within the t complex. Males containing two complementing t haplotypes are sterile. This phenotype is
thought to be a consequence of homozygosity for some or
all of the genes responsible for meiotic drive (Lyon, 1986
).
A variety of recessive lethal mutations are also present in
different t haplotypes, and these result in embryonic death
for many t/t combinations. In spite of these obviously deleterious effects in t/t animals, the t complex is transmitted at
such a high ratio that it is found in ~25% of all feral mice.
Furthermore, although in heterozygotes the t complex reveals no overt phenotype, in the presence of a mutation at
the T (Brachyury) locus it does result in mice that lack
tails. This latter phenotype is apparently caused by a defect in neural tube development (for review see Herrmann
and Kispert, 1994
).
) and Tctex-2 (Huw et al.,
1995
), which are candidates for the proximal Tcd-1t and
Tcd-3t distorters, and the hybrid sterility loci Hst-4 to Hst-6,
which may encode the distal Tcd-2t distorter (Pilder et al.,
1993
). However, none of these proteins have obvious homologies or structural motifs that provide clues as to their
function or to the pathways in which they are involved.
). This has raised the possibility that dysfunction
of intracellular transport as a result of the t-encoded mutations within the Tctex-1 light chain of cytoplasmic dynein
might contribute to meiotic drive. This phenomenon is
thought to occur through direct effects on spermiogenesis
during which the meiotic partners of t haplotype-bearing
spermatids become disabled. Many sperm produced by +/t
males are functionally deficient either in their motility or
acrosome reaction (Olds-Clarke, 1983
; Brown et al., 1989
).
Even though it is not yet clear that these phenotypic effects actually reflect aspects of the meiotic drive phenomenon, they do raise the possibility that defects in sperm dyneins (or other flagellar components) that result in abnormal flagellar beating also might contribute to ratio distortion.
). Therefore, to gain insight into the molecular
structure and function of flagellar dyneins, we have been
examining the outer arm from the Chlamydomonas flagellum as a model system. This microtubule-based molecular motor consists of three heavy chains (
,
, and
dynein
heavy chains [DHCs]; ~520 kD each), two intermediate
chains (IC78 and IC69 of 76 and 63 kD, respectively), and
a series of light chains (10-22 kD) and has a total mass of
~2 MD (Pfister et al., 1982
; Piperno and Luck, 1979
). The
DHCs contain the ATPase and motor domains of the complex (for reviews see Mitchell, 1994
; Witman et al., 1994
),
whereas the ICs are involved in cargo attachment (King et
al., 1991
, 1995) and perhaps also in regulation (Mitchell and Kang, 1993
). Recent molecular studies of the flagellar
LCs from several systems have revealed a variety of intriguing functional attributes associated with these dynein
components including cAMP-dependent phosphorylation
(Barkalow et al., 1994
), Ca2+ binding (King and PatelKing, 1995
a), and sulphydryl oxidoreductase (Patel-King et
al., 1996
) activities. Furthermore, an 8,000-Mr axonemal dynein LC (King and Patel-King, 1995b
) that dimerizes in
situ (Benashski, S.E., A. Harrison, R.S. Patel-King, and
S.M. King, manuscript submitted for publication) has also
been found to occur within both cytoplasmic dynein (King
et al., 1996b
) and the unconventional actin-based motor
myosin-V (Espindola, F.S., R.E. Cheney, S.M. King, D.M.
Suter, and M.S. Mooseker. 1996. Mol. Biol. Cell. 7:372a).
DHC, is a homologue of the mouse t complex protein Tctex-2 and is also more distantly related to Tctex-1
(a cytoplasmic dynein LC; King et al., 1996a
). As Tctex-2
is a strong candidate for the Tcd-3t distorter (Huw et al.,
1995
), this result indicates that defects in outer arm function because of the t-specific mutations within Tctex-2 may contribute to meiotic drive and/or to the male sterility phenotype associated with homozygosity of the genes responsible for transmission ratio distortion of the t complex. The
model for ratio distortion outlined here builds on that proposed by Cebra-Thomas et al. (1991)
and suggests that the
responder acts as a "gatekeeper" for axoneme assembly.
Wild-type sperm become poisoned because the wild-type responder (Tcr+) allows the incorporation of both wildtype and t mutant Tctex-2 into the axoneme. The presence
of the mutant dynein LC is predicted to directly affect dynein function in vivo. The t-bearing sperm are protected
because the t mutant responder (Tcrt) cannot interact with
t mutant distorters (Cebra-Thomas et al., 1991
); i.e., only
wild-type Tctex-2 can be incorporated into the sperm tail.
The predicted result of this model is that t-bearing sperm
show normal motility, whereas wild-type sperm are defective as a result of the presence of mutant dynein.
Materials and Methods
; Witman, 1986
).
Outer arm dynein was subsequently extracted with 0.6 M NaCl, purified
by centrifugation on a 5-20% sucrose density gradient (King et al., 1986
),
and concentrated in a Centricon 30 unit (Amicon Corp., Danvers, MA)
that had been incubated overnight with 5% Tween-20 to reduce nonspecific protein binding.
was used. To investigate the presence of the 19,000-Mr protein in
other axonemal structures, axonemes also were prepared from mutants
lacking the outer (oda9) or different subsets of inner dynein arms (ida1,
ida2, ida4), the radial spokes (pf14), and central pair microtubule complex
(pf18).
rapid amplification of cDNA ends procedure (Frohman et al., 1988
). The
initial reaction used a series of blunt gene-specific primers designed from
peptide sequences derived from several LCs. The reverse primer was the
standard oligo(dT) adaptor primer (5
-GCGCGTCGACTCGAGT20V3
) that contains SalI and XhoI sites at the 5
end. The 100 µl PCR was
performed in 10 mM Tris-Cl, pH 8.85, 25 mM KCl, 5 mM (NH4)2SO4, 2 mM
MgSO4, and 0.2 mM dNTP and contained 1 µg of each primer. A
ZapII
cDNA library made from mRNA derived from cells actively regenerating
flagella (Wilkerson et al., 1995
) was used as the template. 2 U of Pwo
DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis,
IN) was added and, after denaturation at 96°C for 5 min, the sample was
subjected to the following thermal regime: 96°C for 1 min, 50°C for 1 min,
and 72°C for 1 min for 30 cycles. This was followed by a final 10-min incubation at 72°C. Products were reamplified with a fourfold degenerate genespecific primer (5
-GCGCGAATTCAAGGGYCTSTACTACGAG-3
;
based on the peptide sequence [K]GLYYE and incorporating an EcoRI
site and GC clamp at the 5
end) and the oligo(dT) adaptor primer using
Pfu DNA polymerase and were subcloned into pBluescript II SK+ (Stratagene, La Jolla, CA).
ZapII cDNA library made
from mRNA derived from cells actively regenerating flagella (Wilkerson
et al., 1995
). Phagemids were rescued using helper phage and the longest
clone sequenced on both strands using both single- and double-stranded
DNA templates. Northern and Southern blots were prepared by standard
methods (Sambrook et al., 1987) and probed with the 19,000-Mr cDNA
using the conditions described in King and Patel-King (1995a).
). Searches of the GenbankTM and Expressed Sequence
Tag databases were made using BLAST (Altshul et al., 1990
). Pairwise sequence comparisons were generated using GAP (Devereux et al., 1984
);
multiple alignments were constructed with CLUSTALW (Thompson et al.,
1994
); and secondary structure was predicted using PHD (Rost and Sander,
1993
). Helical segments were analyzed using HELICALWHEEL (Devereux et al., 1984
) and COILS (Lupas et al., 1991
). The phylogenetic tree
was calculated with DISTANCES and plotted with GROWTREE.
end and an XbaI site at the 3
end after
the stop codon. This product was subcloned across the XmnI/XbaI sites in
pMal-c2 (New England Biolabs, Beverly, MA) and resulted in the COOHterminal fusion of the LC to maltose binding protein (MBP) via a short
hydrophilic linker that contains a Factor Xa proteolytic cleavage site immediately NH2-terminal to the first Met residue of the LC. Protein expression was induced by addition of isopropyl-1-thio-
-d-galactopyranoside.
The soluble fusion protein fraction was purified by affinity chromatography on amylose resin and eluted with 10 mM maltose in 20 mM Tris-Cl, pH 7.4, 1 mM EDTA, and 200 mM NaCl. The intact purified fusion protein was used as the immunogen for polyclonal antisera production, and a
19,000-Mr LC-specific antibody fraction was obtained by blot purification
using the minor modifications to the method of Olmsted (1986) described
in King et al. (1996b)
.
.
Briefly, blots were blocked with 5% dry milk and 0.1% Tween-20 in Trisbuffered saline and probed with purified antibody diluted ~1:50 and a
peroxidase-conjugated secondary antibody. After a wash with 0.5% Triton X-100 in Tris-buffered saline, antibody reactivity was visualized using
an enhanced chemiluminescent system (ECL; Amersham Corp., Arlington Heights, IL) and RX film (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Results
). Chemical dissection of outer arm dynein has indicated that the 19,000-Mr LC is tightly associated with the
DHC (Mitchell and Rosenbaum, 1986
).
Furthermore, after salt extraction from the axoneme, this
LC cosediments with the
DHC (and other dynein components) at ~18S in sucrose density gradients; it also copurifies with the
DHC after both hydroxylapatite and ion
exchange chromatography. The electrophoretically purified LC was blotted to polyvinylidene difluoride membrane, and the excised band was incubated with trypsin. Peptides eluting from the membrane were purified by reverse
phase chromatography. Two peptides were sequenced and
yielded a total of 23/25 unambiguous residue assignments:
namely, XLXDQTNDNFASEYYENESM and GLYYE.
Note that the thiohydantoin derivatives of Cys and Trp
residues were not readily identifiable under the conditions
used. The shorter peptide (GLYYE) was present in relatively high yield, and the sequence ended abruptly after
the Glu residue, suggesting that it represented the COOH
terminus of the molecule.
ZapII cDNA library made from RNA derived from Chlamydomonas actively regenerating flagella (Wilkerson et al., 1995
). Multiple clones were obtained and the longest one was sequenced.
-untranslated region that contains three in-frame stop codons before the
first Met residue. The reading frame terminates with a single stop codon followed by 445 bp of 3
-untranslated region before the poly A tail. There is a perfect copy of the putative Chlamydomonas polyadenylation signal located
near the end of the clone. Both peptide sequences obtained from the authentic molecule are found within the
predicted protein (23/25 residues correct with two unknown). The two uncalled residues in the longer peptide
are a Cys and a Trp, neither of which would have been identified under our sequencing conditions. This peptide
also is preceded by a basic (Arg) residue as predicted for
the product of a tryptic digest. However, the GLYYE peptide is preceded by a Tyr. Thus, this peptide presumably
derives from tryptic cleavage at a noncanonical site, as was
also found for a peptide from the 16,000-Mr Chlamydomonas LC (Patel-King et al., 1996
). As only the GLYYE
sequence was used to obtain the original PCR product, the
above data fully confirm that this clone indeed encodes
the 19,000-Mr LC.
Fig. 1.
Sequence of the 19,000-Mr LC cDNA clone. The nucleotide and deduced amino acid sequences for the LC are shown.
Residues indicated in bold type were identified by peptide sequencing. A perfect copy of the putative Chlamydomonas polyadenylation signal is underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession number
U89649.
[View Larger Version of this Image (71K GIF file)]
Fig. 2.
Southern and Northern blot analysis. (a) Southern blot
of 10 µg genomic DNA from Chlamydomonas strain S1D2 digested with BamHI, PstI, PvuII, and SmaI. Single bands are observed in the BamHI and SmaI lanes, suggesting that there is only
a single gene for this LC within Chlamydomonas. Standards are
indicated at left (kb). (b) Northern blot of 20 µg total RNA obtained from nondeflagellated cells (NDF) and from cells that had
been deflagellated and allowed to regenerate flagella for 30 min
(30 postDF). Standards are shown at left (kb). A single message
of ~1.6 kb that was greatly upregulated after deflagellation is evident.
[View Larger Version of this Image (47K GIF file)]
). The
NH2-terminal portion of the molecule likely contains three
helical segments, one of which (residues 60-76) is predicted to be amphiphilic (Fig. 3 b). This same region has a
very high probability of forming a coiled-coil when analyzed using the program COILS with window size = 14 (Fig. 3 c). The COOH-terminal region of this LC is predicted to consist of extended sheet structures (Fig. 3 a).
Fig. 3.
Sequence analysis of the 19,000-Mr LC. (a) The secondary structure of the 19,000-Mr LC was predicted using PHD (Rost
and Sander, 1993). E, extended sheet; H, helix. (b) The helical
segment formed by residues 60-76 is amphiphilic and displayed
using HELICALWHEEL. Hydrophobic and hydrophilic residues cluster to opposite sides of the helix. (c) COILS output for
the Chlamydomonas LC using a window size of 14. The plot displays the probability of coiled-coil formation vs residue number.
The region of high probability coincides with the amphiphilic helix predicted for residues 60-76. (d) Sequence comparison between the Chlamydomonas 19,000-Mr LC (U89649) and the
mouse t complex protein Tctex-2 (U21673; Huw et al., 1995
). The
alignment was generated by GAP using the default parameters. These proteins share 35% identity (56% similarity) with the
smallest Poisson probability P(n) = 5.2 × 10
22 (calculated by
BLAST).
[View Larger Version of this Image (29K GIF file)]
)
revealed that the 19,000-Mr LC is a homologue of the mouse
t complex protein Tctex-2 (Huw et al., 1995
). A comparison between the 19,000-Mr LC and mouse Tctex-2 generated by GAP using the default parameters is shown in Fig.
3 d. These proteins share 35% identity and 56% similarity; the smallest Poisson probability P(n) = 5.2 × 10
22 (calculated by BLAST), indicating that the match is highly significant.
), as
well as several other mammalian and nematode proteins, at least one of which (rp3; Roux et al., 1994
) also is a cytoplasmic dynein LC (King, S.M., E. Barbarese, S.E. Benashski, J.F. Dillman III, and K.K. Pfister, manuscript in
preparation). The smallest Poisson probabilities (P(n)) for
the comparisons between the 19,000-Mr LC vs human
Tctex-1 and the nematode open reading frame D1009-5 are 2.2 × 10
7 and 1.8 × 10
10, respectively. An alignment
of these proteins generated by CLUSTALW is shown in
Fig. 4 a. This analysis revealed that the COOH-terminal portions of these molecules are most highly conserved and
that the NH2-terminal regions are much more divergent.
The former region includes a Trp-Asp dipeptide motif that
is invariant in all known members of this protein family
and a number of Cys residues that are conserved between
several of these polypeptides.
Fig. 4.
The 19,000-Mr LC and Tctex-2 are members of the Tctex-1 protein family. (a) Alignment of the Tctex-1 protein family generated by CLUSTALW. Residues conserved between two or more polypeptides are shaded (and boxed where necessary). The Cys residues are marked by black boxes. The invariant Trp-Asp motif is at residues 172/173 of the alignment. Sequences used in the alignment
are: murine Tctex-1 (A32995; Lader et al., 1989), human rp3 (U02556; Roux et al., 1994
), C. elegans ORF T05C12-5 (Z66500), C. elegans
ORF D1009-5 (U40938), Chlamydomonas 19,000-Mr LC (U89649), and murine Tctex-2 (U21673; Huw et al., 1995
). (b) Phylogenetic
analysis of the members of the Tctex-1 protein family. The relationship was calculated with DISTANCES using the Kimura protein option and plotted with GROWTREE (UPGMA option). Murine Tctex-2 is most closely related to the Chlamydomonas outer arm LC.
[View Larger Versions of these Images (39 + 12K GIF file)]
). This assignment was
based on immunological staining of air-dried spermatozoa
and on extraction of Tctex-2 after treatment of intact
sperm with 100 mM Na2CO3 (pH ~10.5). To determine
whether all of the homologous 19,000-Mr protein within Chlamydomonas flagella is actually associated with the
outer dynein arm, the coding region for this molecule was
fused to maltose binding protein, and the entire fusion
protein was used for polyclonal antibody production (antibody R5391). An antibody fraction specific for the 19,000- Mr protein was then obtained by blot purification vs the
recombinant molecule after separation of the LC from
MBP by digestion with Factor Xa (Fig. 5 a). The blot-purified antibody is highly specific for the 19,000-Mr LC and
does not recognize several other members of the Tctex-1
protein family (Fig. 5 b).
Fig. 5.
Distribution of the
19,000-Mr LC in Chlamydomonas flagella. (a) 30 µg of
the MBP/19,000-Mr LC fusion protein and of a similar sample digested with 60 ng
Factor Xa were electrophoresed in a 5-15% acrylamide gradient gel and stained
with Coomassie blue (CBB).
The intact fusion protein
was used for immunizations,
and the resulting antiserum
(R5391) was blot purified using the electrophoretically purified 19,000-Mr LC band.
(b) (Upper panel) Recombinant Chlamydomonas 19,000- Mr LC and two members of
the Tctex-1 family, namely,
murine Tctex-1 and human
rp3, after staining with Coomassie blue. (Lower panel)
Nitrocellulose blot of similar
samples probed with purified R5391 antibody. The antibody
preparation is highly specific
and only recognizes the Chlamydomonas LC. (c) Flagella
(185 µg) were extracted with
1% NP-40 to remove the membrane, and the resulting axonemes were treated with
0.6 M NaCl to solubilize the
outer arm. Equivalent amounts
of each sample were separated in a 5-15% acrylamide
gradient gel and either
stained with Coomassie blue (upper panel) or blotted to
nitrocellulose and probed with the blot-purified R5391 antibody (lower panel). The locations of the Mr markers and the dye front (DF)
are indicated at left. Two immunoreactive bands are evident. Approximately 90% of the upper (Mr19,000) band was extracted with 0.6 M
NaCl, whereas the lower band remained tightly associated with the axonemal remnant. Neither immunoreactive species was solubilized
upon detergent extraction, indicating that these homologues of Tctex-2 are not peripheral membrane proteins.
[View Larger Version of this Image (53K GIF file)]
). After treatment
of Chlamydomonas flagella with detergent to remove the membrane, all of both immunoreactive bands were obtained in the insoluble axoneme fraction. This indicates
that, in Chlamydomonas, the Tctex-2 homologue is not
membrane-associated. After axoneme extraction with 0.6 M NaCl, ~90% of the 19,000-Mr band was solubilized as
expected for an integral component of the outer dynein
arm. Interestingly, the immunoreactive species of lower
Mr was not extracted under these conditions and remained
tightly bound to the axonemal remnants.
). All of the 19,000-Mr
protein precisely comigrated with IC78 as expected for an
integral component of the outer dynein arm (Fig. 6 a).
Fig. 6.
All of the 19,000- Mr protein is associated with
the outer dynein arm. (a)
Proteins extracted from the
axoneme by high salt were
separated by sucrose density
gradient centrifugation. Equal volumes of each fraction were
electrophoresed in a 5-15%
acrylamide gradient gel and
blotted to nitrocellulose. The
bottom of the gradient is at
left. The blot was first probed
with the R5391 antibody and
subsequently with 1878A to
reveal the 19,000-Mr protein and IC78, respectively. Finally, the blot was stained
with Ponceau S to reveal the
location of individual lanes
and the Mr markers. All of
the 19,000-Mr protein comigrates with the outer dynein arm at ~18S. (b) Axonemes (~150 µg) prepared from wild-type and oda9 strains were electrophoresed in a 5-15% acrylamide gradient gel, blotted to nitrocellulose, and probed with the R5391 antibody. The 19,000-Mr protein is completely absent from the outer arm-less axonemes. However, the lower band is present in both samples, indicating that it is not associated with outer arm dynein.
[View Larger Version of this Image (46K GIF file)]
Discussion
), is a flagellar
dynein LC. This is particularly intriguing as the t-encoded
forms of Tctex-2 may thus represent the first defined mammalian flagellar dynein mutations. Furthermore, these data
raise the possibility that flagellar dynein dysfunction contributes to meiotic drive and/or to the male infertility phenotype associated with homozygosity for the t complex.
). Cebra-Thomas et al. (1991)
proposed that this could be achieved if the t-encoded responder protected t-bearing sperm from the t-encoded distorters by failing to interact with those components while
still able to act on its downstream targets. The distorters
are thought to be diffusible between meiotic products and
to act in a concerted fashion such that the degree of ratio
distortion is directly affected by the distorter alleles
present in a given haplotype. However, to achieve distortion, the responder must act only within the cell in which it
is synthesized. If Tctex-2 is indeed the Tcd-3t distorter, it
may well represent the final step in the distortion pathway,
as the t-encoded mutations within Tctex-2 could lead to assembly, regulatory or targeting defects in the outer dynein arm, and, consequently, to the observed aberrant sperm
motility phenotype. This hypothesis readily explains how
different Tcd-3t distorter alleles might result in different
levels of ratio distortion by simply producing differential
effects on sperm dynein and thus having greater or lesser
consequences for functional motility.
) potentially becomes of significance, as it suggests
that alterations in intracellular transport activities during
spermiogenesis also may underlie distortion.
Fig. 7.
A model for dynein-mediated meiotic drive. Recent
studies have revealed that candidates for the Tcd-1t and Tcd-3t
distorters are LCs of cytoplasmic dynein (King et al., 1996a) and
outer arm dynein (this work), respectively. This suggests a model
in which ratio distortion arises as a result of the differential incorporation of wild-type and mutant dynein into sperm axonemes.
The responder (Tcr) is hypothesized to be associated with the
sperm nucleus (possibly at the basal body) and to be involved as a
gatekeeper in determining which components are assembled into
the flagellum. This would satisfy the requirement for Tcr to be retained within the cell in which it is synthesized and also suggests
several mechanisms through which cytoplasmic and flagellar dynein LCs might interact to achieve ratio distortion. (a) The biological consequences are illustrated (1-3 in a indicate the points
at which the different interactions shown in b occur). Distortion arises as a result of two features: (i) the inability of the t-encoded responder (Tcr t ) to incorporate t mutant flagellar dynein into the
axoneme (it thus incorporates only the wild-type enzyme); and
(ii) assembly of both wild-type and t dynein into axonemes by
Tcr+, which leads directly to motility defects. Thus, in this model, axonemes of t haplotype-bearing sperm contain only wild-type
flagellar dynein and would exhibit normal motility, whereas sperm containing the wild-type chromosome would be defective as a result of
axonemal incorporation of t mutant flagellar dynein. (b) The results from three possible interactions between the two putative distorters (Tctex-1 [a cytoplasmic dynein LC] and Tctex-2 [a flagellar dynein LC]) and the responder are shown. In i, cytoplasmic dynein (CD) transports flagellar dynein (FD) to the assembly site and interacts with Tcr to determine which FD (+ or t) is incorporated. In this scenario, wild-type CD+ (the motor) must transport only wild-type FD+ (the cargo) and, likewise, t mutant CDt transports only FDt. (ii)
Immunological analyses of both mouse sperm (O'Neill and Artzt, 1995
) and Chlamydomonas (Harrison, A., and S.M. King, unpublished results) indicate that Tctex-1 (or a close homologue) also may be present within the flagellum. This suggests a second possibility in which CD (i.e., Tctex-1) and FD interact independently with Tcr. In iii, CD is not involved in distortion, which arises simply because
of interactions between FD and Tcr.
[View Larger Versions of these Images (24 + 32K GIF file)]
)
may then simply derive from a low affinity interaction between t mutant Tctex-2 and Tcrt in the absence of the competing high affinity interaction with wild type.
).
We have recently observed that a 14-kD Chlamydomonas
protein recognized by an antibody directed against human
Tctex-1 is an integral axonemal component (Harrison, A.,
and S.M. King, unpublished results). Thus it is possible
that Tctex-1 exerts a direct effect on flagellar function because either flagella contain a "cytoplasmic-like" dynein or
Tctex-1 is also a component of some other flagellar complex. In this case, the incorporation of wild-type and t mutant Tctex-1 and Tctex-2 proteins could be distorted independently simply through differential interactions with
Tcr+ and Tcrt.
) should localize to the base of the growing flagellum within spermatids.
; Lader et al., 1989
; O'Neill and
Artzt, 1995
). Previously, Tctex-2 was considered to be a
peripheral sperm membrane protein (Huw et al., 1995
). This assignment was based on several observations. First,
histochemical staining of air-dried spermatozoa with an
anti-Tctex-2 antiserum yielded a positive signal. However,
the mammalian sperm membrane is a very fragile structure, and this treatment is known to disrupt it, thereby allowing antibodies access to intracellular components (see
Phillips et al. [1991] for discussion of the effects of various treatments on sperm membrane integrity). Moreover, it is
not possible to distinguish between external and internal
sperm antigens at the light microscopic level. Second, Tctex-2 was released after treatment of intact sperm with 100 mM Na2CO3 (pH ~10.5). Again this treatment could have
disrupted membrane integrity as could the multiple centrifugations to which sperm are necessarily subject during
extraction procedures (Phillips et al., 1991
). Finally, high
pH has been found to be very effective in solubilizing
flagellar axonemes and thus releasing both outer and inner
arm dynein components (Gatti, J.-L., S.M. King, and G.B.
Witman, unpublished observations).
, Tctex-2 does not have a
canonical signal sequence that would predict transmembrane passage. Indeed the NH2-terminal domain is very
hydrophilic with 10 charged and 8 polar amino acids within
the first 26 residues and is thus a very unlikely candidate
for passing a hydrophobic barrier. Although there are
mechanisms for transporting specific proteins lacking signal sequences across lipid bilayers such as interleukin-1
and various growth factors (Rubertelli et al., 1990
), the details of how this is achieved remain unclear and are likely
highly specialized for specific molecules. There is no evidence to suggest that Tctex-2 is transported in such a fashion. Our identification of a close homologue of this protein
within the Chlamydomonas outer arm raises the strong
possibility that murine Tctex-2 also is a flagellar dynein
LC and that dysfunction of this protein as a result of the
t-specific mutations contributes to ratio distortion through a direct effect on flagellar motility.
). Together with
several nematode homologues, these proteins form a diverse gene family of dynein LCs (there are at least two additional mammalian members of this protein family exemplified by partial sequences in the Expressed Sequence Tag database). Intriguingly, some of these proteins are differentially expressed in various tissues, suggesting the existence of dynein isoforms based on LC complement and
raising the possibility that these LCs play a role in tissuespecific targeting or regulatory events.
DHC (Mitchell and
Rosenbaum, 1986
). Based on analysis of dynein from a
mutant (oda4-s7) expressing a truncated form of the
DHC, the site of interaction between these molecules
must be located within the NH2-terminal ~160 kD of the
DHC, i.e., in the stem domain of the structure (Sakakibara et al., 1993
). The functional role played by this LC within
outer arm dynein remains unclear at present. However, as
multiple members of this protein family have been identified within both cytoplasmic and flagellar dyneins, they
may be involved in assembly, targeting dyneins to specific
cargoes, or regulating their activity in response to specific
signals.
); intriguingly, this smaller variant was not detected in whole testis protein samples. In Chlamydomonas, only single bands were observed on both Southern and
Northern blots with the 19,000-Mr LC probe. Thus, the
smaller immunoreactive band is either the product of a
separate gene or the result of posttranslational processing
of the 19,000-Mr LC.
Received for publication 21 November 1996 and in revised form 26 February 1997.
1. Abbreviations used in this paper: DHC, dynein heavy chain; IC, intermediate chain; LC, light chain; MBP, maltose binding protein.We thank Dr. John Leszyk for expert assistance with the protein sequencing and Drs. Ann Cowan and Kevin Pfister for helpful discussions.
This study was supported by a New Investigator award from the Patrick and Catherine Weldon Donaghue Medical Research Foundation and by grant GM 51293 awarded by the National Institutes of Health.