(Received for publication, November 8, 1995; and in revised form, January 4, 1996)
From the
We describe here the molecular cloning and analysis of the M 14,000 and 16,000 outer arm dynein light chains
(DLCs) from Chlamydomonas flagella. Within the outer arm, the M
14,000 DLC apparently is associated with the
intermediate chains at the base of the soluble dynein particle; the M
16,000 DLC interacts directly with the
dynein heavy chain. Sequence analysis indicates that both molecules are
novel members of the thioredoxin superfamily and share
30%
sequence identity with thioredoxin from Penicillium. Both DLCs
have a perfect copy of the thioredoxin active site (WCGPCK); the M
16,000 DLC also contains the canonical P-loop
motif (AX
GKS). There is a single gene for both DLCs within Chlamydomonas and only single messages that were up-regulated
more than 10-fold upon deflagellation were observed on Northern blots.
Both recombinant DLCs were specifically eluted from a phenylarsine
oxide matrix with
-mercaptoethanol indicating that they contain
vicinal dithiols competent to undergo reversible oxidation/reduction.
Furthermore, we demonstrate that outer (but not inner) arm dynein may
be purified on the basis of its affinity for phenylarsine oxide
suggesting that the predicted redox-sensitive vicinal dithiols exist
within the native complex.
Flagellar dyneins are molecular motors responsible for generating the force required for interdoublet microtubule sliding which ultimately is converted to flagellar bending (see Warner et al.(1989) for reviews). The related cytoplasmic dyneins are involved in a wide variety of intracellular motile events including axonal transport, endosome and Golgi movement, positioning of the mitotic spindle and nuclear migration (Corthésy-Theulaz et al., 1992; Li et al., 1993; Paschal and Vallee, 1987; Schroer et al., 1989; Xiang et al., 1994). Both the flagellar and cytoplasmic isozymes are highly complex assemblies containing multiple motor subunits associated with a variety of accessory proteins (see Holzbaur et al.(1994) and Witman et al.(1994)). Members of this latter group of polypeptides are thought to target the motor to the appropriate cargo (King et al., 1991, 1995; Paschal et al., 1992) and to modify motor activity to achieve regulated movement (Barkalow et al., 1994; King and Patel-King, 1995b; Stephens and Prior, 1992). However, although considerable progress has recently been made (Gill et al., 1994; Hughes et al., 1995; King and Patel-King, 1995a, 1995b; LeDizet and Piperno, 1995; Mitchell and Kang, 1991, 1993; Ogawa et al., 1995; Paschal et al., 1992; Wilkerson et al., 1995), the molecular identity and function of many of these accessory proteins remains unexplored.
One of the best characterized
dyneins is the outer arm from flagella of Chlamydomonas. This
complex consists of three dynein heavy chains (the ,
, and
DHCs; (
)
520 kDa each) which contain the ATPase
and microtubule motor domains, two intermediate chains of 76.5 and 63
kDa (IC78 and IC69, (
)respectively) and 8 different light
chains (DLCs) of M
8,000-22,000, several of
which are present in multiple copies (reviewed in Witman et
al.(1994)). Structurally, the outer arm appears as three globular
head domains interconnected by stems to a common base (Goodenough and
Heuser, 1984). Each globular head and stem is formed from a single DHC.
Each DHC is also tightly associated with at least one DLC, and we have
recently determined that one of the
DHC-associated DLCs is a
novel Ca
-binding member of the calmodulin superfamily
(King and Patel-King, 1995b). At least for the
DHC, the tightly
bound DLC interacts with the N-terminal 160 kDa of the DHC (Sakakibara et al., 1993) and therefore is predicted to be located in the
stem region rather than the globular head domain. The remaining DLCs
apparently interact with the two ICs in a distinct subcomplex located
at the base of the soluble dynein particle (Mitchell and Rosenbaum,
1986; Witman et al., 1991). Both ICs are known to be essential
for assembly of the outer arm (Mitchell and Kang, 1991, 1993; Wilkerson et al., 1995) and one (IC78) is involved in attachment of this
dynein to its cargo (King et al., 1995). The functional
significance of the various DLCs associated with the ICs is much less
clear. They may be involved in interactions with other axonemal
components located on or near the doublet microtubules or, as dynein
arms overlap in situ, they may interact directly with the
globular head domains of the adjacent dynein particle.
In order to
understand how flagellar dynein functions, it is obviously necessary to
determine the structure and role of all the component polypeptides.
Therefore, we have initiated a molecular analysis of the DLCs within
the Chlamydomonas outer arm. Here we demonstrate that the M 14,000 and 16,000 DLCs, which are associated
with ICs and with the
DHC, respectively, are novel members of the
thioredoxin superfamily containing perfect copies of the thioredoxin
active site. Affinity chromatography on phenylarsine oxide indicates
that both molecules are functional as thioredoxins and we demonstrate
that outer arm (but not inner arm) dynein can be purified on the basis
of redox-sensitive vicinal dithiols. These are the first
redox-sensitive proteins to be found associated with a
microtubule-based molecular motor.
For the M 16,000 DLC, we employed a 3` RACE procedure
(Frohman et al., 1988) utilizing a gene specific primer
(5`-GCGCGAATTCGAYGAYCTSGAGGAGAAC-3`) based on the peptide sequence
DDLEEN and incorporating an EcoRI site, and an oligo(dT)
adaptor primer (5`-GGCCACGCGTCGACTAGTACT
V-3`). The
reaction was performed as described above except that the anneal
temperature was increased to 45 °C. The product was cloned into
pBluescript SK+.
For both DLCs, the cloned regions then were
used to probe a Chlamydomonas cDNA library made in ZapII
from RNA derived from cells that were undergoing flagellar regeneration
(Wilkerson et al., 1995). cDNA containing phagemids were
rescued using helper phage. The longest clones were sequenced (from
double-stranded DNA) on both strands using Sequenase v2.0 (U. S.
Biochemical Corp.) and a 7-deaza-dGTP sequencing kit. Several regions
of high GC content also were sequenced (in both directions) using
single stranded DNA templates. Northern and Southern blots were
prepared and processed by standard methods (Sambrook et al.,
1989) using the conditions described by King and Patel-King (1995a).
Fusion protein expression was induced by the addition of
isopropyl-1-thio--D-galactopyranoside and the soluble
fraction purified by affinity chromatography on amylose resin. For some
experiments, 1 mM
-mercaptoethanol was included
throughout the purification to ensure that thiol groups remained
reduced. DLCs were separated from MBP by cleavage with Factor Xa at a
ratio of 1:1000 (w/w) at room temperature for 12-16 h. For use as
a control protein, the MBP-lacZ fusion that is encoded by the original
pMAL-c2 vector also was purified by amylose affinity chromatography.
Protein concentrations were determined using the bicinchoninic acid
assay (Pierce) with bovine serum albumin as standard.
Dynein extracted from Chlamydomonas flagellar axonemes with 0.6 M NaCl was also subject to PAO affinity chromatography as described above. To distinguish between the various inner and outer arm DHC species, samples were separated in 4% acrylamide, 4 M urea gels and either silver stained using the procedure of Merril et al.(1981) or blotted to nitrocellulose and probed with monoclonal antibodies specific for various outer arm polypeptides (King et al., 1985, 1986).
Figure 1:
Reverse phase
chromatography on an Aquapore RP-300 (C) column of tryptic
peptides obtained from the electrophoretically purified M
14,000 (a) and 16,000 (b) DLCs. The
following peptide sequences were obtained from the indicated peaks. a: a, FYSVSSER; b TEVLETPGTLQVVEVFQSXX. b: a, SVLPTFR; b,
XLTPANADVDDLEENPMYLA.
Figure 2:
Nucleotide sequence of the cDNA clone
encoding the M 14,000 DLC. The deduced amino acid
sequence also is shown. Residues indicated in bold type were
identified by peptide sequencing (see Fig. 1a). A
perfect copy of the putative Chlamydomonas polyadenylation
signal is underlined. This sequence is available under
accession number U43609.
The M 14,000 DLC clone was used to probe genomic DNA obtained from Chlamydomonas strain S1D2 (Fig. 3a). Single
major bands were observed in samples digested with BamHI and SmaI suggesting that there is only one gene for this DLC
within Chlamydomonas. On Northern blots, a single message of
1.34 kb was observed in RNA obtained from nondeflagellated cells (Fig. 3b, NDF). As expected for an integral
axonemal component, the amount of message for this DLC was induced more
than 10-fold upon deflagellation (Fig. 3b, 30` post
DF).
Figure 3:
a, Southern blot of 10 µg of genomic
DNA derived from Chlamydomonas strain S1D2 and digested with BamHI, PstI, PvuII, and SmaI. The
blot was probed with the full-length cDNA for the M 14,000 DLC. Single major bands were observed in the BamHI and SmaI lanes suggesting that there is only a
single gene for this DLC within Chlamydomonas. Standards are
indicated at left (kilobases). b, Northern blot of
total RNA (20 µg) obtained from non-deflagellated cells (NDF) and from cells that had been deflagellated and allowed
to undergo flagellar regeneration for 30 min (30` post DF).
Standards are at left (kb). A single message of
1.34 kb is
evident. As is expected for an integral axonemal component, the amount
of this message was induced >10-fold upon
deflagellation.
Figure 4:
Nucleotide and deduced amino acid sequence
of the cDNA clone encoding the M 16,000 DLC.
Residues shown in bold type were identified by peptide
sequencing (see Fig. 1b). A perfect copy of the
putative Chlamydomonas polyadenylation signal is underlined. This sequence is available under accession number
U43610.
Southern and Northern blot
analysis of the M 16,000 DLC is shown in Fig. 5. Single bands are observed in genomic DNA digested with BamHI and PstI suggesting that there is a single gene
for this protein within Chlamydomonas (Fig. 5a). A single message of
1.33 kb was
evident in RNA samples from cells actively regenerating flagella; this
message was almost undetectable in uninduced cells (Fig. 5b).
Figure 5:
a,
Southern blot of 10 µg of genomic DNA derived from Chlamydomonas strain S1D2 and digested with BamHI, PstI, PvuII, and SmaI. The blot was probed
with the full-length cDNA for the M 16,000 DLC.
Following digestion with BamHI and PstI, only single
bands were observed indicating that there is one gene for this DLC in Chlamydomonas. Standards are indicated at the left (kilobases). b, Northern blot of total RNA (20 µg)
obtained from non-deflagellated cells (NDF) and from cells
that had been deflagellated and allowed to undergo flagellar
regeneration for 30 min (30` post DF). Standards are shown at
the left (kb). A single message of
1.33 kb is evident in the
induced sample.
Figure 6:
a, sequence comparison between the Chlamydomonas M 14,000 and 16,000 DLCs (DLC14 and
DLC16, respectively). The alignment was generated with the program GAP
using the default parameters. b, comparison between the Chlamydomonas DLCs and thioredoxins from chicken (P08629;
Jones and Luk, 1988), rhesus macaque (P29451; An and Wu, 1992), Emericella (P29429; LeMarachal et al., 1992), and Penicillium chrysogenum (P34723; Cohen et al., 1994).
The alignment was generated by the GCG program PILEUP using the default
parameters. Residues conserved in 2 or more sequences are shaded. Those identical in all sequences are indicated by an asterisk (*). c, probability (p
) scores calculated for the comparisons
between DLCs and various thioredoxins by BLAST using the default
matrix.
The secondary structure for
the M 14,000 and 16,000 DLCs and for Penicillium thioredoxin were analyzed using PHD (Rost and
Sander, 1993); the information base for this prediction included the
known structure for thioredoxin from Escherichia coli (Eklund et al., 1984; Jeng et al., 1994) and therefore the
accuracy is likely >70% (Rost and Sander, 1993). The regions of the
three molecules predicted to be in extended or helical conformations
are shown in Fig. 7. Although the M
14,000
DLC is only approximately 30% identical to thioredoxin from Penicillium, the predicted secondary structure is very
similar. The only apparently significant differences are two small
helical regions predicted in the M
14,000 DLC for
residues 29-32 and 63-70 that are not present in the fungal
thioredoxin. The structure prediction for the N-terminal portion of the M
16,000 DLC also is quite similar to thioredoxin.
However, in this case the C-terminal region is significantly different
and is predicted to have an extended section with high helical content.
Figure 7:
Secondary structures for the M 14,000 and 16,000 DLCs and for thioredoxin from Penicillium (P34723) were predicted using PHD (Rost and
Sander, 1993). E, extended sheet; H, helix. The
prediction base contained an experimentally derived thioredoxin
structure and therefore, the expected accuracy is
>70%.
Molecular models of both DLCs based on the known structure of
thioredoxin were built using SWISS-MODEL (Fig. 8). This analysis
emphasizes the structural similarities between these dynein proteins
and thioredoxin and predicts that the active-site thiols within both
DLCs are vicinal (Fig. 8, a and b).
Furthermore, this analysis predicts that the M 16,000 (but not the M
14,000) DLC may
contain a second vicinal dithiol motif (
TVCAEKC
) that is not identified simply from
sequence analysis.
Figure 8:
Molecular models of the M 14,000 (a and c) and 16,000 (b and d) DLCs were built using SWISS-MODEL. Note that the M
16,000 DLC model is truncated as the C-terminal
section is sufficiently divergent from thioredoxin that it cannot be
modeled by homology. In both space filling models (a and b), the dithiol group of the active site motif WCGPCK is shown
in yellow at the lower section of the two molecules. The M
16,000 DLC model (b) also predicts a
second vicinal dithiol within that molecule. This second dithiol
(yellow) is visible in the lower right quadrant of the model. Ribbon
diagrams of the M
14,000 and 16,000 DLC models are
shown in c and d, respectively (red,
helix;
yellow,
sheet; blue, turn; white, other). The predicted
topologies are very similar to that known for thioredoxin (Eklund et al., 1984; Jeng et al.,
1994).
One distinguishing feature of proteins containing vicinal dithiols is that they bind reversibly, but with high affinity, to metals such as arsenic, cadmium, and mercury (Hannestad et al., 1982). To test whether the two DLCs indeed contain vicinal dithiols, we attempted to purify the fusion proteins by affinity chromatography on PAO. The basic reaction scheme is shown in Fig. 9a. In the absence of a reductant, a covalent dithioarsine ring structure is formed. Due to steric constraints, the interaction of monothiols with PAO is weak and unstable (see Kalef and Gitler(1994) for further discussion of dithioarsine chemistry). The addition of high concentrations of either a mono- or dithiol reductant causes release of the vicinal dithiol-containing protein from the affinity matrix.
Figure 9:
a,
scheme describing the reaction of a DLC containing a vicinal dithiol
with 4-nitrophenylarsine oxide (based on Kalef and Gitler(1994)). A
dithioarsine ring is formed between the two sulfydryls and the
arsenical; the stability of the ring depends on the precise ring
geometry. Dithiol-containing proteins may be eluted from PAO by
reduction with mono- or dithiols. b, affinity chromatography
of the MBP-DLC14, MBP-DLC16, and MBP-lacZ fusion proteins on
phenylarsine oxide. The proteins were reduced with 1 mM dithiothreitol. The reductant was removed by dialysis and the
samples incubated with -mercaptoethanol-activated PAO resin. After
extensive washing, bound protein was eluted with 0.5 M
-mercaptoethanol. Equivalent volumes of the initial, unbound,
and eluted fractions were electrophoresed in an 8% acrylamide gel and
stained with Coomassie Blue. c, the reduced DLC fusion
proteins were digested with factor Xa to separate the DLCs from the MBP
moiety (left lanes). Following incubation with PAO, bound
protein was eluted with 0.5 M
-mercaptoethanol. Both DLCs
bind the PAO matrix whereas MBP does not.
Fusion proteins were reduced with 1 mM dithiothreitol and
the reductant (which irreversibly inactivates the arsenical moiety)
removed by extensive dialysis. In some experiments, the proteins were
purified in the presence of 1 mM -mercaptoethanol.
Subsequently, the samples were incubated with the PAO slurry for 60 min
and the matrix then extensively washed. Electrophoretic analysis of the
initial preparations and of the unbound fractions are shown in Fig. 9b (leftmost and central lanes). Proteins
bound via vicinal dithiols then were eluted by incubation of the PAO
resin with 0.5 M
-mercaptoethanol. The composition of
those eluates is shown in Fig. 9b (rightmost
lanes). Only a very small fraction of the MBP-lacZ fusion protein
bound to, and was subsequently eluted from, the PAO matrix. As MBP-lacZ
does not contain vicinal dithiols, this presumably represents either
nonspecific association with the agarose support or, possibly, a weak
interaction with PAO via monothiols. In contrast,
50% of the
MBP-DLC14 protein was purified by this procedure; somewhat lesser
amounts of MBP-DLC16 were obtained. To ensure that interaction of the
fusion proteins was due to the DLC moiety of each, both fusions were
digested with Factor Xa to separate the DLCs from MBP. The interaction
of these samples with PAO is shown in Fig. 9c. In both
cases, the separated DLC shows affinity for the PAO matrix whereas MBP
does not. Thus, the MBP-DLC fusion proteins contain vicinal dithiols
that can undergo reversible oxidation/reduction; this is a property of
the DLC, rather than the MBP, portion of the recombinant molecules.
To determine whether redox-sensitive dithiols occur within flagellar
dynein, we extracted both inner and outer arms from Chlamydomonas axonemes with 0.6 M NaCl and assessed the ability of the
various dynein species to specifically bind to PAO. Electrophoretic and
immunological analysis of the resulting fractions is shown in Fig. 10, a-c. Approximately 50% of the ,
,
and
outer arm DHCs were found to specifically bind to and be
eluted from the PAO matrix with
-mercaptoethanol. In contrast,
inner arm DHCs belonging to arms I2 and I3 (see Table I in
Piperno(1995)) and a high molecular weight flagellar membrane protein
show no affinity for the matrix. Inner arm DHCs 1a and 1b which
comprise inner arm I1 are not resolved from the outer arm DHCs in this
gel system. However, inner arm I1 also contains an IC of
140 kDa;
no component of this approximate mass in the high salt extract showed
significant affinity for PAO (not shown).
Figure 10:
a, a high salt extract of flagellar
axonemes was incubated with PAO resin. The initial and unbound
fractions and the proteins specifically eluted with 0.5 M -mercaptoethanol are shown following electrophoresis in a 4%
acrylamide, 4 M urea gel. Only the high molecular weight
region of the silver-stained gel is shown. b, samples shown in a were blotted to nitrocellulose and probed with monoclonal
antibody 12
B which specifically binds the
DHC (King et
al., 1985). Approximately 50% of the
DHC is eluted from the
PAO matrix. c, the DHCs in the high salt extract were probed
with either antibody 12
B or with 12
B plus 18
B (a
monoclonal antibody recognizing the
DHC; King et al. (1986)). The 18
B antibody also reacts with the minor band
migrating just faster than the
DHC. Therefore, this band is not
an inner arm DHC but rather the endogenous proteolytic fragment of the
DHC that has been referred to previously as ``band 11''
(Pfister et al., 1982; King and Witman,
1987).
In this report, we describe the molecular analysis of the M 14,000 and 16,000 DLCs from the outer arm of Chlamydomonas flagella. These molecules are novel thioredoxins
containing perfect copies of the redox-active site that is the major
distinguishing feature of this class of sulfhydryl oxidoreductases.
When expressed as fusion proteins, both DLCs can be purified on the
basis of a high affinity interaction with arsenic indicating that they
contain vicinal dithiol groups (i.e. thiols so arranged within
the DLC tertiary structure that they can be oxidized to form a
disulfide); these vicinal dithiols are present in outer arm but not
inner arm dyneins from Chlamydomonas flagella. This
constitutes the first demonstration of redox-sensitive polypeptides as
integral components of a microtubule-based molecular motor.
Thioredoxins act as sulfhydryl oxidoreductases (for review, see Holmgren(1985)) and have now been identified in a very wide range of organisms from bacteria to vertebrates. Members of this class of redox-active enzyme are known, or predicted based on in vitro studies, to be involved in a wide variety of regulatory activities. For example, thioredoxin derived from the E. coli host is a component of T7 DNA polymerase that is required for high processivity of the enzyme along the DNA strand (Tabor et al., 1987). Lack of this subunit also has significant consequences for the fidelity of replication (Kunkel et al., 1994). In Chlamydomonas, thioredoxin is intimately involved in the light regulated translation of chloroplast mRNA by manipulating redox potential (Danon and Mayfield, 1994). A Drosophila thioredoxin homologue, encoded at the locus deadhead, is essential for female-specific meiosis, preblastoderm mitosis, and for early embryonic development (Salz et al., 1994). The phenotypes observed suggest that the deadhead protein is not a general protein disulfide reductase but rather is required for post-translational modification of proteins involved in specific developmental processes. Thioredoxin has also been shown to interact with microtubules in vivo (Stemme et al., 1985) and to affect the rate of microtubule assembly in vitro by reducing intratubulin disulfides (Khan and Ludueña, 1991).
In almost
all cases, thioredoxins contain the active site sequence WCGPCK.
Outside of this, the overall amino acid conservation between members of
this superfamily is relatively low (30-40% identity), although
there apparently is a much greater level of similarity at the secondary
and tertiary structure levels. The dynein-associated thioredoxins we
describe here follow this same pattern. Within Chlamydomonas,
thioredoxins have been identified from both the chloroplast and cytosol
(Decottignies et al., 1991; Stein et al., 1994). ()Pairwise comparison between these proteins and the
flagellar DLCs (not shown) indicate that the Chlamydomonas DLCs are no more related to bona fide Chlamydomonas thioredoxins than they are to the chicken isozyme. This suggests
an evolutionarily ancient origin for the flagellar proteins. Note that
comparison between the chloroplast and cytosolic Chlamydomonas isozymes reveals only 33% sequence identity and emphasizes the
fact that members of this superfamily are highly divergent. Molecular
modeling, however, suggests that both DLCs share very significant
structural homology with thoredoxin.
Our identification of two DLCs
as novel thioredoxins poses the obvious question of whether these
molecules have a role in manipulating the redox state of functionally
important thiol groups on dynein. Previous studies of the effects of
sulfhydryl modifying reagents on dynein ATPase activity have indicated
that thiol groups play a pivotal role in enzymatic activity (Gibbons
and Fronk, 1979; Ogawa and Mohri, 1972; Shimizu and Kimura, 1974).
Treatment of Tetrahymena outer arm dynein with submicromolar
concentrations of N-ethylmaleimide caused a 2-3-fold
activation of ATPase activity, whereas that activity was completely and
irreversibly abolished by exposure to higher levels. In contrast, the
activity of the 14 S inner arm dyneins simply decreased following
sulfhydryl modification. The activation of outer arms was blocked by
the addition of nucleotide, leading to the hypothesis that the thiols
modified by N-ethylmaleimide and p-chloromercuribenzene sulfonate reside at or near the active
site (Shimizu and Kimura, 1974). In Chlamydomonas also,
sulfhydryl oxidation by low concentrations of dithionitrobenzoic acid
activates axonemal ATPase activity. ()
The accessory
proteins for inner arms I2 and I3 have now been identified by molecular
cloning (see LeDizet and Piperno(1995)); none are thioredoxins. As the
outer arms but not inner arms may be purified on PAO, this implies that
the redox-sensitive interaction is likely due to accessory proteins
(such as the DLCs described here) rather than to the DHCs which
obviously are related to each other in the two dynein classes and might
be expected to behave similarly in such an assay. In this regard, it is
intriguing that the DHC is readily purified on PAO. This DHC has
generally been considered to dissociate from the remainder of the outer
arm upon high salt extraction (Pfister et al., 1982) and does
not interact directly with either the M
14,000 or
16,000 DLCs. As the binding appears to be quite specific, the
DHC
may reassociate with the
dimer upon the latter's
binding to PAO. Alternatively, the
DHC might bind an additional
thioredoxin-like subunit. This DHC is known to interact with three DLCs
(Pfister et al., 1982): a M
18,000
protein that we have recently shown to be a novel homologue of
calmodulin (King and Patel-King, 1995b), and two copies of a M
22,000 polypeptide. By analogy with the
subunit (which contains the
DHC and the M
16,000 DLC), it is possible that the M
22,000 DLC also is a thioredoxin homologue.
Assuming that the thioredoxin homologues we have identified within the outer arm indeed undergo a redox interaction with other thiols in the flagellum, this necessarily implies the presence of a thioredoxin reductase and a flagellar source of reducing equivalents. Alternatively, these DLCs might function to regulate enzymatic activity while the outer dynein arm is being assembled and stored in the cytoplasm. In this scenario, the DLCs would presumably be able to tap the same stores of reducing equivalents as does the cytosolic thioredoxin.
The M 16,000 DLC contains a perfect copy of the canonical P-loop motif
for nucleotide binding sites (Walker et al., 1982), raising
the possibility that this protein binds nucleotide. There are, however,
several reasons for caution in this interpretation. Photoaffinity
labeling of purified outer arm dynein with photoactive analogues of ATP
consistently failed to show significant label associated with any of
the DLCs (King et al., 1989; Pfister et al., 1985).
Also, we note that this region of the M
16,000 DLC
in fact forms part of a relatively well conserved primary structural
element within the thioredoxin superfamily. On balance, it seems more
likely that the presence of this motif is serendipitous rather than
indicating a specific functionality.
Quantitative densitometry of
Coomassie Blue-stained gels gave a stoichiometry of 1.46-1.65
copies of the M 14,000 DLC per outer arm dynein
particle (data quoted in King and Witman(1989)). Thus it remains
unclear as to whether the outer arm contains one or two M
14,000 DLCs. Two-dimensional electrophoretic
analysis revealed two distinct M
14,000
polypeptide species (Pfister et al., 1982). Although the
sample buffer used for that analysis contained some
-mercaptoethanol, it may not have been sufficient either to
inhibit a substantial fraction of this redox-sensitive DLC from
becoming oxidized during electrophoresis or to reduce previously
oxidized protein. The observation that only
50% of outer arm
dynein could be purified on PAO supports the latter hypothesis.
Therefore, the two M
14,000 spots observed on
two-dimensional gels may represent oxidized (protein-S
) and
reduced (protein-{SH}
) forms of this DLC. Our
identification of both a single message and a single gene for this
protein also is completely consistent with this interpretation.
In
conclusion, we have identified the M 14,000 and
16,000 outer arm DLCs from Chlamydomonas flagella as novel
members of the thioredoxin superfamily. These molecules both contain a
redox-sensitive vicinal dithiol and thus may be involved in regulating
the redox state of functionally important thiol groups within dynein.
Further analysis of these DLCs and of the effects of sulfhydryl
oxidation on dynein enzymatic properties will enable us to define the
role these proteins play in outer arm function.