Download Molecular analysis of the γ heavy chain of

497
Journal of Cell Science 107, 497-506 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Molecular analysis of the γ heavy chain of Chlamydomonas flagellar outerarm dynein
Curtis G. Wilkerson, Stephen M. King* and George B. Witman†
Cell Biology Group, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, Massachusetts 01545, USA
*Present address: Department of Biochemistry, The University of Connecticut Health Center, Farmington, CT 06032, USA
†Author for correspondence
SUMMARY
We report here the complete sequence of the γ dynein heavy
chain of the outer arm of the Chlamydomonas flagellum,
and partial sequences for six other dynein heavy chains.
The γ dynein heavy chain sequence contains four P-loop
motifs, one of which is the likely hydrolytic site based on
its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein
heavy chain sequences reveals regions that are highly
conserved in all dynein heavy chains sequenced to date,
regions that are conserved only among axonemal dynein
heavy chains, and regions that are unique to individual
dynein heavy chains. The presumed hydrolytic site is
absolutely conserved among dyneins, two other P loops are
highly conserved among cytoplasmic dynein heavy chains
but not in axonemal dynein heavy chains, and the fourth P
loop is invariant in axonemal dynein heavy chains but not
INTRODUCTION
Dyneins are molecular motors involved in many different types
of microtubule-based intracellular movements, including
flagellar movement, organelle transport, mitotic spindle
placement, and possibly certain types of chromosomal
movement during cell division. The best characterized dyneins
are the outer arms of flagella, which consist of two or three
different heavy chains (depending on species) together with
several intermediate and light chains (Witman, 1992; Witman
et al., 1994), and cytoplasmic dynein, which consists of two
copies of a single heavy chain plus intermediate and light
chains (Paschal et al., 1987). In both cases the heavy chains
contain the ATP-hydrolytic site(s) and are responsible for force
production. However, axonemal dyneins differ from cytoplasmic dyneins in a number of important characteristics (Shpetner
et al., 1988). Moreover, recent cell biological and genetic
studies indicate that the different heavy chains of outer-arm
dynein have different functions (Moss et al., 1992a,b; Sakakibara et al., 1991, 1993).
An understanding of the underlying bases for the various
activities of the different dynein heavy chains (DHCs) will
require detailed structural information on the individual chains.
To date, full sequences are available only for the β DHC from
in cytoplasmic dynein. One region that is very highly
conserved in all dynein heavy chains is similar to a portion
of the ATP-sensitive microtubule-binding domain of
kinesin. Two other regions present in all dynein heavy
chains are predicted to have high α-helical content and
have a high probability of forming coiled-coil structures.
Overall, the central one-third of the γ dynein heavy chain
is most conserved whereas the N-terminal one-third is least
conserved; the fact that the latter region is divergent
between the cytoplasmic dynein heavy chain and two
different axonemal dynein heavy chains suggests that it is
involved in chain-specific functions.
Key words: dynein, ATP-binding site, microtubule-binding domain,
Chlamydomonas, flagellum
sea urchin (Gibbons et al., 1991; Ogawa, 1991), and for the
cytoplasmic DHC from Dictyostelium (Koonce et al., 1992)
and rat (Mikami et al., 1993). The midregions of both chains
contain four glycine-rich domains, which conform more or less
to the P-loop consensus sequence GXXXXGKS/T that has
been shown to be involved in nucleotide binding in
many.proteins (Walker et al., 1982; Saraste et al., 1990). In the
outer-arm β DHC, one of the P loops appears to correspond to
the V1 site, which is cleaved by UV light in the presence of
ATP and vanadate; this site is presumed to be the ATPhydrolytic site (Lee-Eiford et al., 1986). This P loop is highly
conserved between the outer-arm β and cytoplasmic DHCs.
The other P loops are less highly conserved and their function
is unknown. It has been suggested that one may correspond to
the β DHC’s single V2 site, which is cleaved by UV light in
the presence of oligomeric vanadate and the absence of ATP
(Tang and Gibbons, 1987). In addition, the β DHC contains a
fifth P-loop motif near its N terminus, which is not present in
the cytoplasmic DHC. Sequence information for additional
flagellar DHCs will be necessary to determine if any of these
sites is highly conserved among the axonemal dyneins and
therefore likely to be particularly important in the generation
of flagellar movement.
There are also regions of overall structural similarity and
498
C. G. Wilkerson, S. M. King and G. B. Witman
divergence between the outer-arm β and cytoplasmic DHCs.
There are two short regions of high α-helical content in the Cterminal third of both chains, which may be important in the
formation of tertiary or quaternary structure (Koonce et al.,
1992; Mikami et al., 1993). In contrast, the N-terminal onethird of the chains exhibit considerable sequence divergence,
leading to speculation that this region may be important in the
performance of axonemal vs cytoplasmic dynein functions
(Koonce et al., 1992; Mikami et al., 1993). Again, information
on additional axonemal DHCs will be important for determining which regions are conserved among all dyneins, which are
conserved only between axonemal dyneins, and which are
specific to individual DHCs.
Ultimately, identification of functional domains in DHCs
will benefit from in vitro mutagenesis of sites of interest,
followed by introduction of the altered gene into a cell having
a null mutation for that gene. For cytoplasmic dynein, such
experiments could be carried out most readily in yeast as long
as the null mutation is not lethal. For flagellar dynein, the
organism most amenable to such an approach is the flagellated
green alga Chlamydomonas. Thirteen different loci have been
identified that affect the structure or assembly of the outer arm
of the Chlamydomonas flagellum (Kamiya, 1988; Sakakibara
et al., 1991). Several of these loci encode structural components of the outer arm, including the α and β DHCs (Sakakibara et al., 1991, 1993). A physical map of the Chlamydomonas genome has been developed, allowing the tentative
identification of cloned genes with specific mutations by RFLP
mapping (Ranum et al., 1988). Chlamydomonas is efficiently
transformed (Kindle et al., 1989), so cloned genes can be
unequivocally identified with loci by complementation of
mutants (Diener et al., 1990; Mitchell and Kang, 1991), and
genes carrying specific mutations could be introduced into the
nuclear genome and expressed in vivo. The outer-arm dynein
of Chlamydomonas can be readily isolated and its subunits
separated from each other (Pfister et al., 1982; Pfister and
Witman, 1984), so the properties of individual dynein subunits
having specific defects can be examined in vitro.
Here we present the complete sequence of the γ DHC of
Chlamydomonas. Our approach allowed unequivocal identification of the P loop corresponding to the V1 site of this chain.
A comparison of the sequence with other available DHC
sequences confirms that this P loop is highly conserved among
all dyneins. Two other P loops are conserved among cytoplasmic DHCs but not axonemal DHCs, whereas the fourth P loop
is invariant among axonemal but not cytoplasmic DHCs. The
comparison also reveals a region that is highly conserved
among all cloned dyneins and has structural similarity to a
portion of the ATP-dependent microtubule-binding domain of
kinesin. Finally, the N-terminal one-third of the γ DHC is
divergent from that of the sea urchin β and cytoplasmic DHCs,
suggesting that this region carries out chain-specific functions
rather than cytoplasmic vs axonemal functions. These results
provide the basic structural information necessary for functional analysis of these regions using molecular genetic techniques.
MATERIALS AND METHODS
cDNA cloning
RNA was isolated from vegetative Chlamydomonas (strain 1132D−)
30 minutes after pH-induced deflagellation (Witman et al., 1972) as
follows: 10 ml of packed cells (concentrated by centrifugation at
10,000 g for 5 minutes) were vortexed to make a paste and then added
slowly and with rapid stirring to 100 ml of 5% SDS, 20 mM Tris-Cl,
pH 8.0, 20 mM EDTA and 1 mg/ml proteinase K at 50°C. The
resulting mixture was incubated at 50°C for 4 hours. One-fifth volume
of 7.5 M ammonium acetate, pH 7.5, was added and the solution was
extracted with an equal volume of phenol/chloroform (50:50). An
equal volume of isopropanol was added to precipitate the RNA. RNA
was separated from DNA by LiCl precipitation. Poly-A+ RNA was
prepared by oligo-dT chromatography. To enrich for large mRNAs
such as would encode DHCs, 100 µg of the poly-A+ RNA was sizefractionated on a methyl mercury hydroxide sucrose gradient
(Sambrook et al., 1989). Fractions of this gradient were used to
prepare a northern blot, which was subsequently probed with a γ DHC
genomic clone. Fractions containing γ DHC sequences with sizes in
the 12-14 kb range were pooled and ethanol precipitated. Doublestranded cDNA was prepared using a random 9-mer as the primer
(Gubler and Hoffman, 1984). Synthetic EcoRI adapters were added
and the cDNA was ligated to EcoRI and alkaline phosphatasedigested λgt10 DNA. The ligation mixture was packaged into λ phage
particles using Stratagene’s Gigapack Gold in vitro packaging system
(Stratagene, La Jolla, CA).
cDNA clones were isolated by screening with a genomic clone
picked from a genomic expression library using the monoclonal
antibody 12γB. The first cDNA clone isolated, pcγ1, was then used to
rescreen the size-selected cDNA library. Phage isolated in this manner
vere subcloned into pBluescript SK+. Primers from the T7 and T3
RNA polymerase promoter regions of this plasmid were used to
obtain sequence data from the ends of these clones. From the size of
a cDNA clone and the sequence of its ends the amount of overlap was
calculated. From each round, clones that contained the most new
sequence were used to rescreen the cDNA library. This was repeated
until the entire coding region of the γ DHC mRNA was obtained. A
combination of ExoIII-generated nested deletions and synthetic
oligonucleotide primers was used to obtain the sequence of both
strands of the clones shown in Fig. 3. The sequencing was accomplished using the 7-deaza dGTP Sequenase Kit from USB (United
States Biochemical, Cleveland, OH).
Peptide sequence
The γ DHC was obtained from a high-salt extract of Chlamydomonas
axonemes and purified by sucrose density gradient centrifugation
(King et al., 1986) followed by ion exchange chromatography on a
MonoQ column (Pharmacia, Piscataway, NJ) (Goodenough et al.,
1987). The sample was reduced, alkylated and subsequently cleaved
with cyanogen bromide in 70% formic acid. Peptides were separated
by reverse-phase chromatography on a Vydac C18 column. The
peptides were sequenced using an Applied Biosystems 477A protein
sequencer.
Northern and Southern blots
For northern blots, 10 µg of poly-A+ RNA were separated on
formaldehyde-containing agarose gels and blotted to nylon
membranes. Blots were probed at 65°C overnight with 32P-labeled
DNA in 7% SDS, 0.25 M disodium phosphate, pH 7.2, and 1 mM
EDTA. The blots were washed to a final stringency of 0.2× SSC and
0.1% SDS at 70°C. For Southern blots, 5 µg of genomic DNA were
digested overnight with the appropriate restriction enzyme and electrophoresed on a 0.8% agarose gel in TBE buffer. Subsequent manipulations were as described for northern blots.
PCR cloning
The following primers were used in obtaining new DHC clones
directly from the size-selected cDNA library:
(1)
CGCGAATTC(CG)GC(CTG)GG(CT)AC(CT)GG(CT)AA,
derived from the protein sequence PAGTGK;
Chlamydomonas γ dynein heavy chain
499
Fig. 1. Linear map of the γ DHC. V1, V2a, V2b,
sites cleaved by UV light in the presence of
vanadate; Box with B, region containing the
epitope recognized by monoclonal antibody 12γB.
The scale is in kDa and is based on SDS-PAGE
analysis (King and Witman, 1988).
(2) GCGCGAATTCTGCTT(CT)GA(CT)GAGTT(CT)AACCG
derived from the protein sequence CFDEFNR; and
(3) GCGCCTCGAGCC(GCA)GGGTTCAT(GA)AA derived from
the protein sequence ITMNPG.
A 1 µl sample of a phage lysate from the size-selected cDNA
library was used in a 100 µl reaction mixture containing 20 mM TrisCl, pH 8.8, 10 mM KCl, 6 mM (NH4)2SO4, 1.5 mM MgCl2, and 0.1%
Triton X-100. The reaction mixture was boiled for 5 minutes and
quenched on ice. The tube containing the reaction mixture was placed
in a Coy thermal cycler (Coy Laboratory Products, Inc., Ann Arbor,
MI) that was at 96°C. Four units of Pfu DNA polymerase were added
and 30 cycles of a three-step program (1 minute at 96°C, 1 minute at
30°C, 1 minute at 75°C) were executed. A final cycle of 10 minutes
at 75°C was performed to ensure that most of the product was doublestranded. The PCR products were extracted with phenol/chloroform
(50:50), ethanol precipitated, digested with EcoRI and XhoI, and
ligated to pBluescript SK+.
Computational methods
The GCG suite of programs (Devereux et al., 1984) was used for
sequence assembly, dot plot comparisons and protein structure predictions. The ALIMAT and FILTER programs (Vingron and Argos,
1991) were used to identify regions of similarity between dyneins and
kinesins. The PHYLIP suite of programs (Felsenstein, 1989) were
used to generate parsimony trees. PHD (Rost and Sander, 1992) was
used to predict secondary structure of the potential microtubulebinding domains. The program NEWCOILS (Lupas et al., 1991) was
used to predict regions of coiled-coil structure.
Fig. 2. (a) Northern blot prepared with 10 µg of poly-A+ RNA
isolated from Chlamydomonas cells that were regenerating their
flagella. The probe pcγ1 hybridized with a single band of ~13.5 kb.
(b) Southern blot probed with pcγ1. Single bands were detected in
Chlamydomonas DNA (5 µg) cleaved with, in order left to right,
PvuII, PstI, NotI and HindIII. Size standards (kb) are shown to the
right of each blot.
RESULTS
In previous studies of the γ DHC from Chlamydomonas, we
had mapped the location of its V1 cleavage site, which is
believed to correspond to the chain’s functional ATPhydrolytic site, as well as the locations of two V2 cleavage
sites, which are of unknown function but possibly related to
ATP binding (Fig. 1) (King and Witman, 1988). Because we
ultimately wished to unambiguously identify the V1 cleavage
site within the dynein sequence, we used monoclonal antibody
12γB, which binds within 16 kDa of the V1 cleavage site (Fig.
1) (King and Witman, 1988), to screen a genomic expression
library constructed in λgt11. A clone (p12γB3) producing an
immunoreactive fusion protein was obtained and used to select
several large cDNA clones from a λgt10 library prepared from
size-selected mRNA isolated from cells that were regenerating
flagella. One of the clones, pcγ1 (corresponding to amino acid
residues 1659-2132 in the full sequence), hybridized to an ~13
kb mRNA, which is of an appropriate size to encode a DHC
(Fig. 2A). Southern blotting of genomic DNA cleaved with
PvuII, PstI, NotI and HindIII revealed that the sequence of this
clone is present once in the Chlamydomonas genome (Fig. 2B).
Clone pcγ1 was used to select overlapping cDNA clones until
the complete coding sequence was obtained. The relationship
of these clones is shown in Fig. 3. Each of these overlapping
clones was sequenced and the derived protein sequence is
shown in Fig. 4. The deduced sequence contains 4486 amino
acids and predicts a polypeptide mass of 513 kDa. The N
terminus was identified as the first methionine following a
region with stop codons in all three reading frames; the C
terminus corresponds to the next stop codon, beyond which
there are no long open reading frames.
Confirmation that this sequence was that of the outer-arm γ
DHC was obtained by comparison with peptide sequences
obtained directly from cyanogen bromide fragments of the
purified γ DHC. In Fig. 4 the single underlines show seven
sequences (two of the sequences are contiguous) that were an
exact match for those obtained from direct protein sequencing.
These matching sequences were spread throughout virtually
the entire γ DHC.
The immunoreactive fusion protein produced by the original
genomic clone p12γB3 ended at a stop codon in an intron. The
sequence from the λgt11 EcoRI cloning site to the donor splice
site for that intron encoded only 24 amino acids, which are
shown with filled circles in Fig. 4. Therefore, the epitope recognized by monoclonal antibody 12γB is contained within the
region bound by Q1735 and Q1758.
Sixty-one amino acids (~7 kDa) towards the C terminus
from the region containing the 12γB epitope is a P-loop
500
C. G. Wilkerson, S. M. King and G. B. Witman
Fig. 3. Relationship of cDNA clones used to
generate the sequence of the γ DHC. The
numbers represent the positions of the end of
each clone in the full nucleotide sequence.
consensus sequence GPAGTGKT. Because our previous
mapping data showed that the 12γB epitope was located within
16 kDa of the V1 site, this P loop almost certainly corresponds
to the V1 cleavage site and therefore the ATP-hydrolytic site.
Three other P-loop consensus sequences are present in the γ
DHC, with the next one being 32 kDa towards the C terminus
from the first P loop. This second P loop is very near the
position mapped for the V2a site, which was estimated to be
~35 kDa from the V1 cleavage site (King and Witman, 1988).
The third and fourth P loops are 69 and 112 kDa from the first
P loop. Although biochemical studies indicated that the γ
DHC’s V2b site was located ~70 kDa from the V1 site, the discrepancy between the mass of the chain estimated by SDSPAGE (415 kDa) and the actual mass calculated from the
sequence (a discrepancy noted for all DHCs whose sequences
have been determined) precludes unequivocal identification of
the third or fourth P loop as the V2b site.
In the initial screen for γ DHC cDNAs, a clone (pcDHC5)
was isolated and sequenced and found to be similar to but
clearly different from the γ DHC sequence. Subsequently it
was learned that this new sequence is identical to that of a
portion of the α DHC of the Chlamydomonas outer arm (D.
Mitchell, SUNY Health Science Center, Syracuse, NY;
personal communication). Comparison of the new sequence
(not shown) with that of the γ DHC and the published sequence
for the sea urchin β DHC allowed us to identify regions of similarity between these three outer-arm DHCs. The sequence
around the first P loop was very highly conserved, as would be
expected if this is the ATP-hydrolytic site.
Another very highly conserved region began with the
sequence FITMNP (double underline in Fig. 4). These and
several adjacent residues are nearly invariant in all DHCs
whose sequences have been published (Fig. 5). Visual inspection suggested that this region was similar to a portion of the
microtubule-binding domain of kinesin. To test the significance of this similarity between the dyneins and kinesin, three
axonemal dyneins were compared with four members of the
kinesin superfamily using an objective alignment procedure
that relies on dot-plot filtering and is useful for comparing
distantly related sequences (Vingron and Argos, 1991). Due
to the size of the dynein peptide and the limitations of
computer memory only the regions containing the first two Ploop motifs from the DHC sequences were compared. The
technique aligned five short regions between the two groups
of proteins (Fig. 6A). As expected, the two P loops in the
dyneins were aligned with the single P loop in the kinesins.
Two additional conserved sequences (FEY and FNC) on
either side of the first P loop in the dyneins were aligned with
an invariant sequence (FAY) contiguous to the P loop in the
kinesin sequences. Since the region of similarity is short and
the surrounding regions are not conserved between dyneins
and kinesins, these aligments are unlikely to be of functional
significance. The fifth alignment was between the conserved
dynein sequence FITMNP and a similar sequence within the
ATP-sensitive microtubule-binding motif of the kinesin
superfamily. Examination of the surrounding region revealed
that the invariant glutamic acid and leucine in the kinesin
superfamily are also present in the same positions in the
axonemal dyneins (Fig. 5), although the glutamic acid is
replaced by an asparagine in the Dictyostelium cytoplasmic
DHC. Several other residues appear to represent conservative
substitutions between the dyneins and members of the kinesin
superfamily. A similar secondary structure, consisting of a β
sheet, loop and an α helix (Fig. 6B), is predicted for this region
for all of the proteins. The loop includes those residues that
exhibit the greatest variation between the dyneins and kinesins;
this may reflect lack of structural constraints in such regions.
When those residues predicted to be part of an α helix are
displayed using the GCG program HELICALWHEEL,
hydrophilic and hydrophobic residues are clustered on opposite
sides of the helix (Fig. 6C,D,E), indicating that the α helix
would be amphiphilic (Kaiser and Kézdy, 1984) in both the
dyneins and kinesins. This region also has one of the highest
surface probabilities in the entire γ DHC (not shown).
Secondary structure analysis of the entire γ DHC predicts
short regions of α helix and β sheet scattered throughout the γ
DHC (not shown). The only features that stand out are two
regions predicted to be almost exclusively α helix (residues
~3025-~3150 and ~3200-~3350); these correspond to the
similar regions previously noted for both the β DHC of sea
urchin and the cytoplasmic DHC (Koonce et al., 1992; Mikami
et al., 1993). The NEWCOILS program (Lupas et al., 1991)
predicts several short regions of high coiled-coil probability in
both the N-terminal and C-terminal thirds of the chain; two of
the latter correspond to the regions of high α-helicity noted
above. A similar coiled-coil structure is predicted for the
equivalent region of the cytoplasmic DHC (Koonce et al.,
1992; Mikami et al., 1993) and the β DHC of both sea urchin
and Chlamydomonas (Mitchell and Brown, 1993). The fact that
this pattern of predicted structure is retained throughout the
DHC family suggests that these regions are important in some
aspect of dynein assembly or function.
Pairwise comparison between the γ DHC and the sea urchin
β DHC is shown in Fig. 7B. The greatest degree of homology
is in the middle one-third of the protein, which contains the Ploop motifs and the potential microtubule-binding region. The
N-terminal one-third is the least homologous region. Fig. 7A
shows a comparison of the Chlamydomonas γ DHC with the
rat cytoplasmic DHC. Again, the middle of the chains are most
conserved and the N-terminal regions least conserved.
Using the conserved sequences from the first P loop
(PAGTGKT) and the site similar to the microtubule-binding
domain of kinesin (ITMNPG), we designed degenerate
oligonucleotides, which were used in the polymerase chain
reaction to amplify additional DHC sequences from our λgt10
library prepared from size-selected mRNA. Three new
sequences - pcr 1, pcr 2, and a sequence identical to that of a
portion of the β DHC (Mitchell and Brown, 1993) - were
Chlamydomonas γ dynein heavy chain
501
Fig. 4. Deduced amino acid sequence of the γ DHC. The seven amino acid sequences obtained from cyanogen bromide fragments of the γ DHC
are indicated by single underlines (the underline from residues 3163 to 3199 represents two cyanogen bromide fragments). The region that
includes the epitope for monoclonal antibody 12γB is indicated by filled circles. The four P-loop motifs are indicated by asterisks. The potential
microtubule-binding region is indicated by the double underline.
502
C. G. Wilkerson, S. M. King and G. B. Witman
*
*
*
αDHC
βDHC
βS.U.
γDHC
cytoDHC
kinesin
ncd
kar3
bimc
cut7
hydrophobic
polar
charged
glycine
obtained (Fig. 8A). An additional sequence, pcr 3, was
obtained from the same library using degenerate oligonucleotides based on the conserved sequences CFDEFNR and
ITMNPG. A fifth new sequence, pcr 4, was obtained from a
genomic library using the latter primers. Our success in
obtaining new DHC sequences indicates that the FITMNP
sequence is a common one in DHCs. An alignment of all the
Chlamydomonas DHC sequences is shown in Fig. 8A. Because
the Pfu polymerase used in the polymerase chain reaction has
a 3′ to 5′ exonuclease activity, the primer can be digested and
then extended to reveal the sequence of the DNA under the
original primer. This has occurred in pcr 1 where Y has
replaced F in the FITMNP sequence, and in pcr 4 where V has
replaced I in the same sequence. Both of these replacements
should preserve the secondary structure and perhaps the
function of this region.
Fig. 8B shows an unrooted parsimony tree of the α, β and γ
outer-arm DHCs from Chlamydomonas, pcr 1 and pcr 2 from
Chlamydomonas, the β outer-arm DHC from sea urchin, and
three cytoplasmic DHCs. The tree is divided into three major
branches representing: (1) the yeast cytoplasmic DHC; (2) the
rat and Dictyostelium cytoplasmic DHCs; and (3) the outerarm DHCs plus pcr 1 and pcr 2. Within the latter branch, pcr
1 and pcr 2 are most closely associated with each other. The
fact that pcr 1 and pcr 2 are grouped with known axonemal
DHCs further suggests that they represent inner-arm DHCs. As
was expected from their biochemical similarities (Pfister and
Witman, 1984), the Chlamydomonas β DHC and the sea urchin
β DHC are grouped together.
DISCUSSION
In this study we have obtained the complete sequence of the γ
heavy chain of Chlamydomonas outer-arm dynein, and the
partial sequence of the α heavy chain of the same dynein. The
γ DHC sequence, like that of the cytoplasmic DHC (Koonce
et al., 1992; Mikami et al., 1993) and the β DHC from Chlamydomonas (Mitchell and Brown, 1993), lacks the P-loop motif
present near the N terminus of the sea urchin β DHC (Gibbons
Fig. 5. Alignment of the potential microtubulebinding domains of the Chlamydomonas outer-arm
α DHC (αDHC), the Chlamydomonas outer-arm β
DHC (βDHC) (Mitchell and Brown, 1993), the sea
urchin outer-arm β DHC (βS.U.) (Gibbons et al.,
1991; Ogawa, 1991), the Chlamydomonas outerarm γ DHC (γDHC), the Dictyostelium
cytoplasmic DHC (cytoDHC) (Koonce et al.,
1992), Drosophila kinesin (kinesin) (Yang et al.,
1989), Drosophila ncd (ncd) (Endow et al., 1990;
McDonald and Goldstein, 1990), Saccharomyces
KAR3 (kar3) (Meluh and Rose, 1990), Aspergillus
bimC (bimc) (Enos and Morris, 1990), and
Schizosaccharomyces cut7 (cut7) (Hagan and
Yanagida, 1990). Residues are grouped according
to Branden and Tooze (1991). The asterisks
indicate amino acids that vary in at most one
sequence.
et al., 1991; Ogawa, 1991). Therefore, this P loop is unlikely
to be necessary for generic dynein function. The four
remaining P loops (here termed P1-P4) in the midregion of the
chain are present in all DHCs sequenced to date (Fig. 9).
P1 is absolutely conserved in all available DHC sequences.
This P loop was previously proposed to correspond to the ATPhydrolytic site of the sea urchin β DHC because both it and the
β DHC’s V1 site were located ~70 kDa C-terminal to a tryptic
cut site (Gibbons et al., 1991; Ogawa, 1991). Our finding that
Fig. 6. (A) Filtered dot plots of three axonemal DHCs and four
members of the kinesin superfamily. The program ALIMAT
(Vingron and Argos, 1991) was used to generate pairwise
comparisons between amino acids 1660 through 2130 of the γ DHC
and comparable regions of the Chlamydomonas α DHC and the sea
urchin β DHC. This region included the first and second P-loop
motifs of these proteins. Pairwise comparisons were also generated
between these partial sequences and the full sequences of Drosophila
kinesin, Aspergillus bimC, Saccharomyces KAR3, and Drosophila
ncd. Finally, pairwise comparisons were generated among the 4
members of the kinesin superfamily. Comparisons between dynein
pairs and between kinesin pairs were performed using a cutoff of six
standard deviations; comparisons between a dynein and a kinesin
were performed with the cutoff at three standard deviations. The
program FILTER was then used to extract the consistently alignable
regions of the dot plots. All plots contained the same points after
filtration. The plot shown is that for the Chlamydomonas γ DHC and
Aspergillus bimC. (B) Secondary structure prediction using the
program PHD (Rost and Sander, 1992). The line labeled γ is the
amino acid sequence of the potential microtubule-binding domain of
the Chlamydomonas outer-arm γ DHC. The lines labeled 1-6 are the
secondary structure predictions for the Chlamydomonas γ DHC, the
sea urchin β DHC, the Dictyostelium cytoplasmic DHC, Aspergillus
bimC, Saccharomyces KAR3, and Drosophila kinesin, respectively.
E, residues predicted to be part of a β sheet; H, residues predicted to
be part of an α helix; −, residues predicted to be part of a loop
region. (C-E) The GCG program HELICALWHEEL was used to
display a 10-amino acid segment predicted to be α helical in the
putative microtubule-binding domains of the Chlamydomonas γ
DHC, Drosophila kinesin, and the Dictyostelium cytoplasmic DHC.
The program illustrates how the amino acids would be positioned if
they were in an α helix. In each case, the hydrophobic residues
(boxed) are clustered on one side of the helix.
Chlamydomonas γ dynein heavy chain
in the γ DHC this P loop and the V1 site are both located within
16 kDa of the epitope recognized by monoclonal antibody
12γB provides even stronger evidence that P1 and the V1 site
are identical. This assignment is consistent with detailed biochemical evidence that UV-induced vanadate-dependent photocleavage occurs in the P loops of both myosin and adenylate
kinase (Cremo et al., 1989, 1992).
P2 and P3 are highly conserved in the cytoplasmic DHC
sequences reported to date. However, neither of these P loops
are highly conserved in axonemal DHCs. In P2 of the Chlamydomonas α DHC, an R is substituted for the K in the consensus
sequence. Since the K is important for the conformation of the
P loop and may interact directly with the β- and γ-phosphates
of the bound nucleotide (Saraste et al., 1990), this P loop may
not function in ATP hydrolysis. Moreover, the region around
this P loop is not particularly well conserved, suggesting that
the region does not have a critical function in all DHCs. In P3
of the Chlamydomonas γ DHC, an A is substituted for the
second G of the consensus sequence, suggesting that this P
loop also may be incapable of hydrolysing ATP.
503
In contrast to P2 and P3, P4 is absolutely invariant in the
axonemal DHCs sequenced to date. Interestingly, this
sequence differs from the generally accepted P-loop motif in
that a Q has replaced the consensus S or T. Ogawa (1991) noted
that the P4 sequence resembles the ATP-binding sequence of
some adenylate kinases. However, it does not conform to the
consensus sequence (GXPGXGKGT) for that family of
proteins (Saraste et al., 1990). Therefore, it is unclear whether
this P loop could be involved in ATP binding or hydrolysis.
Nevertheless, the fact that P4 is so highly conserved in
different axonemal DHCs, and in the same axonemal DHC
from different species, indicates that it must have a very
important role in outer-arm DHC function. Consistent with
this, the region around this P loop is also very highly conserved
between axonemal dyneins.
The cytoplasmic DHC differs from the axonemal DHCs in
that all four of its P loops conform to the canonical consensus
sequence. Moreover, in the cytoplasmic DHC, P1 and P3 are
identical between species, with the regions around P1, P3 and
P4 being highly conserved. This is in contrast to the situation
for the axonemal dyneins, where P1 and P4 are invariant and
the regions around those two sites are much more highly
conserved than the regions around the other two sites. Analysis
of the functions of the individual P loops will be necessary to
determine if these differences are responsible for some of the
distinctive characteristics of axonemal vs cytoplasmic dynein.
A
B
Fig. 7. Dot-matrix analysis of (A) the Chlamydomonas γ DHC and
the Dictyostelium cytoplasmic DHC and (B) the Chlamydomonas γ
DHC and the sea urchin β DHC using the GCG program COMPARE
with a window size of 50 and a stringency of 30.
504
C. G. Wilkerson, S. M. King and G. B. Witman
A
B
Fig. 8. (A) Alignment of
Chlamydomonas dynein heavy
chain PCR products. Seven
different PCR products have
been isolated and are shown
aligned by the GCG program
BESTFIT. Asterisks indicate
amino acids that are invariant in
these sequences. Dashed lines
represent gaps inserted to align
the sequences. (B) Unrooted
parsimony tree for two PCR
products and the corresponding
regions of the Chlamydomonas
α, β, and γ DHCs, the sea
urchin β DHC and the yeast (Li
et al., 1993), rat and
Dictyostelium cytoplasmic
DHCs. One hundred randomly
resampled alignments generated
using the bootstrap algorithm of
the program SEQBOOT were
analyzed using the program
PROTPARS. The average of
the results was determined with
the program CONSENSE. The
program DRAWTREE was
used to generate the unrooted
tree diagram. The number at
each node represents the
percentage of bootstrapped data
sets that produced identical
branch nodes.
Fig. 9. Sequence alignment of the four P loops of the
Chlamydomonas α, β and γ DHCs, the sea urchin β
DHC, and the cytoplasmic DHC of rat and
Dictyostelium. Asterisks indicate amino acids that
are invariant in these sequences. The data for P loops 3 and 4 of the Chlamydomonas α DHC and P loops 1-4 of the Chlamydomonas β DHC
are courtesy of Dr D. Mitchell (SUNY Health Science Center, Syracuse, NY).
Earlier studies comparing cytoplasmic and axonemal DHC
sequences noted that the N-terminal one-third of the cytoplasmic DHC was conserved between species but had little similarity to the N-terminal one-third of the sea urchin β DHC
(Koonce et al., 1992; Mikami et al., 1993). It was suggested
that this region of the DHC specified functions unique to cytoplasmic vs axonemal dyneins. In light of our finding that the
outer-arm β and γ DHCs are also most divergent in this region,
it seems likely that the region carries out functions that are
chain-specific rather than class-specific. One such function
would be chain:chain interaction. The region probably allows
each DHC to bind a different set of dynein subunits and to
interact with different DHCs. In support of this, in the Chlamydomonas mutant oda4-s7, a truncated β DHC lacking the Cterminal two-thirds of the molecule is still able to bind dynein
intermediate and light chains, and to associate with the α and
γ DHCs (Sakakibara et al., 1993). Therefore, the N-terminal
one-third of the molecule must contain the binding sites for
these interactions. Because cytoplasmic dynein contains two
identical DHCs that interact with each other in a parallel orientation, it might be expected that the rate of evolutionary
change in the region of interaction would be slower than in the
equivalent regions of axonemal dynein, where a change in one
DHC could be compensated for by a change in another DHC.
We have also identified a region that is very highly
conserved among DHCs and that has some similarity to the
ATP-dependent microtubule-binding domain of kinesin (Yang
et al., 1989; Stewart et al., 1993). Although there are substantial amino acid differences between the dyneins and kinesins
in this region, most of the amino acids that are highly
conserved in the kinesins are also present in the dyneins, and
many of the other amino acids appear to be conservative substitutions. Moreover, the predicted secondary structure of this
region - a beta sheet, loop, and amphiphilic α helix - is very
Chlamydomonas γ dynein heavy chain
similar in both the dyneins and kinesins. Finally, this region is
in the same direction and at approximately the same distance
from the ATP-hydrolytic site in all DHCs and kinesins
examined. Although one cannot conclude from such comparisons alone that this region is involved in ATP-dependent
microtubule binding in dynein, the fact that the region is so
highly conserved among DHCs indicates that it has an
important role in a generic dynein function. It would be of considerable interest to substitute this region of the dynein
sequence for the equivalent region of kinesin clones and
determine if the bacterially expressed protein is still able to
bind and move microtubules (Yang et al., 1990). The kinesin
heavy chain generates force in the opposite direction from
dynein and ncd, so such chimeric molecules might reveal how
the polarity of force generation is determined.
Using the polymerase chain reaction and primers based on
highly conserved regions of the known DHCs, we have
obtained PCR and subsequently cDNA clones for several additional DHCs. Two of these (pcr 1 and pcr 2) group with known
axonemal DHCs in a parsimony tree, suggesting that they
represent inner arm dyneins. Recent biochemical studies
indicate that the Chlamydomonas axoneme may contain as
many as eight different inner arm DHCs (Kagami and Kamiya,
1992), and it has been proposed that some of these inner arm
DHCs are differentially distributed along the axoneme
(Piperno and Ramanis, 1991). However, the lack of specific
antibodies for distinguishing between the different inner-arm
dyneins has made both biochemical and localization studies
difficult. It now should be possible to generate antibodies to
chain-specific peptides deduced from these PCR sequences,
and to use these antibodies to determine where the corresponding DHCs are located in the axoneme.
In collaboration with Drs C. Silflow and P. Lefebvre (University of Minnesota), we have used restriction fragment length
polymorphism (RFLP) analysis to map the γ DHC gene within
the Chlamydomonas genome. Preliminary results indicate that
the gene encoding the γ DHC is located on linkage group XI
within a few map units of the locus defined by the mutation
pf28/oda2, which results in loss of the outer dynein arm
(Mitchell and Rosenbaum, 1985; Kamiya, 1988). Therefore,
we have tentatively assigned the γ DHC gene to the pf28/oda2
locus. We are currently trying to confirm this assignment by
rescuing the pf28/oda2 mutant by transformation with the
wild-type γ DHC gene. If pf28/oda2 is a null mutant for the γ
DHC, it should be possible to carry out in vitro mutagenesis
of the P loops and the potential microtubule-binding domain,
and then introduce the altered gene into the mutant by transformation to determine how modification of the sites affects
the functioning of the dynein both in vivo and in vitro.
We thank Dr D. Mitchell for sending us his sequence of the
Chlamydomonas β DHC prior to publication. This work was
supported by NIH grant GM30626 and a grant from the W. M. Keck
Foundation for the WFEB Protein Chemistry Facility.
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(Received 11 October 1993 - Accepted 13 December 1993)