Báo cáo khoa học: A 49 kDa microtubule cross-linking protein from Artemia franciscana is a coenzyme A-transferase pdf

A 49 kDa microtubule cross-linking protein from
Artemia franciscana
is a coenzyme A-transferase
Mindy M. Oulton
1
, Reinout Amons
2
, Ping Liang
3
and Thomas H. MacRae
1
1
Department of Biology, Dalhousie University, Halifax, NS, Canada;
2
Department of Molecular Cell Biology, Sylvius Laboratory,
Leiden, the Netherlands;
3
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA
Embryos and larvae of the brine shrimp, Artemia francis-
cana, were shown previously to possess a protein, now
termed p49, which cross-links microtubules in vitro.
Molecular characteristics of p49 were described, but the
protein’s identity and its role in the cell were not determined.
Degenerate oligonucleotide primers designed on the basis of
peptide sequence obtained by Edman degradation during
this studywere usedto generate p49 cDNAs byRT-PCR and
these were cloned and sequenced. Comparison with archived
sequences revealed that the deduced amino acid sequence of
p49 resembled the Drosophila gene product CG7920, as well
as related proteins encoded in the genomes of Anopheles and
Caenorhabditis. Similar proteins exist in several bacteria but

no evident homologues were found in vertebrates and plants,
and only very distant homologues resided in yeast. When
evolutionary relationships were compared, p49 and the
homologues from Drosophila, Anopheles and Caenorhabditis
formed a distinct subcluster within phylogenetic trees.
Additionally, the predicted secondary structures of p49,
4-hydroxybutyrate CoA-transferase from Clostridium ami-
nobutyricum and glutaconate CoA-transferase from Acid-
aminococcus fermentans were similar and the enzymes may
possess related catalytic mechanisms. The purified Artemia
protein exhibited 4-hydroxybutyrate CoA-transferase acti-
vity, thereby establishing p49 as the first crustacean CoA-
transferase to be characterized. Probing of Western blots
with an antibody against p49 revealed a cross-reactive pro-
tein in Drosophila that associated with microtubules, but to
a lesser extent than did p49 from Artemia.
Keywords: CoA-transferase; microtubule cross-linking pro-
tein; Artemia franciscana.
Cell shape and polarity are regulated by microtubules,
which serve as key structural elements of mitosis and
provide tracks for intracellular transport. Microtubules are
polar structures [1] and most are unstable, undergoing
assembly and disassembly predominately from the plus end
by a process called dynamic instability [2,3]. The formation
of microtubules is modulated by tubulin isotypes [4] and
microtubule-associated proteins (MAPs), a heterogeneous
family defined simply by coassembly with tubulin and
adherence to microtubules. Structural MAPs stabilize
microtubules and modulate dynamic instability [5–8],
whereas dynamic MAPs, or molecular motors, hydrolyse

ATP as a prerequisite for vectorial movement of cells and
their components [9]. Molecular motors are important
during mitosis [10], and the Kin1 kinesin subfamily mediates
ATP-dependent microtubule depolymerization [2,11].
Many proteins in addition to those just mentioned associate
with microtubules in vivo and in vitro. For example, enzymes
involved in tubulin post-translational processing [12,13] and
glycolysis [14,15], affiliate with microtubules. Rho family
GTPases, their kinases, and Ras GTPases interact with
microtubules, seemingly as integral components of cell
signaling mechanisms [16,17].
Incubation of cell-free extracts from the brine shrimp
Artemia franciscana with paclitaxel (taxol) yielded cross-
linked microtubules [18], and in this context, a 49 kDa
microtubule interacting protein was isolated [19]. The
protein, herein referred to as p49, failed to react with
antibodies to structural MAPs such as MAP2 and tau, was
moderately heat resistant and consisted of several develop-
mentally invariant isoforms [19–21]. GTP, ATP and their
analogues, at final concentrations of 10 m
M
, disrupted p49
binding to microtubules and weak microtubule-independent
nucleotidase activity was detected [20]. In this study, p49
was sequenced and its molecular properties examined,
showing that the protein is an acetyl CoA-transferase, the
first described for a crustacean. A related protein was
observed in Drosophila.
Experimental procedures
Preparation of

Artemia
and
Drosophila
cell-free extracts
Sixty grams (dry weight) of Artemia franciscana cysts
(Sanders Brine Shrimp Co. or INVE Aquaculture, Inc.,
Ogden, UT, USA) were hydrated in distilled water at 4 °C
for a minimum of 5 h, collected on a Buchner funnel and
washed with cold distilled water. The embryos were divided
among six 2 L flasks, each containing 1000 mL of Hatch
Medium [22] and incubated with shaking at 220 r.p.m. for
either 6 or 12 h. The cysts were suction-filtered on a
Correspondence to T. H. MacRae, Department of Biology,
Dalhousie University, Halifax, NS, B3H 4J1, Canada.
Fax: + 1 902 4943736; Tel.: + 1 902 4946525;
E-mail:
Abbreviation: MAP, microtubule-associated protein.
(Received 3 September 2003, revised 20 October 2003,
accepted 24 October 2003)
Eur. J. Biochem. 270, 4962–4972 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03898.x
Buchner funnel, washed with cold distilled water followed by
Pipes buffer [100 m
M
1,4-piperazine-N,N¢-bis(2-ethanesulf-
onic acid) as free acid, 1 m
M
EGTA, 1 m
M
MgCl
2

, pH 6.5],
and homogenized with a Retsch motorized mortar and pestle
(Brinkman Instruments Canada, Rexdale, ON, Canada) in
Pipes buffer for 5 min in three 60 g (wet weight) batches.
Cysts developed for 12 h were homogenized as described
except that 200 lg of each proteolytic inhibitor, leupeptin,
soybean trypsin inhibitor, pepstatin A and phenylmethyl-
sulfonyl fluoride (Sigma Chemical Co., St. Louis, MO,
USA), were added to 60 g (wet weight) of cysts during
homogenization. The homogenate was centrifuged at
40 000 g for 30 min at 4 °C. The upper two-thirds of each
supernatant was removed, placed in a fresh tube, and
centrifuged under the same conditions for 20 min. The
supernatant was either used immediately or frozen at )70 °C.
Drosophila melanogaster embryos developed for 12 h
were harvested from grape juice agar plates supplemented
with yeast paste and washed with Ringer’s solution (10 m
M
Tris/HCl, 182 m
M
KCl, 46 m
M
NaCl, 3 m
M
CaCl
2
,pH,
7.2). Larvae were collected from Drosophila culture medium
plates after 12–36 h of incubation and washed with Ringer’s
solution. Pupae and adults were collected from culture

bottles and rinsed with Ringers solution. All Drosophila
samples were homogenized in Dounce homogenizers in
Pipes buffer containing leupeptin, soybean trypsin inhibitor
and pepstatin A, each at a final concentration of
0.004 lgÆmL
)1
and phenylmethylsulfonyl fluoride at
0.008 lgÆmL
)1
. The homogenates were centrifuged at
16 000 g for 10 min at 4 °C and the supernatants trans-
ferred to fresh tubes before centrifugation at 40 000 g for
20 min at 4 °C. The supernatants were placed in fresh tubes,
recentrifuged at 40 000 g for 20 min and these supernatants
were either used immediately or stored at )70 °C. Droso-
phila were obtained from Vett Lloyd, Department of
Biology, Dalhousie University, Halifax, NS, Canada.
Purification of p49, gel electrophoresis and protein
immunodetection
p49 was prepared from Artemia MAPs as described
previously [19] with paclitaxel [23] generously provided by
the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Bethesda, MD, USA.
Protein concentrations were determined by the method of
Lowry et al. [24] using bovine serum albumin (Sigma) as
standard. To assess p49 purity, protein fractions were
electrophoresed in 12.5% (w/v) SDS/polyacrylamide gels
overlaid with 4% (w/v) stacking gels [25]. Gels were stained
with Coomassie blue and protein size was determined by

comparison to molecular weight markers (Bio-Rad Labor-
atories, Mississauga, ON, Canada).
Proteins in SDS/polyacrylamide gels were transferred
overnight to nitrocellulose (Bio-Rad) at 100 mA, and
membranes were stained with 0.2% (w/v) Ponceau S (Sigma)
in 3% (w/v) trichloroacetic acid to verify transfer. Mem-
branes were blocked by incubation with gentle shaking in
5% (w/v) Carnation skimmed milk powder in TBS/Tween
[10 m
M
Tris/HCl, 140 m
M
NaCl, 0.1% (v/v) Tween 20,
pH 7.4] for 45 min, then incubated in primary antibody
diluted in TBS/Tween for 15 min. Polyclonal antibodies
raised in rabbits included an anti-peptide antibody to the
N-terminal 15 residues of p49 [19] and an antibody prepared
to native p49 during this study. Rabbits were obtained from
Charles River Canada (St. Constant, QC, Canada) and
cared for in accordance with guidelines in ÔGuide to the Care
and Use of Experimental AnimalsÕ available from the
Canadian Council on Animal Care. The blots were washed
twice for 3 min each in TBS/Tween and HST (10 m
M
Tris/
HCl, 1
M
NaCl, 0.5% Tween 20, pH 7.4) followed by 3 min
in TBS/Tween. The membranes were incubated for 15 min
with goat anti-(rabbit IgG) IgG horseradish-peroxidase

conjugated secondary antibody (Jackson ImmunoResearch
Laboratories, Inc., Bio/Can Scientific, Mississauga, Ontario,
Canada). The enhanced chemiluminescence technique
(PerkinElmer Life Sciences, Boston, MA, USA) was used
for detection of antibody-reactive proteins.
Co-assembly of
Artemia
p49 and tubulin
Purified p49 at 0.5–1.0 lgÆmL
)1
and Artemia tubulin at
1.0 lgÆmL
)1
[26], were incubated for 30 min at 37 °Cwith
1.8 m
M
GTP and 10 l
M
paclitaxel in final volumes of either
50 or 100 lL. Assembly conditions were the same for Artemia
cell-free extract which was used at a final concentration of
approximately 35 mgÆmL
)1
. Assembly mixtures were centri-
fuged at 40 000 g for 30 min at 22 °C after overlaying on
either 500 or 1000 lL 15% sucrose cushions in Pipes buffer.
Pellets were rinsed gently with Pipes buffer at 37 °C,
resuspended in 18 lL of the same buffer, and examined for
tubulin and p49 by SDS/polyacrylamide gel electrophoresis
followed by Western blotting. Microtubule cross-linking was

detected by transmission electron microscopy with 5 lL
samples of assembly mixtures fixed in 4% (v/v) glutaralde-
hyde applied to formvar-covered, carbon-coated, 200-mesh
copper grids for 1 min. Excess liquid was removed by blotting
with filter paper and grids were negatively stained with 1%
(w/v) uranyl acetate for 30 s. Specimens were examined in a
Philips Tecnai transmission electron microscope and images
were captured with
ANALYSIS
, version 2.1.
Detection of a
Drosophila
p49 analogue
Drosophila cell-free extract was examined for p49 analogues
by electrophoresis in SDS polyacrylamide gels and immu-
noprobing of Western blots using procedures described for
Artemia. Drosophila tubulin was induced to assemble by
paclitaxel addition to cell-free extracts and microtubules
were collected by centrifugation through sucrose cushions.
Pellets were rinsed, resuspended in Pipes buffer and
processed for SDS/PAGE, immunoprobing of Western
blots and electron microscopy as described earlier.
4-Hydroxybutyrate CoA-transferase assay
The presence of 4-hydroxybutyrate CoA-transferase activity
was detected by formation of thiophenolate anion [27].
Reaction mixtures of 1.0 mL contained 100 m
M
KH
2
PO

4
,
pH 7.0, 200 m
M
sodium acetate, 1 m
M
oxaloacetic acid,
1m
M
5,5¢-dithiobis(2-nitrobenzoate), 0.1 m
M
butyryl
CoA, 0.5 U citrate synthase (Sigma) and p49. Absorbance
increaseat412 nmwasmeasuredat20 °Candenzymeactivity
is reported in arbitrary units as DA
412
min
)1
Æmg protein
)1
.
Ó FEBS 2003 Artemia CoA-transferase (Eur. J. Biochem. 270) 4963
Sequencing of p49 peptides
For sequencing by Edman degradation purified p49 was
electrophoresed in 12.5% (w/v) SDS/polyacrylamide gels
overlain with 4% (w/v) stacking gels and transferred to
Immobilon poly(vinylidene difluoride) membrane (Milli-
pore, Mississauga, ON, Canada) at 100 V for 1 h in 10 m
M
3-(cyclohexylamino)-1-propane-sulfonic acid (CAPS) buffer,

pH 10.5 containing 20% (v/v) methanol. The membranes
were stained with Coomassie blue for 2 min, destained with
90% methanol/10% acetic acid (v/v), and rinsed with
deionized water before drying. Edman sequencing was
performed in a Hewlett Packard Model G1005A protein
sequencer using the
ROUTINE
3.1 PVDF program and
analysis of PTH amino acids on line with a Hewlett Packard
Model 1100 HPLC. When the sequence became difficult to
read the sequencing cartridge contents were treated in situ
with acetic anhydride to block partially degraded proteins at
the amino terminus [28]. The acetylated proteins were cleaved
at methionine residues with BrCN, excess reagent and
reaction products were removed, and sequencing resumed.
Cloning and sequencing of p49 cDNA
Approximately 1.5 g (wet weight) of Artemia nauplii were
homogenized in 0.5 mL TRIzolÒ reagent (Life Technol-
ogies, Boston, MA, USA) for 1 min in a glass homogenizer
and RNA was recovered. Polyadenylated mRNA was
purified with an mRNA Purification Kit (Amersham
Fig. 1. Cloning of p49 cDNA. p49 cDNA was cloned in three sections
called clones p49-1, p49-2 and p49-3. Primer locations and names are
indicated on the schematic and arrows indicate the 5¢ to 3¢ direction of
each primer. Primer sequences are listed.
Fig. 2. Purification of p49. Protein fractions obtained during p49 purification were electrophoresed in 12.5% (w/v) SDS/polyacrylamide gels and
either stained with Coomassie blue (A) or blotted to nitrocellulose and immunostained with anti-(native p49) by enhanced chemiluminescence (B).
(A) Lane 1, 60 lgofArtemia cell-free extract; lane 2, 35 lgofMAPs;lane3,35lg of heated MAPs; lane 4, 2.5 lgof0.2
M
NaCl fraction from P11;

lane 5, 1 lg of purified p49. (B) Lanes 1–3 each received 10 lgofprotein;lane4,2.5lgofthe0.2
M
NaCl fraction from P11; lane 5, 1 lgofpurified
p49. Because low yields for the final two purification steps precluded accurate determination, protein amounts reported for lanes 4 and 5 were
estimates based on staining intensity of bands. Lane M, molecular mass markers · 10
)3
; arrows, p49; arrowhead, cross-reactive protein. (C)
Purified tubulin was assembled in the presence of p49, microtubules were collected by centrifugation, resuspended in Pipes buffer, applied to grids
and negatively stained with 1% (w/v) uranyl acetate. Arrows, p49 microtubule cross-linking particles. The bar represents 200 nm.
4964 M. M. Oulton et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Biosciences, Baie d’Urfe, QC, Canada) and used with
Ready-To-Go
TM
RT-PCR beads (Amersham Biosciences)
to synthesize cDNA and amplify p49 cDNA. Fifty-micro-
liter reaction mixtures contained 900 ng of poly(A)
+
mRNA, 0.5 lgofoligod(T)
18
primer, 2 l
M
of each gene
specific primer and 2.0 m
M
MgCl
2
. The mixtures were
Fig. 3. Nucleotide and amino acid sequences of p49. The nucleotide sequence of p49 was obtained as described in Experimental procedures, and from
this the amino acid sequence was deduced. Amino acid residues determined by Edman degradation are in bold. The termination codon (TAA) is
underlined and the polyadenylation signal is boxed. Putative phosphorylation motifs are underlined in the deduced amino acid sequence. The first

six amino acid residues, revealed only by peptide sequencing, are in brackets.
Ó FEBS 2003 Artemia CoA-transferase (Eur. J. Biochem. 270) 4965
topped with 50 lL of mineral oil and reverse transcription
was carried out at 42 °Cfor30minfollowedby5minat
95 °C. PCR amplification was performed immediately as
follows: 95 °Cfor45s,48°C for 60 s, 72 °Cfor90s,
sequentially for 35 cycles, and 10 min at 72 °C. Samples of
reaction mixtures were diluted 1 : 100 in RNase-free water
and amplified by nested PCR. Clone p49-1 was amplified by
using degenerate primers 1a and 2 designed from peptide
data, followed by seminested PCR using primers 1b and 2
(Fig. 1). The remaining 3¢-p49 cDNA sequence, represented
by Clone p49-2, was amplified with gene-specific primer 3a
and an adaptor-coupled oligo d(T) primer, 5a, followed by
seminested PCR using primer 3b and adapter primer 5b
(Fig. 1). Clone p49-3 was amplified with primers 4a and 5a,
followed by seminested PCR with primers 4b and adapter
primer 5b (Fig. 1). PCR products were sized in 1% (w/v)
agarose gels in TAE buffer (40 m
M
Tris/HCl, 20 m
M
acetic
acid, 1 m
M
EDTA, pH 8.5) with a 1000 bp marker set
(MBI Fermentas, Burlington, Ontario, Canada), then
cloned with the pGEMÒ-T Easy Vector Systems Kit
(Promega, Madison, WI, USA) and Escherichia coli JM109.
Plasmid DNA was isolated using WizardÒ Plus SV

Minipreps DNA Purification System (Promega), followed
by DNA digestion with EcoR1 and agarose gel electro-
phoresis to confirm insert size. Cloned DNA was sequenced
at least twice in both directions at the DNA Sequencing
Facility, Centre for Applied Genomics, Hospital for Sick
Children, Toronto, ON, Canada.
Sequence analysis of p49
Sequences obtained for p49 were compared to archived
sequences at the National Center of Biological Information
(NCBI) using Basic Local Alignment Search Tool (BLAST)
[29], including
BLASTX
for DNA and
BLASTP
for proteins.
Motif searches were performed with PROSITE database
[30] using
PREDICT PROTEIN
at the EMBL website, Heidel-
berg, Germany. Secondary structure predictions were made
with Prof_ s, accessible via
PREDICT PROTEIN
. Multiple
alignments were performed with
CLUSTALX
[31] with output
files formatted by
BOXSHADE
( />software/BOX_form.html). To examine evolutionary rela-
tionships, all 46 sequences in fasta format from the NCBI

nonredundant protein database showing a high similarity
to p49 were collected using the arbitrary cutoffs of
E-value ¼ 1e)50, and greater that 35% identity based on
the observed distinct classes of similarity among all matches.
Twenty-nine sequences remained after eliminating redund-
ant entries representing partial sequences and splicing
variants of the same gene. Sequence alignments and
neighbor-joining trees were generated with
CLUSTALX
using
the Gonnet protein comparison matrix and 1000 bootstrap
trials. The tree was viewed and printed with
TREEVIEW
[32].
Results
Purification of p49
Purification of p49 to apparent homogeneity was
obtained from Artemia cysts developed for either 6 or
12 h. Briefly, Artemia tubulin and MAPs, the latter
defined by their ability to coassemble in vitro with
tubulin [19], were induced to form microtubules by
addition of paclitaxel and GTP to cyst cell-free extracts.
After centrifugation of assembly mixtures through
sucrose cushions, MAPs were recovered by incubating
microtubule pellets in Pipes buffer containing 0.5
M
NaCl. Enrichment of p49 was by heating MAPs to
50 °C for 5 min followed by centrifugation to remove
precipitated proteins, chromatography on phosphocellu-
lose P11 and (NH

4
)
2
SO
4
fractionation. Only one weakly
staining band of 49 kDa was observed in Coomassie blue
stained gels after (NH
4
)
2
SO
4
fractionation (Fig. 2A) and
it interacted strongly with an antibody to native p49 even
though there was almost no reaction in equivalent
positions in lanes containing cell-free extract (Fig. 2B).
A higher molecular mass protein of unknown identity
that reacted with anti-p49 was observed routinely on
Western blots containing cell-free extract, MAPs and
heated MAPs, but only occasionally in more purified
fractions. Approximately 0.2 mg of pure p49 was
obtained from 2970 mg of starting protein. When the
49 kDa protein was incubated with Artemia tubulin at
37 °C in the presence of GTP and taxol, the resulting
microtubules were cross-linked by irregularly shaped,
randomly distributed particles (Fig. 2C).
Sequencing of p49
Fifty eight cycles of Edman degradation were performed on
p49 blotted to poly(vinylidene difluoride) membrane. The

first 50 cycles yielded the sequence FYSYSQEPFHP
IQGRSPKWTSLEDSVKAVRSGDTVFVHsaaxtpxxxlxa
with some residues either not determined (x), or assigned
tentatively (lower case). Residues 51–58 could not be
assigned. The protein sample, after sequential treatment
with acetic anhydride and BrCN, gave a readable sequence
Table 1. Kinase recognition motifs in p49. Protein motif searches were
performed with
GENE RUNNER
(Hastings, Inc) and the PROSITE
database (40) using the
PREDICT PROTEIN
E-mail server at EMBL,
Heidelberg, Germany.
Kinase class
Motif sequence
and position
cAMP/cGMP-dependent
protein kinase
56 KKSS 59
Protein kinase C 16 SPK 18
25 SVK 27
59 SLK 61
198 TTK 200
284 SKK 286
371 TTK 373
391 TTR 393
412 SLR 414
Casein kinase II 20 TSLE 23
134 SPPD 137

172 TFGD 175
181 SHFD 184
202 TDVE 205
207 TIGE 210
322 SCIE 325
4966 M. M. Oulton et al. (Eur. J. Biochem. 270) Ó FEBS 2003
from the seventh Edman cycle onward, although the
identity of some residues was uncertain QVD
FLRGAAIxPEAGXPILALPATTxRGES.
cDNA for p49 was cloned in sections with PCR
amplification of first strand cDNA achieved by the use of
degenerate oligonucleotide primers 1a and 2 (Fig. 1)
designed on the basis of peptide sequence. Seminested
PCR was then performed with primers 1b and 2, giving
Clone p49-1, a 1107 bp DNA fragment encoding 369 amino
acid residues (Fig. 3). Peptides identified by Edman degra-
dation were encoded by Clone p49-1. Clone p49-2,
containing the rest of the p49 cDNA, was obtained by
PCR amplification. The sequences of these clones were
partially confirmed by analysis of Clone p49-3. The
assembled p49 cDNA, deposited in GenBank under the
accession number AY304544, was 1411 bp and it contained
an ORF of 1320 bp encoding 440 amino acid residues. The
ORF was flanked by a stop codon (TAA) composed of
Fig. 4. Sequence alignment of p49 and related invertebrate proteins. The deduced amino acid sequence of p49 was aligned by
CLUSTALW
with the
following proteins: D_mel (NP_651762.1 Drosophila melanogaster), A_gam (EAA09276.1 Anopheles gambiae str. PEST with three residues, ÔSEKÕ,
at the N-terminal removed based on the alignment and annotation practice by Celera of not defining the start codon), C_ele1 (AAN63431.1
representing partial sequence of NP_495409.2 which has 261 additional residues at the C-terminus, Caenorhabditis elegans), C_ele2 (CAA87047.1

Caenorhabditis elegans). Black, identical residues; grey, similar residues; no shading, different residues.
Ó FEBS 2003 Artemia CoA-transferase (Eur. J. Biochem. 270) 4967
nucleotides 1321–1323, followed by a 3¢ noncoding region of
70 nucleotides containing the poly adenylation signal
AAGTAAA and a poly(A) tail of 18 nucleotides. The
combined cDNAs represent the complete p49 sequence,
except for six N-terminal residues determined only by
Edman degradation (Fig. 3). The initiator methionine was
not observed, indicating removal of the residue during
protein maturation. The calculated molecular mass of the
protein was 48.3 kDa, in agreement with SDS/PAGE.
Motif searches with PredictProtein E-mail Server and
GENE
RUNNER
displayed several putative phosphorylation motifs
recognized by different classes of kinases, but no typical
microtubule binding regions (Fig. 3, Table 1).
Identification of p49 as a CoA-transferase by sequence
analysis
p49 has significant sequence similarity to CoA-transferases
encoded in the genomes of D. melanogaster, Anophe-
les gambiae,andCaenorhabditis elegans (Fig. 4). C. elegans
hastwomembersinthisgenefamilywith52%identityto
one another. Analysis with the Conserved Domain Data-
base disclosed a region of p49 beginning at residue 84 with
64.3% similarity to the acetyl CoA hydrolase/transferase
domain, indicating that p49 belongs to this family. Phylo-
genetic analysis exposed evolutionary relationships between
p49 and other proteins and revealed distinctive phylogenetic
protein groups (Fig. 5). p49 and the homologous inverteb-

rate sequences formed a subcluster within a main branch
with p49 positioned between C. elegans and insect proteins,
this in line with established lineage relationships. Highly
similar p49 homologs exist in many prokaryotic species,
including the archaebacteria and eubacteria (Fig. 5). Homo-
logues of lower similarity levels are present in many of the
species represented in Fig. 5, in several other bacteria species
andintheSaccharomyces cerevisiae and Schizosaccharo-
myces pombe genomes, suggesting multiple subfamilies within
the large hydrolase/CoA-transferase family. All purification
fractions displayed 4-hydroxybutyrate CoA-transferase
activity ranging from 0.22 units for the cell-free extract, to
0.88 units for heated MAPs and 0.51 units for purified p49.
Higher order structure of p49
The secondary structures of p49, 4-hydroxybutyrate
CoA-transferase from Clostridium aminobutyricum and
Fig. 5. Phylogenetic tree of p49-related CoA-transferases. The following bacterial proteins were used, in addition to the proteins in Fig. 5:
M_sp. (ZP_00042556.1, Magnetococcus sp. MC-1), C_tep (AAM71277.1, Chlorobium tepidum TLS), L_int (AAN51814.1, Leptospira interrogans
serovar), SS_one (ANN54762.1, Shewanella oneidensis), C_klu (AAA92344.1, Clostridium kluyveri), C_ami (CAB60036.1, Clostridium aminobu-
tyricum), C_tet (AAO35111.1, Clostridium tetani), F_nuca (NP_603518.1, Fusobacterium nucleatum ssp. nucleatum ATCC 25586), F_nucb
(EAA24344.1, Fusobacterium nucleatum ssp. vincentii ATCC 49256), S_sp. (BAA17706.1, Synechocystis sp.), G_met1 (ZP_00079959.1, Geobacter
metallireducens), G_met2 (ZP_00082143.1, Geobacter metallireducens), G_met3 (ZP_00082133.1, Geobacter metallireducens), G_met4
(ZP_00080028.1, Geobacter metallireducens), T_10 (AAM23830.1, Thermoanaerobacter tengcongensis), D_haf1 (ZP_00099788.1, Desulfitobacte-
rium hafniense), D_haf2 (ZP_00099512.1, Desulfitobacterium hafniense), D_hal3 (ZP_00098805.1, Desulfitobacterium hafniense), A_ful1
(AAB90101.1 Archaeoglobus fulgidus), A_ful2 (AAB89400.1 Archaeoglobus fulgidus), N_aro (ZP_00095224.1, Novosphingobium aromaticivorans),
Y_pes (CAA21375.1, Yersinia pestis), S_ent (AAK97550.1, Salmonella enteritidis), R_pal (ZP_00009324.1, Rhodopseudomonas palustris), B_jap
(BAC52055.1, Bradyrhizobium japonicum). Bootstrap values above 700 (70%) out of 1000 trees are indicated at the nodes. The branch length is
proportional to distance. The subbranch for sequences from invertebrate species is shaded.
4968 M. M. Oulton et al. (Eur. J. Biochem. 270) Ó FEBS 2003
glutaconate CoA-transferase from Acidaminococcus fermen-

tans were predicted with Prof_s (Fig. 6). Notwithstanding
limited sequence similarity, p49 and the C. aminobutyricum
4-hydrodybutyrate CoA-transferase have related secondary
structure predictions, an observation which correlates with
their structural and functional similarities. A striking
congruence between the two proteins is alternation of
relatively short stretches of a-helical and b-sheets through-
out much of their lengths. Additionally, the glutaconate
CoA-transferase, for which the crystal structure has been
determined, is only weakly related in sequence to p49 and
C. aminobutyricumm 4-hydroxybutyrate CoA-transferase,
but predicted secondary structures are similar. The tertiary
structure of p49 is uncertain, although secondary structure
predictions suggest similarities between the CoA-transfer-
ases. The size of microtubule cross-linking particles in
concert with molecular mass measurements indicate p49
forms a homomultimeric complex of 10–20 subunits, but
how monomers self-associate is not apparent.
Drosophila
contain a p49 analogue
A protein of 49 kDa was detected on Western blots
containing cell-free extract from Drosophila adults but not
from embryos, larvae and pupae (Fig. 7). The Drosophila
49 kDa protein often appeared as a doublet and reaction
with anti-p49 was stronger than for Artemia p49 in cell-
free extract. As demonstrated by immunoprobing of blots,
the Drosophila p49 analogue coassembled with taxol-
induced microtubules, albeit in reduced quantity as
compared to Artemia microtubules (Fig. 8). Small
amounts of Drosophila tubulin and the p49 analogue

were detected under control conditions, even when
reaction mixtures were centrifuged prior to assembly
and incubation time was shortened, perhaps due to
limited tubulin polymerization. The mAb DM1A, directed
against tubulin, detected tubulin and a polypeptide lower
in molecular mass than tubulin thought to be a proteo-
lytic degradation product. Microtubules assembled in
Drosophila cell-free extract were distributed sparsely on
grids with no evidence of cross-linking.
Discussion
A 49 kDa protein, termed p49, was purified to apparent
homogeneity from Artemia developed either 6 or 12 h,
and cross-linked microtubules were produced when the
protein was incubated with Artemia tubulin. This obser-
Fig. 6. Predicted secondary structures of p49 and bacterial CoA-transferases. The secondary structures of p49 from A. franciscana (p49), hyd-
roxybutyryl CoA-transferase from C. aminobutyricum (HBCoA) predicted according to Profile network prediction HeiDelberg (Prof_ s) accessible
via PredictProtein, and glutaconate CoA-transferase A chain (GlutCoA) from A. fermentans derived from its crystal structure (NCBI Structure
entry POIA) were compared. The number of residues for each sequence is indicated on the right side of the figure. Single underline, a-helix; double
underline, b-sheet.
Ó FEBS 2003 Artemia CoA-transferase (Eur. J. Biochem. 270) 4969
vation, coupled with the finding that an antibody raised
previously to a 49 kDa microtubule cross-linking protein
recognized p49 (not shown), and the N-terminal 15 amino
acid residues of both proteins were identical, demonstra-
ted p49 is the protein described by Zhang and MacRae
[19–21]. Sequencing by Edman degradation yielded amino
terminal and internal peptides essential to primer design
for PCR amplification of p49 cDNA. Clone p49-1, a
cDNA fragment of 1107 bp beginning near the
N-terminus and representing about 84% of p49, was

obtained initially. Clone p49-2, which overlapped with the
3¢ end of clone p49-1 and contained the remaining p49
sequence, included the polyadenylation signal, AAGT-
AAA. Clone p49-3 overlapped partially with clones p49-1
and p49-2, confirming a portion of each sequence.
According to motif analysis, p49 has several phosphory-
lation sites, in line with the presence of two phosphoryl-
ated p49 isoforms [21], and suggesting how protein
function is regulated. p49 lacks microtubule binding
domains typical of MAP2, MAP4 and tau.
Comparison of the deduced amino acid sequence to
archived sequences demonstrated p49 is a CoA-transferase
Fig. 7. Detection of a Drosophila p49 analogue. Cell-free extracts from
Drosophila and Artemia were electrophoresed in 12.5% SDS poly-
acrylamide gels and either stained with Coomassie blue (A) or blotted
to nitrocellulose and probed with anti-native p49 antibody (B). Panels
A and B, lane 1, 60 lgofArtemia cell-free extract protein; lane 2,
1.0 lg of purified p49; lanes 3–6, 60 lgofDrosophila cell-free extract
from embryos, larvae, pupae and adults, respectively. Molecular mass
markers are shown in lane M and represent 97, 66, 43, 31 and 22 kDa.
Arrow, p49; arrowhead, cross-reactive high molecular mass protein.
Fig. 8. Coassembly of Drosophila tubulin and the p49 analogue. Tubulin
in Artemia and Drosophila cell-free extracts was assembled by the addi-
tion of taxol and GTP. Microtubules were collected by centrifugation
through sucrose, resuspended in Pipes buffer, electrophoresed in 12.5%
SDS polyacrylamide gels and either stained with Coomassie blue (A) or
blotted to nitrocellulose and stained with anti-tubulin mAb, DM1A (B),
or anti-p49 (C). In all panels, lane 1, complete assembly reaction with
Artemiacell-freeextract;lane2,assembly reactionlackingGTPandtaxol
with Artemia cell-free extract; lane 3, complete assembly reaction with

Drosophila cell-free extract; lane 4, assembly reaction lacking GTP and
taxol with Drosophila cell-free extract. All assembly mixtures contained
630 lg of protein in a final volume of 50 lL. Molecular mass markers
represent 97, 66, 43, 31, 22 and 14 kDa. Arrow, p49; tub, tubulin;
arrowhead, cross reactive high molecular mass protein.
4970 M. M. Oulton et al. (Eur. J. Biochem. 270) Ó FEBS 2003
family member. Representatives of this family in the
anaerobic bacteria C. aminobutyricum and Clostridium
kluyveri [27,33], catalyze the formation of 4-hydroxybutyryl
CoA from 4-hydroxybutyrate, using either butyryl-CoA or
acetyl-CoA as coenzyme A donors in a fully reversible
process thought to be important in meeting the energy needs
of these anaerobic organisms. The signature motif EXG,
located near the C-terminus of CoA-transferases, and
encompassed by residues 402–404 in p49, may play a critical
role in the catalytic formation of a thiol ester between
glutamate and the substrate CoAS-moiety [34]. Propionate
CoA-transferase from Clostridium propionicum was rapidly
inactivated by borohydride mediated modification of
Glu324 in the presence of propionyl CoA [35]. Glu324
corresponds to p49 Glu402, suggesting residues 402–404 of
p49 are important catalytically and both proteins have
similar reaction mechanisms. Purified p49 exhibited
4-hydroxybutyrate CoA-transferase activity, reinforcing
the conclusion that the protein belongs to the CoA-trans-
ferase family. Because the intent was to demonstrate enzyme
activity and low yields precluded extensive analysis, assays
were not optimized nor were other potential substrates
determined. Of interest, however, purified p49 was less active
than heated MAPs, suggesting the loss during purification of

a cofactor required for maximal enzyme activity. No other
descriptions of CoA esters and their hydrolysis products are,
to our knowledge, available for Artemia.
Secondary structure predictions for p49, 4-hydroxybuty-
rate CoA-transferase from A. aminobutyricum and glutaco-
nate CoA-transferase from A. fermentans resemble one
another and CoA-transferases are generally thought to have
similar tertiary structures even though sequence identity is
limited. As one example, the crystal structure of glutaconate
CoA-transferase indicates a globular protein accommoda-
ting many secondary structural elements, in which b-strands
form a barrel-like structure [36]. The quaternary structure of
p49 probably includes 10–20 subunits, an estimate based on
microtubule cross-linking particle size and monomer
molecular mass. In comparison, C. aminobutyricum
4-hydroxybutyrate CoA-transferase is a homodimer [27],
and other bacterial CoA-transferases organize into hete-
rooctomers [37].
Most species displayed in the phylogenetic tree (Fig. 5)
have a single gene, but the bacterium Geobacter metalliredu-
cens has four CoA-transferase genes, the largest number
known. Three family members arose by recent gene dupli-
cations, as indicated by identities P 77% and membership
in the same phylogenetic tree subbranch. The large gene
family may relate to the ability of G. metallireducens to live in
extraordinarily high iron concentrations, suggesting a role
for CoA-transferases in metal metabolism or detoxification.
Desulfitobacterium hafniense, capable of reductive dechlo-
rination of hydrocarbons and use of sulfite and thiosulfate as
terminal electron acceptors, has three gene family members,

with two probably from a recent duplication.
The Drosophila genome encodes 4-hydroxybutyrate
CoA-transferase that is analogous to p49 but the protein
has not been characterized. Drosophila cell-free extract from
adult flies contains a protein that reacts strongly with anti-
p49, indicating it is 4-hydroxybutyrate CoA-transferase, but
it is lacking from embryos, larvae and pupae. The results
contrast with the situation in Artemia where p49 is expressed
in encysted embryos and early larvae. Microtubules assem-
bled in Drosophila cell-free extract associate with a 49 kDa
protein, but to a lesser degree than for Artemia and they are
distributed sparsely on grids with no evident cross-linking.
These differences, perhaps reflecting protein sequence
variation, indicate the Drosophila analogue is less dependent
than p49 on microtubules for spatial organization. p49
displays a weak nucleotide-independent nucleotidase [20]
and there are no intact nucleotide binding sites in p49. The
Drosophila gene product has a putative nucleotide recogni-
tion site encompassing residues 79–86, and it may bind GTP
efficiently, thus causing protein dissociation from micro-
tubules. This is the first time Drosophila 4-hydroxybutyrate
CoA-transferase has been shown to associate with micro-
tubules.
Microtubules organize many proteins in the cytoplasm,
and one example is the nucleotide–dependent association of
enolase with these polymers [14]. Glyceraldehyde-3-phos-
phate dehydrogenase, involved in transporting vesicular
tubular clusters between the endoplasmic reticulum and
Golgi [38,39], binds to microtubules in a phosphorylation-
dependent mechanism [15]. Hexokinase, a key enzyme in

glucose metabolism, associates with brain microtubules [40].
Signaling molecules such as the Rho family of kinases engage
microtubules [16,17], as does the tumor suppressor protein
p53 [41] and the transcriptional coordinator P/CIP [42].
Clearly, microtubules recognize many cytoplasmic proteins
in addition to those classically designated as MAPs, and
some associations have functional implications, as may be
reflected in the relationship between p49 and microtubules.
Acknowledgements
The authors thank Dr Robert Schultz, National Cancer Institute,
Bethesda, MD, USA, for the generous gift of paclitaxel and Dr Vett
Lloyd, Dalhousie University, for supplying Drosophila. The work was
supported by a Natural Sciences and Engineering Research Council of
Canada Discovery Grant to THM.
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