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

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 o

A 49 kDa microtubule cross-linking protein from Artemia franciscana

is a coenzyme A-transferase

Mindy M Oulton1, Reinout Amons2, Ping Liang3and Thomas H MacRae1

1

Department of Biology, Dalhousie University, Halifax, NS, Canada;2Department of Molecular Cell Biology, Sylvius Laboratory, Leiden, the Netherlands;3Department 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 study were used to generate p49 cDNAs by RT-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-nobutyricumand glutaconate CoA-transferase from Acid-aminococcus fermentanswere 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 mM, 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 ofArtemia 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: tmacrae@dal.ca

Abbreviation: MAP, microtubule-associated protein.

(Received 3 September 2003, revised 20 October 2003,

accepted 24 October 2003)

Buchner funnel, washed with cold distilled water followed by

Pipes buffer [100 mM

1,4-piperazine-N,N¢-bis(2-ethanesulf-onic acid) as free acid, 1 mMEGTA, 1 mMMgCl2, 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 mM

Tris/HCl, 182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 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 mM Tris/HCl, 140 mM 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 Guide to the Care and Use of Experimental Animals available from the Canadian Council on Animal Care The blots were washed twice for 3 min each in TBS/Tween and HST (10 mMTris/ HCl, 1MNaCl, 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 ofArtemia 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
C with 1.8 mMGTP and 10 lMpaclitaxel 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 withANALYSIS, version 2.1

Detection of aDrosophila p49 analogue Drosophilacell-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 mMKH2PO4,

pH 7.0, 200 mM sodium acetate, 1 mM oxaloacetic acid,

1 mM 5,5¢-dithiobis(2-nitrobenzoate), 0.1 mM butyryl CoA, 0.5 U citrate synthase (Sigma) and p49 Absorbance increaseat412 nmwasmeasuredat20
Candenzymeactivity

is reported in arbitrary units as DA min)1Æmg protein)1

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 mM

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 TRIzol 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 lg of Artemia cell-free extract; lane 2, 35 lg of MAPs; lane 3, 35 lg of heated MAPs; lane 4, 2.5 lg of 0.2 M NaCl fraction from P11; lane 5, 1 lg of purified p49 (B) Lanes 1–3 each received 10 lg of protein; lane 4, 2.5 lg of the 0.2 M NaCl fraction from P11; lane 5, 1 lg of purified 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.

Biosciences, Baie d’Urfe, QC, Canada) and used with

Ready-To-GoTMRT-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 lg of oligo d(T)18primer, 2 lMof each gene specific primer and 2.0 mM MgCl 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.

topped with 50 lL of mineral oil and reverse transcription

was carried out at 42
C for 30 min followed by 5 min at

95
C PCR amplification was performed immediately as

follows: 95
C for 45 s, 48
C for 60 s, 72
C for 90 s,

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 mMTris/HCl, 20 mMacetic

acid, 1 mM EDTA, pH 8.5) with a 1000 bp marker set

(MBI Fermentas, Burlington, Ontario, Canada), then

cloned with the pGEM-T Easy Vector Systems Kit

(Promega, Madison, WI, USA) and Escherichia coli JM109

Plasmid DNA was isolated using Wizard 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], includingBLASTX for DNA andBLASTPfor proteins

Motif searches were performed with PROSITE database

[30] usingPREDICT PROTEINat the EMBL website,

Heidel-berg, Germany Secondary structure predictions were made

with Prof_ s, accessible via PREDICT PROTEIN Multiple

alignments were performed withCLUSTALX[31] with output

files formatted by BOXSHADE (http://www.ch.embnet.org/

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-value¼ 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 withCLUSTALXusing

the Gonnet protein comparison matrix and 1000 bootstrap

trials The tree was viewed and printed withTREEVIEW[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 0.5M 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 (NH4)2SO4 fractionation Only one weakly staining band of 49 kDa was observed in Coomassie blue stained gels after (NH4)2SO4 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

25 SVK 27

59 SLK 61

198 TTK 200

284 SKK 286

371 TTK 373

391 TTR 393

412 SLR 414

134 SPPD 137

172 TFGD 175

181 SHFD 184

202 TDVE 205

207 TIGE 210

322 SCIE 325

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, SEK,

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.

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 andGENE

RUNNERdisplayed 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, and Caenorhabditis elegans (Fig 4) C elegans

has two members in this gene family with 52% identity to

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 and in the Saccharomyces cerevisiae and Schizosaccharo-myces pombegenomes, 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, DesulfitobacteDesulfitobacte-rium hafniense), D_hal3 (ZP_00098805.1, DesulfitobacteDesulfitobacte-rium 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.

glutaconate CoA-transferase from Acidaminococcus

fermen-tanswere 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.

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 lg of Artemia cell-free extract protein; lane 2,

1.0 lg of purified p49; lanes 3–6, 60 lg of Drosophila 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 centrifugaaddi-tion 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,assemblyreactionlackingGTPandtaxol 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.

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 Drosophilagene 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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