From the Molecular and Cell Biology Program, University of Texas at Dallas, Richardson, Texas 75083-0688
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ABSTRACT |
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Previous investigators have reported the presence of two dominant proteins, tectonin I (25 kDa) and tectonin II (39 kDa), in nuclei and nuclear matrix from plasmodia of Physarum polycephalum. We demonstrate, by a modification of the nuclear isolation protocol and by protease sensitivity, that the tectonins are not nuclear proteins but rather are located on the exterior surface of the plasma membrane.
We report the sequences of cDNAs of tectonins I and II, which
encode 217 and 353 amino acids, respectively. Tectonin I is homologous
to the C-terminal two-thirds of tectonin II. Both proteins contain six
tandem repeats that are each 33-37 amino acids in length and define a
new consensus sequence. Homologous repeats are found in L-6, a
bacterial lipopolysaccharide-binding lectin from horseshoe crab
hemocytes. The repetitive sequences of the tectonins and L-6 are
reminiscent of the WD repeats of the -subunit of G proteins,
suggesting that they form
-propeller domains. Tectonin II has an
additional N-terminal domain that includes a 47-residue sequence highly
similar to the galactoside-binding sequence of the B-chain of ricin.
The tectonins may be lectins that function as part of a transmembrane
signaling complex during phagocytosis.
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INTRODUCTION |
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In its plasmodial form, the myxomycete Physarum polycephalum exists as a multinucleated syncytium that feeds on bacteria and organic detritus by phagocytosis. The many nuclei within a single plasmodium progress through the cell cycle synchronously, and at the end of the G2 phase undergo closed mitosis. Because of these characteristics, several investigators have examined the P. polycephalum nuclear matrix (1-4) and reported that, as with mammalian nuclear matrix, the P. polycephalum matrix contained a number of proteins ranging from approximately 40 kDa to more than 100 kDa but that it differed from mammalian nuclear matrix by having two dominant proteins with reported molecular masses of 23-28 and 35-38 kDa as determined by SDS-PAGE.1 The same proteins have also been found associated with purified rDNA chromatin (5). We have termed these proteins tectonins I and II, respectively.
In the present study we report the cloning and sequencing of the cDNAs for the tectonins, and we use trypsin digestion of cell fractions to assess their localization in the plasmodium. We find that the tectonins are not nuclear proteins but instead are located on the plasmodial surface, and we report a method for purifying P. polycephalum nuclei not contaminated with the tectonins.
The tectonins share with lectin L-6 of horseshoe crab hemocytes (6) six
repeats of a novel consensus sequence that may form a -propeller
structure. Additionally, tectonin II contains in its N-terminal region
a sequence similar to the galactose-binding domain of the B-chain of
the plant toxin ricin (7). The tectonins may share with lectin L-6 the
ability to recognize the outer membrane lipopolysaccharide of
Gram-negative bacteria for phagocytosis and utilize an additional
affinity for galactose to expand the number of ligands that they
recognize.
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MATERIALS AND METHODS |
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Strain and Culture of P. polycephalum-- P. polycephalum M3, a diploid subline of the natural isolate Wisconsin I (8), was used. Microplasmodia were cultured in the semi-defined liquid medium of Daniel and Baldwin (9) at 27 °C in the dark with shaking.
Preparation of Nuclei, Nucleoli, Matrices, and Mitochondria-- Initially, nuclei were isolated from exponentially growing microplasmodia by the method of Mohberg and Rusch (10) which uses 250 mM sucrose, 10 mM Tris-HCl, pH 7.6, 10 mM CaCl2, and 0.1% Triton X-100 as the homogenization buffer (ST buffer). These nuclei were further purified by sedimentation (1000 × g, 30 min, 4 °C) through 50% Percoll (Pharmacia Biotech Inc.) in ST buffer.
Later, nuclei were isolated by the following procedure that was developed to remove contaminating tectonins from nuclei. Microplasmodia were collected by centrifugation (200 × g, 2 min, 23 °C), washed briefly with deionized water at 23 °C, and immediately homogenized in 5-10 volumes of 4 °C isolation buffer (200 mM glycerol, 20 mM Tris-HCl, pH 7.5, 40 mM KCl, 10 mM NaCl, 7 mM MgCl2, and 0.1% Triton X-100) in a Waring blender at low speed using three 15-s pulses. Large debris were removed by passing the homogenate through a milk filter. The nuclei were sedimented from the homogenate (700 × g, 10 min, 4 °C) and suspended in 4 volumes of isolation buffer. The suspension was made 14% in Percoll and centrifuged (700 × g, 10 min, 4 °C), yielding a pellet of tectonin-free nuclei and a tectonin-rich pellicle which formed on top of the buffered Percoll. Entrapped nuclei were recovered from the pellicle by transferring it along with the Percoll supernatant to a new centrifuge tube, mixing well, and centrifuging. After two to three repetitions, nearly all the nuclei were collected in the pellet. Nucleoli were obtained by suspending microplasmodia 1:5 (v/v) in 25 mM sucrose, 3 mM EGTA, 10 mM Tris-HCl, pH 7.2, and disrupting them with a French pressure cell (10,000 p.s.i., 4 °C). As the lysate was collected, aliquots of 1 M CaCl2 were added to bring the Ca2+ concentration to 10 mM. Nucleoli were sedimented from the lysate (1000 × g, 15 min, 4 °C), washed 3-4 times in ST buffer before being sedimented through 50% Percoll in ST buffer (1200 × g, 30 min, 4 °C), and finally washed and resuspended in ST buffer. Nucleolar matrix was prepared by mixing equal volumes of the resuspended nucleoli and 200 mM NaCl, 20 mM MgCl2, 74 mM Tris-HCl, pH 7.4, and digesting with 50 µg/ml RNase A, 150 µg/ml DNase I, and 50 µg/ml micrococcal nuclease (3-5 h, 37 °C). After being sedimented (800 × g, 15 min), washed, and resuspended in ST buffer, the nucleoli were dialyzed against 2.5 M NaCl, 25 mMTectonin Purification-- Plasmodial tectonins I and II were purified from urea-solubilized nuclear matrices by isoelectric focusing followed by preparative SDS-PAGE (11).
Additionally, purified tectonins were obtained after cloning the cDNAs of both tectonins into expression vectors in Escherichia coli as follows. The coding sequences and 3'-untranslated regions of cDNAs encoding tectonin I and II were inserted between the NdeI and BamHI sites of pET-3a (12) to create pHKI and pHKII, respectively. The constructions involved reverse transcriptase-polymerase chain reaction (PCR) with total plasmodial RNA as template for tectonin I and PCR with aPreparation of Antibodies-- New Zealand White rabbits were immunized against either plasmodial tectonin II or tectonins I and II expressed in E. coli. Freund's complete adjuvant and 20-100 µg of purified tectonin were used for the first injection. Booster doses contained Freund's incomplete adjuvant and 20-50 µg of tectonin. All antibody titers were greater than 1:1000.
IgG antibodies were separated from the anti-plasmodial tectonin II serum by diluting the serum 1:10 with 15 mM sodium phosphate, pH 6.3, and passing it through a DEAE 250 capsule (Cuno Laboratory Products). IgG in the flow-through was precipitated by 40% (NH4)2S04 and then dissolved in 10 mM sodium phosphate, pH 6.8. Antisera against the bacterially expressed tectonins were used without fractionation of the immunoglobulins.Immunoblots-- For Western blots, proteins were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes by semi-dry electroblotting (13), blocked for 30 min with 3% bovine serum albumin or saturated non-fat milk in phosphate-buffered saline, and incubated for 1 h with anti-tectonin diluted in phosphate-buffered saline. Bound antibodies were detected with 125I-labeled protein A (ICN) and autoradiography or with horseradish peroxidase-conjugated protein A (Amersham Life Science, Inc.) and a chemiluminescent substrate (ECL, Amersham Life Science, Inc., or Super Signal, Pierce) with exposure to x-ray film (Kodak X-Omat AR).
Selection of Tectonin cDNAs--
A gt11 expression
library carrying P. polycephalum plasmodial cDNAs
inserted at the EcoRI site of the vector (gift of Volker Vogt) was plated at 100 plaques per cm2 and immuno- or
hybridization-screened by standard methodology (14). Anti-tectonin II
IgG and 125I-labeled protein A were used to detect clones
expressing tectonin II. Tectonin I clones were identified by low
stringency hybridization to the tectonin II cDNA labeled with
32P by nick translation (Life Technologies, Inc., kit).
Hybridization was for 24-48 h at 37 °C in 6× SSC, 50% formamide,
0.05% sodium pyrophosphate, 0.1% SDS, 0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin, and 100 µg/ml
sheared E. coli DNA. After hybridization, the filters were
subjected to three 15-min washes in 2× SSC, 0.1% SDS at room
temperature, followed by two 30-min washes in 0.1× SSC, 0.1% SDS at
37 °C.
Primer Extension-- Primer extensions to determine the 5' ends of tectonin mRNAs were performed by the procedure described for first strand cDNA synthesis, using primers that had been 5'-32P-labeled with polynucleotide kinase (Promega). The primers were complementary to nucleotides 74-94 of the tectonin I coding sequence and nucleotides 121-140 of the tectonin II coding sequence.
RNA Preparation and Blots-- Total RNA was isolated from microplasmodia solubilized with guanidinium thiocyanate using a commercial kit (CLONTECH). For Northern blots, total RNA (15 µg per lane) was electrophoresed in a 1% agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized to 32P-labeled tectonin cDNA under the conditions described for cDNA library screening. The final washes were at 37 °C for low stringency and at 65 °C for normal stringency.
Circular Dichroism-- The purest fractions of tectonins I and II from DEAE-cellulose chromatography were dialyzed against 2 mM phosphate buffer, pH 7.2, 200 mM NaCl, and circular dichroism (CD) spectra were measured at 20 °C using a Jasco J-500A spectropolarimeter and a 0.2-mm cell. SDS-PAGE with Coomassie Blue stain demonstrated that the preparations used for CD analysis contained less than 2% contaminating proteins.
Protease Digestions-- Digestion with V8 protease (Miles Scientific) at 5 µg/ml was in 60 mM Tris-HCl, pH 7.5, 10 mM NH4Cl at 37 °C. Free trypsin and trypsin attached to acrylic beads were purchased from Sigma. Digestions with trypsin were in ST buffer without Triton X-100. At intervals, aliquots were removed and heated with SDS-containing Laemmli sample buffer to stop the digestion. The products were separated by SDS-PAGE and analyzed by protein staining and by Western blotting.
DNA Sequencing and Analysis--
Single-stranded copies of
pIBI24 and pIBI25 carrying the tectonin cDNAs were prepared using
M13KO7 helper phage (14). Sequence analysis was performed by the
dideoxy chain termination method using the single-stranded phagemid DNA
as template, deoxyadenosine 5'-(-[35S]thio)triphosphate for labeling, and a
Sequenase kit from U. S. Biochemical Corp. Products were analyzed
by electrophoresis in 5-6% polyacrylamide/8 M urea
gels.
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RESULTS |
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Cloning Tectonin II--
Tectonin II cDNAs were identified by
immunoscreening the gt11 expression library carrying P. polycephalum microplasmodial cDNAs, using antibodies raised
against tectonin II that had been purified from P. polycephalum. In Western blots, this antibody reacted strongly
with the 39-kDa tectonin II and weakly with a 25-kDa protein,
indicating the presence of a shared epitope in the smaller protein
(Fig. 1). Among 32,000 recombinant phage
screened, four immunopositive clones were found; all contained the same 1.13-kilobase pair insert, based on restriction enzyme digestion patterns. Sequencing revealed a single open reading frame of 1059 nucleotides that was fused in frame at its 5' end to the
lacZ gene of the vector. Although the protein predicted from
the reading frame of the cDNA was equivalent in size to tectonin
II, the reading frame did not contain an initiating methionine codon.
Therefore, to establish the N terminus of the cDNA-encoded protein,
anchored reverse transcriptase-PCR using total plasmodial RNA, a primer complementary to nucleotides 498-518 downstream from the
lacZ junction, and poly(A)-tailing of the first strand
cDNA were performed to obtain the complete 5' end of the cDNA.
Cloning and sequencing of six of these cDNAs showed that the only
nucleotide missing from the reading frame of the original cDNA was
A of the AUG initiation codon (Fig.
2A). Preceding the initiation
codon was a leader 19-25 nucleotides long, with the shortest leader
being present in half the clones. Primer extension from nucleotide 121 of the coding sequence using total plasmodial RNA confirmed that the
19-nucleotide 5' leader represented the major mRNA species with a
minor species two nucleotides longer (Fig.
3A). Among the six sequenced
cDNAs, there were only three nucleotide variances, and these were
all in the noncoding 5' and 3' ends (Fig. 2A), suggesting
that they were transcripts of different alleles in the diploid
organism.
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Tectonin II mRNA and Gene-- To ascertain that the major species of tectonin II mRNA was represented by the cloned cDNA, a Northern blot of plasmodial RNA was probed with the tectonin II cDNA (Fig. 4). A single mRNA species of 1,200 nucleotides corresponding in size to the full-length tectonin II cDNA was observed. With low stringency hybridization conditions, weak labeling of a band below the tectonin II mRNA was observed. This will be shown to correspond to tectonin I mRNA.
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Cloning Tectonin I--
Because tectonin I appeared to be related
to tectonin II on the basis of the Northern blot of total plasmodial
RNA, the gt11 cDNA expression library was screened for tectonin
I clones by low stringency hybridization to the tectonin II cDNA.
Of four selected clones, three were incomplete tectonin II cDNAs,
but the fourth carried a 582-nucleotide open reading frame 73%
identical to the C-terminal portion of tectonin II. Anchored reverse
transcriptase-PCR with the same primer used to obtain the 5' end of the
tectonin II cDNA and cloning in pIBI24 yielded two identical
independent cDNAs that overlapped the putative tectonin I reading
frame by 21 nucleotides and extended it for an additional 102 nucleotides. Combined, the cDNA sequences encode a tectonin I-sized
25-kDa protein (Fig. 2B). There is a single methionine codon
33 nucleotides from the 5' end. As was the case with the tectonin II
sequence, the AUG initiation codon is preceded by an A at
3 and has a
G at +4 nucleotides in accordance with the initiation codon rule of
Kozak (16). A poly(A) signal sequence AATAAA is located 11 nucleotides
upstream from the 3' poly(A) tail. Primer extension from nucleotide 74 within the coding body using total cellular RNA confirmed that the 5'
leader of the complete cDNA corresponds in length to that of the
tectonin I mRNA (Fig. 3B).
Tectonin I mRNA-- A Northern blot of plasmodial RNA was probed with tectonin I cDNA (Fig. 4) to determine if the major species of tectonin I mRNA was represented by the cloned cDNA. A single mRNA species of 900 nucleotides corresponding in size to the full-length tectonin I cDNA was observed.
Tectonins I and II Contain a Repeated Sequence-- The deduced amino acid sequences for the tectonins show that tectonin I and the C-terminal two-thirds of tectonin II are 73% identical and are comprised of six similar repeats that vary from 33 to 37 residues in length. An alignment of the repeats is shown in Fig. 5A. The tectonins appear to have diverged from each other after the set of six repeats was established because the sequences are more conserved between corresponding repeats of the two tectonins than between repeats within the individual proteins.
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Circular Dichroism of the Tectonins--
The circular dichroism
(CD) spectra of pure solutions of bacterially expressed tectonins I and
II were compared with reference spectra (21) to determine the relative
contents of -helix and
-sheet (Fig. 7).
However, both tectonins contain such large amounts of aromatic amino
acids, which absorb strongly below 200 nm, that the spectra could not
be measured below 200 nm where the signature spectrum of
-sheet is
found. Thus no direct estimation of the amount of
-sheet secondary
structure was possible. The CD spectrum expected for an
-helical
protein should have two large negative peaks of similar magnitude at
208 and 222 nm (21). Although both tectonins I and II have negative CD
values near 208 nm, their molar ellipticities at that wavelength are
only 6 and 9% of the values for a pure
-helical protein,
respectively, implying that less than 10% of the total secondary
structure of either tectonin is likely to be
-helix. Furthermore,
the negative CD peak at 222 nm is lacking in the spectra of both
tectonins. Thus, neither the magnitudes nor the shapes of the CD
spectra support the presence of a significant amount of
-helix
secondary structure in either tectonin I or II.
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Distribution of Tectonins among Cellular Compartments-- To assess the reported presence of the tectonins as major components of the nuclear/nucleolar matrix, nuclei were isolated from microplasmodia by the procedure of Mohberg and Rusch (10), which has been used by most investigators of the P. polycephalum matrix. Nucleoli were isolated after disrupting the nuclei in a French press, and nucleolar matrix was obtained by repeated nuclease digestion and extraction with 2.5 M NaCl. SDS-PAGE showed that tectonins I and II were enriched in the nuclei and nucleoli, and they dominated the nucleolar matrix profile (Fig. 8). We also found that mitochondria prepared by differential centrifugation of a plasmodial homogenate contained tectonins, and the tectonins dominated the proteins present in mitochondrial nucleoids obtained by sucrose gradient centrifugation of Nonidet P-40 lysed mitochondria.
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Removal of Tectonin Contaminants from Nuclei-- Because the tectonins were apparently contaminants in preparations of nuclei, we sought to devise a method to remove them. We succeeded by modifying the isolation procedure of Mohberg and Rusch (10). Some changes were made in the homogenization buffer, such as substituting 7 mM Mg2+ for Ca2+ and 0.2 M glycerol for the 0.25 M sucrose, and including 40 mM KCl, but the critical difference was to eliminate sedimentation of the nuclei through 1 M sucrose. Instead, a crude nuclear pellet was collected by centrifuging the homogenate at 700 × g, followed by suspension and sedimentation from a 14% Percoll solution at 700 × g. A viscous, tectonin-rich fraction collected at the top of the tube while the nuclei sedimented to the bottom forming a pellet. SDS-PAGE and Western blot of the proteins in the homogenate, the supernatant (S700), and crude nuclear pellet (P700) after the first centrifugation and the tectonin fraction and purified nuclei after the Percoll centrifugation are shown in Fig. 11. Although a significant portion of the tectonins remained in the S700 supernatant, they were still the dominant components in the P700 nuclei. However, nearly all of both tectonin I and II was removed by Percoll centrifugation of the P700 pellet, confirming that the tectonins are not nuclear proteins.
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DISCUSSION |
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The amino acid sequences of tectonins I and II deduced from cloned cDNAs show that the two proteins are closely related, with 73% of the amino acid residues in tectonin I and in the C-terminal two-thirds of tectonin II being identical. The tectonin genes are transcribed to yield poly(A)-tailed mRNAs of 870 and 1200 nucleotides for tectonin I and II, respectively. The only variations within either mRNA were extension of a few nucleotides at the 5' end for minor species of the tectonin II mRNA and isolated base changes in the untranslated leaders and 3' regions of both mRNAs, which indicate transcription of both alleles of the genes in the diploid plasmodium that was used as the source of mRNA in constructing the cDNA library.
The calculated pI for tectonin I is 6.49 and for tectonin II is 6.27. By using two-dimensional electrophoresis, we have determined pI values of 7.2 for tectonin I and 6.7 for tectonin II, whereas Denovan-Wright and Wright (3) reported 7.4 for tectonin I and 6.70-6.85 for isoforms of tectonin II which they isolated from nuclear matrix preparations. These values all show reasonable agreement, with tectonin II being somewhat more acidic then tectonin I.
The tectonin homologue lectin L-6 has been shown to have affinity for bacterial lipopolysaccharide, and it appears to participate in the anti-bacterial defense system of the horseshoe crab (6). By virtue of the similarity between lectin L-6 and the tectonins, we suggest that the tectonins may perform a similar bacterial recognition function and thus aid P. polycephalum in identifying and phagocytosing its food.
The N-terminal domain of tectonin II seems likely to have an additional
lectin activity, -galactoside binding. A segment of 47 amino acids
(residues 47-94) shares 31% identity with part of the B-chain of
ricin and an additional 20% conservative substitution of amino acids.
The B-chain of ricin contains two similar galactoside-binding sites
created by gene duplication. The two halves of the B-chain have a 32%
identity in amino acids (7). It is the N-terminal half of ricin that is
most similar to the tectonin II segment, but the putative
galactoside-binding site in tectonin II shares features of each of the
ricin B-chain sites. Specifically, x-ray crystallography of a
ricin-lactose complex (20) has shown that the N-terminal site uses Asp,
Gln, Trp, Lys, and Asn to bind galactose, whereas at the second site
there is no Gln and Tyr replaces Trp. Tectonin II has Asp, Leu, Tyr,
Lys, and Asn at the equivalent positions in the sequence. Lectin
activity resident in this segment of tectonin II could augment its
breadth of specificity or its affinity for potential foodstuff. The
surface slime of P. polycephalum, which is a galactan (25),
does not appear to be a ligand of the tectonins, as we have not
detected either tectonin in slime recovered from culture medium.
Denovan-Wright and Wright (3) have reported that tectonin II is a glycoprotein, with up to four isomers of the protein having been detected by concanavalin A. Perhaps the apparent isomers were the result of tight binding of the tectonins to oligosaccharides. If there are covalently linked saccharide residues, their number must be low since we find that plasmodial tectonin II co-migrates with tectonin II expressed in E. coli, which should not be glycosylated.
Originally described as possible nuclear matrix proteins, our results indicate that the tectonins are instead principally cell-surface proteins that are accessible to digestion by trypsin in unpermeabilized plasmodia. In other experiments using anti-tectonin antibodies for immunoelectron and fluorescence microscopy,2 we have confirmed the surface location of the tectonins and found that they also are bound to the inner surface of certain cytoplasmic vesicles. On the external membrane surface, the tectonins are concentrated in crowns similar to the structures believed to be involved in phagocytosis and macropinocytosis in Dictyostelium discoideum (27, 28). At the crowns of D. discoideum, coronin accumulates on the cytoplasmic side of the plasma membrane and interacts with actin. The crowns subsequently enclose bacteria or fluid in a vesicle for digestion. The aggregation of tectonins on the external surface of similar crowns supports a role for tectonins in such endocytoses by binding food and perhaps triggering vesicle formation.
How newly synthesized tectonins are transported and bound to the plasmodial surface is not clear. Their cDNAs do not encode precursor forms with N-terminal signal sequences, and neither protein has recognizable sequences for membrane transport or attachment of lipids.
We are able to isolate P. polycephalum nuclei lacking the tectonins with mild conditions, which confirms that they are not nuclear proteins. By sedimentation through buffered 14% Percoll, tectonin-free nuclei are separated from contaminating membranes, and both tectonins are found in a viscous, membranous pellicle at the top of the Percoll solution as would be expected for fragments of the plasmodial surface. Unless the tectonin-rich membranes are separated from the nuclei or nucleoli that are used to prepare matrix, we confirm that they appear as dominant components as reported by other investigators (1-3). We have also observed that the tectonins can contaminate mitochondrial preparations, and Amero et al. (5) have reported that they were present as possible contaminants of P. polycephalum rDNA chromatin. Why are the tectonins such ubiquitous contaminants? A likely answer is that they are part of complexes on the surface of plasmodia, which are associated with the viscous polysaccharide slime that coats the exterior surface. When fragmented by homogenization, surface fragments enriched in tectonins could entrap organelles and large molecular complexes via the associated polysaccharide. The purification method of Mohberg and Rusch (10), which relies on sedimentation through 1 M sucrose, may actually have driven the entrapment due to the sucrose concentration. The fact that tectonins can be separated from nuclei under low ionic strength and with 0.1% Triton X-100 in the buffer shows that their apparent association with nuclei is not a matter of strong interactions that must be disrupted. The buffered 14% Percoll that we used for purification of nuclei provides the density differential and low solute concentration needed to separate nuclei from tectonin-containing fragments of the cell membrane.
Together with limulus lectin L-6, the tectonins establish a new family
of proteins based on the repeated consensus sequence: pWpX(V/I)pGpLXpVpnX3-5pVWGVNpXppIY.
Including the additional 4-7 residues that connect the six tandem
repeats of this sequence in the tectonins and L-6, the repeat units are
33-38 amino acids long. Conserved residues in the consensus sequence
define two blocks of 13-14 amino acids that are connected by 3-5
residues in the middle of the consensus sequence. The second or third
residue at the beginning of each block is tryptophan or another
aromatic amino acid, and the penultimate residue at the end of the
second block is tyrosine. Valine or isoleucine is found at four rather evenly spaced intervals across the consensus sequence. In lectin L-6, a
tight three-residue turn is locked in place between every other repeat
of the consensus by a disulfide bond between cysteines that bracket the
turn (6); this feature is absent from the sequences that link the
tectonin repeats.
Collectively, the tectonin and L-6 repeat sequences predict that each
repeat is comprised of four similarly sized short -strands connected
by turns or random coils. Thus, each of the repeats appears to form a
four-stranded
-sheet. This pattern of multiple repeats 23-40 amino
acids long, each containing 4 short
-strands typifies
-propeller
proteins (17). Furthermore, the tectonin consensus sequence is similar,
particularly in its latter half, to the repeated WD sequences of the
-subunits of G proteins, where Y
sometimes replaces the WD
residues (18). Much is known regarding the position of G
on the inner surface of plasma membranes and its function in
transmembrane signaling (26). The apparent similarity of tertiary
structure of the tectonins may reflect their proposed function as
external components of a signal system for response to food.
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ACKNOWLEDGEMENTS |
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We thank Volker Vogt for the cDNA library, Franklin Yau for help with restriction mapping, and Donald Gray for help in evaluating the CD spectra. The late David Schwartz also helped in mapping and subcloning the tectonin II cDNA. Protein sequencing was performed at the Texas A&M Sequencing Center.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant PCM-8303010 (to R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF041455 and AF041456 for the tectonin I and II cDNA sequences, respectively.
Present address: Medical Laboratory Sciences Dept., University of
Texas Southwestern Medical Center, Dallas, TX 75235-8878.
§ To whom correspondence should be addressed. Tel.: 972-883-2511; Fax: 972-883-2409; E-mail: rmarsh{at}utdallas.edu.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
2 J. E. Aldrich and R. Marsh, submitted for publication.
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REFERENCES |
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