Molecular cloning of profilin from Tetrahymena thermophila

Gene 246 (2000) 295–301 www.elsevier.com/locate/gene

Molecular cloning of profilin from Tetrahymena thermophila David E. Wilkes, Joann J. Otto * Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA Received 22 November 1999; received in revised form 7 February 2000; accepted 15 February 2000 Received by S. Salzberg

Abstract The actin-binding protein profilin was isolated from Tetrahymena thermophila by affinity chromatography, and the peptide sequence was determined for part of the protein. The cDNA sequence was obtained by using the peptide sequence, reverse transcription-PCR and 5∞ and 3∞ RACE. The cDNA coded for a profilin of 16 680 Da, which made it among the largest known profilins, and it had a predicted isoelectric point of 8.27. The deduced amino acid sequence was divergent from other profilins, having more than 26% identity only with profilin from Tetrahymena pyriformis. The sequence contained insertions that are also present in profilins from Tetrahymena pyriformis and Trypanosoma brucei. There appeared to be only a single profilin gene and one transcript from this gene. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Actin-binding protein; cDNA

1. Introduction Profilins are small ubiquitous proteins originally described as actin-binding proteins (Carlsson et al., 1976). Characterizations of the effects that profilin has on actin in vitro reveal that profilin can either inhibit or enhance polymerization of actin filaments. Profilin can prevent polymerization by sequestering actin monomers (reviewed in Sohn and Goldschmidt-Clermont, 1994). Stimulation of actin polymerization can occur because the profilin–actin complex has a lower critical concentration for assembly at the barbed end of the filament when compared with actin alone. This stimulation of polymerization is amplified by the presence of thymosin-b4 (reviewed in Sun et al., 1995). Profilin appears to affect actin dynamics in vivo (reviewed in Sun et al., 1995). Disruption of profilin in Schizosaccharomyces pombe results in a failure to undergo cytokinesis, which is mediated by a filamentous Abbreviations: cDNA, DNA complementary to mRNA; F-actin, filamentous actin; PIP , phosphatidylinositol 4,5-bisphosphate; PLP, 2 poly--proline; PVDF, polyvinylidene difluoride; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VASP, vasodilator-stimulated phosphoprotein. * Corresponding author. Tel.: +1-765-4948171; fax: +1-765-4940876. E-mail address: [email protected] (J.J. Otto)

( F )-actin ring in these cells (Balasubramanian et al., 1994). In addition, overexpression of profilin results in a decrease in F-actin as well as a failure to undergo cytokinesis. In Dictyostelium, disruption of both profilin genes causes an increase in F-actin content and a failure to undergo cytokinesis (Haugwitz et al., 1994). Profilin is encoded by the chickadee gene in Drosophila. Alleles of chickadee with reduced expression of profilin result in the lack of actin filament bundles in nurse cells and abnormal bristles that contain increased numbers of actin bundles ( Verheyen and Cooley, 1994). Profilin also interacts with several ligands other than actin. These include poly--proline (PLP) ( Tanaka and Shibata, 1985), the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP ) (Lassing and 2 Lindberg, 1985), an actin-related protein complex (Machesky et al., 1994), vasodilator-stimulated phosphoprotein ( VASP)-like proteins (Reinhard et al., 1995), and formin-related proteins (Chang et al., 1997; Watanabe et al., 1997). The functions of these interactions are not clear, but the identity and function of the ligands suggest that profilin may be involved in signal transduction. The three-dimensional structures have been determined for profilins as diverse as bovine (Schutt et al., 1993), Acanthamoeba ( Vinson et al., 1993) and Arabidopsis (Thorn et al., 1997). Although there is low conservation in the amino acid sequences of these pro-

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teins, the tertiary structures are all very similar. These structures consist of an antiparallel beta sheet flanked by alpha helices on either side. Two long alpha helices on one side consist of the amino- and carboxyl termini. Based on the primary sequence, the most divergent profilin is that from Tetrahymena pyriformis ( Edamatsu et al., 1991). Tetrahymena actin is also poorly conserved compared to other actin sequences (Cupples and Pearlman, 1986; Hirono et al., 1987). Profilin from T. pyriformis has been isolated through its binding to poly-proline. Characterization of this profilin in vitro shows that it inhibits polymerization of T. pyriformis actin and, to a lesser degree, inhibits polymerization of rabbit skeletal muscle actin ( Edamatsu et al., 1990). Tetrahymena contain relatively low concentrations of actin (Hirono et al., 1987), and most of the F-actin present is in a stable form (Ohba et al., 1992). However, F-actin that makes up the contractile ring in the cleavage furrow of dividing cells is dynamic (Ohba et al., 1992). Cell division in T. pyriformis is associated with an increase in the level of actin mRNA ( Kimura et al., 1991), which suggests that actin levels may be regulated transcriptionally in this cell type. Although localization of profilin in T. pyriformis shows that it is in the cleavage furrow of dividing cells ( Edamatsu et al., 1992), the function of this profilin is not known. Because of the divergency of Tetrahymena profilin, these cells may provide a useful system to analyze functions of profilin that are not apparent in other cell types. Techniques for disruption of protein function are not practical with T. pyriformis but have been well developed for T. thermophila (Gaertig and Gorovsky, 1995; Sweeney et al., 1996; Cassidy-Hanley et al., 1997). As an initial step in the determination of the function of profilin in Tetrahymena, we have isolated the cDNA for T. thermophila profilin and have characterized the gene and the predicted protein sequence.

2. Materials and methods 2.1. Purification of profilin Profilin was purified from Tetrahymena thermophila strain CU428 (from Dr Peter Bruns, Cornell University, Ithaca, NY ) with a method similar to that described by Edamatsu et al. (1990). All steps were either at 4°C or on ice. Cells were washed with NKC solution (34 mM NaCl, 1 mM KCl, 1 mM CaCl ), resuspended in three 2 volumes of Buffer A (10% sucrose, 0.1 M KCl, 2 mM MgCl , 1 mM ATP, 0.5 mM dithiothreitol, 1 mM phe2 nylmethylsulfonyl fluoride), and disrupted with a Dounce homogenizer. The homogenate was centrifuged at 200 000×g for 1 h, and the supernatant was applied to a poly--proline Sepharose 4B column prepared as described by Rozycki et al. (1991). The column was washed with a high-salt column buffer (0.6 M NaCl,

1 mM NaN , 5 mM b-mercaptoethanol, 10 mM Tris, 3 pH 7.4), followed by column buffer (150 mM NaCl, 1 mM NaN , 5 mM b-mercaptoethanol, 10 mM Tris, 3 pH 7.4). Profilin was eluted from the column with 3.5 M urea in column buffer. 2.2. Proteolytic digestion and peptide sequencing To generate proteolytic fragments, 50 pmol of profilin were incubated with 3.3 ng/ml of V8 protease (Miles Scientific, Naperville, IL) overnight at 37°C. The resulting fragments were separated by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Scha¨gger and von Jagow, 1987) and transferred to polyvinylidene difluoride (PVDF ) membrane (Bio-Rad, Hercules, CA) by wet blotting overnight at 4°C (Otto, 1993). A fragment of 10 kDa was cut from the PVDF membrane, and the amino-terminal 25 amino acids were sequenced by Edman degradation by the Purdue University Laboratory for Macromolecular Structure. 2.3. Isolation of profilin cDNA The 25 amino acid sequence obtained from the 10 kDa fragment was used to design a degenerate primer [5∞ - AACAACGGT (A/G) (C/T ) (C/T )CC (C/T ) AC(C/T )G-3∞] for use in reverse transcription-polymerase chain reaction (RT-PCR) amplification along with a degenerate primer based on a region near the C-terminal end of the T. pyriformis profilin sequence [5∞-GT(A/G)GC(A/G)GC(A/C/G/T )A(A/G) (A/G)GATTC(A/G)AC-3∞]. The RNA for RT-PCR was isolated by the method of Chirgwin et al. (1979). From the RT-PCR product, both 5∞ and 3∞ RACE (rapid amplification of cDNA ends) reactions were performed with the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). Overlapping clones were obtained to verify the sequence. 2.4. Analysis of predicted protein sequence The predicted molecular mass and predicted isoelectric points were determined by the method of Wilkins et al. (1998). Sequence alignment was created by MAP Multiple Sequence Alignment analysis (Huang, 1994). The sequences were obtained with the following Accession Nos (SwissProt unless noted otherwise): Homo sapiens I, P07737; Betula verrucosa, P25816; Drosophila melanogaster, AAA28419.1 (GenBank); Strongylocentrotus purpuratus, P32006; Schizosaccharomyces pombe, P39825; Dictyostelium discoideum I, P26199; Trypanosoma brucei, Q26734; Tetrahymena pyriformis, P23412; and Tetrahymena thermophila, AF174306 (GenBank). The percentage identities were determined by comparison of the amino acid sequences after MAP alignment.

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256–509 of the cDNA (see Fig. 2) and washed twice for 15 min each at 50°C with a low-stringency buffer 5× SET (0.75 M NaCl, 10 mM EDTA, 0.15 M Tris, pH 8.0), 0.1% NaPP , 25 mM phosphate buffer i (12.5 mM Na HPO , 12.5 mM NaH PO ), 0.1% SDS]. 2 4 2 4 Poly(A)+ RNA was isolated with the PolyATract System 1000 (Promega, Madison, WI ). For Northern analysis, 2 mg of poly(A)+ RNA were separated by electrophoresis and transferred to a nylon membrane. The membrane was probed with a fragment identical to that used for Southern analysis and then washed twice for 15 min each at room temperature with Wash I (150 mM NaCl, 15 mM Na C H O , 0.1% SDS) and 3 6 5 7 once for 30 min at 55°C with Wash II (37.5 mM NaCl, 3.75 mM Na C H O , 0.1% SDS). 3 6 5 7 Fig. 1. Tricine-SDS gel of purified native T. thermophila profilin. The cell lysate was centrifuged at 200 000×g for 1 h. The supernatant was applied to a poly--proline affinity column, and profilin was eluted with a 3.5 M urea buffer solution.

2.5. Southern and Northern analyses Southern and Northern blots were performed by standard methods (Sambrook et al., 1989). For Southern analysis, 10 mg of genomic DNA were digested with EcoRI, EcoRV, HindIII or XbaI, separated by electrophoresis and transferred to a nylon membrane (NEN Research Products, Boston, MA). The membrane was probed with a 32P-labeled fragment consisting of bases

3. Results and discussion 3.1. Isolation of profilin cDNA Profilin was purified by poly--proline affinity chromatography (Fig. 1). The amino terminus of the native protein was blocked so the protein was digested with V8 protease. Peptide sequence was obtained from a 10 kDa fragment of the digest (Fig. 2). Part of this sequence was used to design a degenerate primer. The T. thermophilabased primer was used for RT-PCR along with a primer based on the T. pyriformis profilin sequence. From this product, both 5∞ and 3∞ RACE reactions were performed.

Fig. 2. Tetrahymena thermophila profilin cDNA sequence and predicted amino acid sequence. The cDNA sequence is shown on the top line, and the predicted amino acid sequence is on the bottom line. Underlined amino acids correspond to peptide sequence determined from the V8 protease cleavage fragment, and the underlined bases show the HindIII restriction site. The asterisk indicates the stop codon.

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Fig. 3. Amino acid sequence alignment of selected profilins. The alignment was created by MAP Multiple Sequence Alignment analysis. Asterisks indicate residues identical in at least six of nine of the selected profilins. The top line indicates the location of secondary structures based on human profilin (Metzler et al., 1993). Open boxes correspond to alpha helices, and shaded boxes correspond to beta strands. The organisms and profilin isoforms are Hs, Homo sapiens I; Bv, Betula verrucosa (white birch); Dm, Drosophila melanogaster; Sp, Strongylocentrotus purpuratus; Spo, Schizosaccharomyces pombe; Dd, Dictyostelium discoideum I; Tb, Trypanosoma brucei; Tp, Tetrahymena pyriformis; Tt, Tetrahymena thermophila.

The products from these procedures provided the fulllength cDNA for T. thermophila profilin (Fig. 2). The predicted molecular mass from the sequence was 16 680 Da, which was close to the size of about 17 000 Da obtained from tricine-SDS gels (Fig. 1). The predicted isoelectric point for the protein was 8.27. Most profilins are in the 12–15 kDa range (reviewed in Sun et al., 1995). The T. thermophila profilin was substantially larger than the majority of profilins, which is also the case for the T. pyriformis profilin. The T. thermophila profilin had a basic predicted isoelectric point. Many organisms contain both acidic and basic

isoforms of profilin (Haugwitz et al., 1991; Pollard and Rimm, 1991; Gieselmann et al., 1995). However, the functional significance of these isoforms is not clear. T. pyriformis contains only one profilin, but the predicted isoelectric point for this profilin is acidic (5.70). 3.2. Analysis of predicted protein sequence Alignment of T. thermophila profilin with profilins representative across phyla showed that most of the residues highly conserved among the selected profilins were present for T. thermophila ( Fig. 3). Of the 32

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D.E. Wilkes, J.J. Otto / Gene 246 (2000) 295–301 Table 1 Conserved profilin residues with known functionsa Conserved residue

Function

Residue conserved in T. thermophila?

W4 Y7 A20 122 G24 W31 A32 E56 G76 G81 K83 K98 G134 Y146 L147

PLP binding PLP binding Fold conservation PLP binding PLP binding PLP binding PLP binding Fold conservation Fold conservation Fold conservation Conserved (+) patch Actin binding Actin binding PLP binding PLP binding

Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No Yes

a Adapted from Thorn et al. (1997). Residue numbering corresponds to position in T. thermophila sequence.

amino acids conserved among at least six of the nine selected profilins, 24 of these were present in the T. thermophila sequence. Many of the conserved residues are known to be involved in specific functions ( Thorn et al., 1997), and most of these residues were present in the T. thermophila sequence ( Table 1). The alignment also showed that the T. thermophila profilin sequence contained two insertions that are also present in the T. pyriformis sequence. These insertions make the Tetrahymena profilins longer than other profilins. From alignment of the Tetrahymena sequences with the known secondary structures of human profilin I (Metzler et al., 1993), the locations of the two insertions appear to be between beta strand 2 and alpha helix 2 and between the last beta strand and the carboxyterminal alpha helix (Fig. 3). The insertions may result in functions for profilin in Tetrahymena that do not exist in other organisms or are not apparent. An insert similar to that before the carboxy-terminal alpha helix is present in Trypanosoma brucei profilin ( Wilson and Seebeck, 1997). Trypanosoma brucei, like Tetrahymena, contains low concentrations of actin (Ben Amar et al., 1988). Thus, these inserts may have a function that does not involve actin. Comparison of profilin amino acid sequences showed that T. thermophila profilin was most closely related to that from T. pyriformis (58% identity; Table 2). The other selected profilins had between 19 and 26% identity to T. thermophila profilin.

Table 2 Percentage identities between selected profilinsa

Bv Dm Sp Spo Dd Tb Tp Tt

Hs

Bv

Dm

Sp

Spo

Dd

Tb

Tp

19 19 23 26 22 20 21 24

39 28 35 42 31 23 23

27 36 41 29 25 21

26 25 33 28 26

42 25 24 21

33 28 24

20 19

58

a Percentage identities were determined by comparison of amino acid sequences after MAP alignment. The organisms and profilin isoforms are Hs, Homo sapiens I; Bv, Betula verrucosa (white birch); Dm, Drosophila melanogaster; Sp, Strongylocentrotus purpuratus; Spo, Schizosaccharomyces pombe; Dd, Dictyostelium discoideum I; Tb, Trypanosoma brucei; Tp, Tetrahymena pyriformis; and Tt, Tetrahymena thermophila.

and probed with the 32P-labeled fragment described in Section 2. The cDNA does contain a HindIII restriction site (Fig. 2) but does not contain sites for the other three restriction enzymes. Under low-stringency conditions, only one band was observed for the EcoRI, EcoRV and XbaI digests, while two bands were observed for the HindIII digest (Fig. 4). These results are consistent with the conclusion that the profilin gene exists as a single copy in T. thermophila. However, it is possible that a highly divergent isoform exists that was not detected. To determine the size of the profilin transcript, Northern blot analysis was performed. Poly(A)+ RNA was probed with the same fragment used for the Southern

3.3. Southern and Northern analyses Southern blot analysis was performed to determine the number of profilin genes in T. thermophila. Genomic DNA was digested with EcoRI, HindIII, EcoRV or XbaI

Fig. 4. Southern blot analysis of T. thermophila genomic DNA. For each lane, 10 mg of genomic DNA were digested with EcoRI, HindIII, EcoRV or XbaI and hybridized to a 32P-labeled probe consisting of bases 256–509 of the T. thermophila profilin cDNA.

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This work was supported by National Science Foundation grant MCB-9224083 and a Purdue Research Foundation grant to J.J.O.

References

Fig. 5. Northern blot analysis of T. thermophila poly(A)+ RNA. Two micrograms of poly(A)+ RNA were hybridized to a 32P-labeled probe consisting of bases 256–509 of the T. thermophila profilin cDNA.

blot anlaysis. Only one band of about 0.66 kb was recognized (Fig. 5). This was similar to the size of 653 bp obtained for the profilin cDNA. Thus, there appears to be only one transcript from the single profilin gene. 3.4. Conclusions We have accomplished the first step toward the determination of the function of profilin in Tetrahymena. The cDNA sequence for T. thermophila profilin was determined, and the predicted protein sequence was found to be most similar to that from T. pyriformis. The T. thermophila profilin did contain most of the conserved profilin residues that have known functions. However, both Tetrahymena profilins contain two insertions within their sequences that are not present in most other profilins. Thus, it is important to determine whether profilin in Tetrahymena performs similar functions as in other cell types. It was necessary to determine the T. thermophila sequence because molecular manipulation techniques to disrupt protein functions have been described much more extensively in T. thermophila (Gaertig and Gorovsky, 1995; Sweeney et al., 1996; Cassidy-Hanley et al., 1997) than in T. pyriformis. The T. thermophila profilin gene appears to exist as a single copy and to encode a single transcript.

Acknowledgements We thank Drs David Asai, James Forney, and Christopher Staiger for their suggestions and advice.

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