Vol.

175,

March

No.

2, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

15, 1991

Pages

THE PRIMARY Masaki

Institute

STRUCTURE

Edamatsu,

OF TETRAHYMENA

Masafumi

Watanabe+

Sciences,

University

Ibaraki Bank,

Received

RIKEN

February

305,

Institute

of

Tsukuba,

Ibaraki

PROFILIN*

Tohru

and Yoshio of Biological

'Cell

Hirono',

543-550

Takemasa

of

Tsukuba,

Tsukuba,

Japan

Physical

and Chemical

305,

Research,

Japan

5, 1991

The cDNA of Tetrahymena profilin was cloned and seThe deduced product has a molecular mass of 16,785 Da the largest among profilins known so far. Tetrahymena shows higher homologies with lower eukaryotic profilins mammalian profilins. Although the homologies with mammalian and lower eukaryotic profilins are only 20-29% which is the lowest one among lower eukaryotic profilins, the N- and Cterminal regions of Tetrahymena profilin are considerably conserved as those in other profilins, suggesting that these regions are responsible for the essential properties common to profilins. SUMMARY: quenced. which is profilin than with

0 1991

Academic

Press,

Inc.

Profilin weight

is

(12-15

have

been

urchin

Acanthamoeba it

kDa) (3)

that

and

actin

was known

ly,

succeeded

in

*The nucleotide sequence appear in the DDBJ, EMBL Databases under the accession +To whom all PAGE,

correspondence

The abbreviations sodium dodecyl

eukaryotic

(1,

profilin

(6). of

In

low

Very

Tetrahycellular recent-

Tetrahymena

in this Nucleotide

and

paper will Sequence

be addressed.

used are: DNase, sulfate-polyacrylamide

Deoxyribonuclease; gel electrophoresis. 0006-291X/91

543

as

maintaining the

(7).

sea

such

because from

data reported and GenBank number D00813.

2),

organisms capable

be extremely

should

molecular Profilins

tissues

existed

isolating

low

polymerization.

proteins

as profilin to

a

and Saccharomyces

whether

such

of

(5)

with

mammalian

lower

Physarum interest

actin

several

various

pool

content

protein

inhibits

from

(4),

was of

a G-actin

we

actin-binding

isolated

egg

mena,

an

SDS$1.50

Cop.vright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

showing in

BIOCHEMICAL

175, No. 2, 1991

that

the

Tetrahymena

same fashion Tetrahymena

actin

structure

to

some unusual

have

tory

activity

actinin mena

lack

study,

cDNA was isolated is

such

(10,

11). should

be

investigate

(7,

DNase

I

studies in the

The sequence

learning

with

those

9)

and

inhibia-

about

proteins. of

profilin,

data

its

Tetrahy-

on

properties

Tetrahymena,

in

muscle

phalloidin,

and actin-binding

in

comparison

of

helpful

further

in

actins

Therefore,

and sequenced.

presented

with

known

polymerization divergent

as lack

actin

protein

greatly

ubiquitous

between to

actin-binding

be

with binding

actin

(8).

to

of

proteins

interactions this

known

properties,

and

RESEARCH COMMUNICATIONS

inhibited

profilins

as compared

actin-binding In

profilin

is

and tropomyosin

molecular only

profilin

as other

primary

AND BIOPHYSICAL

of

the the

Tetrahymena

of profilins

known

so far. MATERIALS

AND METHODS

Amino Acid sequence analysisTetrahymena profilin was purified as described previously (8). Five hundred ug of the protein in 500 pl of 6 M guanidine/HCl, 0.2 M Tris/HCl, pH 8.5, 10 mM EDTA, and 10 mM dithiothreitol was placed at room temperature for 1 h and dialyzed against 7 M urea. An equal volume of 10 solution which was then mM Tris/HCl pH 9.5 was added to this treated with 4 pg/ml lysyl endopeptidase (Wako Pure Chemicals) at 37-C for 4 h. The resulting fragments were separated by reversephase HPLC and one of the fragments was sequenced using a gasphase sequencer (model 477A; Applied Biosystems Inc.) equipped with a on-lined PTH-amino acid analyzer (model 120A; Applied Biosystems Inc.) Oligonucleotide Probe SynthesisBased on the amino acid sequence of the proteolytic fragment composed of 25 amino acids and the biased codon usage of Tetrahymena (12), an oligonucleoon a DNA synthesizer tide probe was synthesized (model 380A; Applied Biosystems Inc.). cDNA library construction and screeningThe cDNA was synthesized from the poly(A)+ RNA of Tetrahymena pyriformis by the method of Gubler and Hoffman (13). The resulting cDNA was cloned into the EcoRI site of ;LgtlO. The cDNA library was plated out and incubated at 37-C overnight. Recombinant phages were screened by plaque hybridization with 32P-labeled oligonucleotide probe under the same conditions as described by Takemasa, et al (14). DNA SequencingEcoRI inserts of the positive clones were subcloned into the EcoRI site of pUC18, and the nucleotide sequence was determined by the dideoxy chain termination method (15). Southern and Northern blot analysisSouthern and Northern hybridization were carried out under the same conditions as described by Takemasa, et al (14). Determination of the molecular weight of Tetrahymena profilinSDS-PAGE was performed by the method of Weber and Osborn and gel filtration chromatography was performed with a (161, 544

Vol.

175,

No.

BIOCHEMICAL

2, 1991

AND

BIOPHYSICAL

Superose 6 column (Pharmacia) which guanidine/HCl, 50 mM sodium phosphate,

RESULTS Since are

the

other

N-terminus

known

proteolytic

of

profilins

fragments

COMMUNICATIONS

was equilibrated pH 8.0, buffer.

with

6 M

blocked

as

AND DISCUSSION Tetrahymena

(17, (see

RESEARCH

profilin

20-22),

"MATERIALS

we

was

sequenced

AND METHODS")

one with

of

an amino

A Y N N TAG AAC AAC

Y Q I D V E G TAC CAA ATC GAT GTT GAA GGT T C C

+ +

4 +

100 bp C GAAAAAAATTAAATAAAATA

GGAAGAACAAACAAACAAACAATCAAACAATCACA

60

ATAATGAGCGGCTGGGATCAATACGTCTAATACCTTACCTTACTGCCAACTAACAAGTTG~TAC MSGWDQYVQYLTANQQVEY

120 10

GGTCTTATCTTGGGTAAGACTGACGGTACCATCTGGGCTTCCAATGTTGGTTT~CCACT GLILGKTDGTIWASNVGLTT

180 30

CTTTACAACAATTACTAAATCGATGTTGAAGGTCAAAAGGCCGCT L Y .._. N ____ N__.. ?! A ____T __...0 . P . .E . .G. . Q K

240 50

A

NVNETA

AACTTGCTTGCTGCCATGAACAACAACGGTGTGTCCCCACTGACCCTCTCTGCGGTATCAGA NLLAAMNNNGVPTD PLCGIR

300 70

ATTATGAACCAAAAGTACTACACCGTCAAGTACGATGCTGACTCCC~GTCTGGTATCTT IMNQKYYTVKYDADSQVWYL

360 00

AAGAAGGATCACGGTGGTGCCTGTATTGCCATTACC~CCAAGCTTTGGTCATCGGTACC KKDHGGACIAITNQALVIGT

420 110

TTCGATATTACTAAAAMTAATAGAACGGTGTTGCTCAAAG FDITKKQQNGVAQNI'GQVNK

480 130

GTCGTCGAAAGTCTCGCTGCCACCTTAAAGCAAGCTGGTTCTTCATGCTC V V E SLAATLKQAGY*

540 153

TGATCGCAACTTCACTAATATAAATTTTAATTATTATT~ACTTAAGTTCCCTTT~ATATG

600

TTTTATTTGTGTTTCCAATTCGTATCCACCTCTATCCTTTTTTTAGM~TACGACTCAC

660

Fig.1

Sequencing strategy and nucleotide sequence Jf the Tetprofilin cDNA. (A) synthetic oligonucleotide probe (lower) with corresponding amino acid sequence of the proteolytic fragment (upper). Nucleotide mixtures were used at three positions. (B) Restriction map of the Tetrahymena profilin cDNA and sequencing strategy. The coding region is indicated by an open box. (C) Nucleotide sequence and the deduced amino acid sequence of the Tetrahymena profilin cDNA. Amino acid sequence of proteolytic fragment determined by amino acid sequencer is underlined, and the region corresponding to oligonucleotide probe is indicated by a broken line.

rahymena

545

its

Vol.

BIOCHEMICAL

175, No. 2, 1991

acid

We then

sequencer.

based

on the

(Fig.

lA),

200 out

sequence

and

100,000

Inserts

from

the

strategy

one

of 153

amino

of

the

analyzed

be

the

cDNA

homology

Fig.

except

This

for

is

those

12.8

method

of

Laemmli

(Fig.

2A)

2B).

From

these

A kD

this

Tetrahymena

probe,

About

and sequenced

by

complete

DNA sequence

of

Fig.

1C.

frame

The

the

insert

which the (Fig.

profilin.

No

sequences

the

from

protein the

kDa as estimated (8).

contains

codes

for

sequence

lC),

it

is

EMBL and

is

calculated

as

that

thought

significant

in

660

a protein

same

by gel

previously by gel

However, by the

the

method

filtration

results,

prof 'ilin

Testrahymena

of

to

sequence GenBank

data-

of profilins.

estimated and

probe

subcloned

includes

Tetrahymena

with

usage

using

The

fragment

different

of those

it

mass of

weight with

in

reading

Since

was found

oligonucleotide

codon

were 1B.

shown

proteolytic

The molecular Da.

clones in

is

of

biased

RESEARCH COMMUNICATIONS

were positive.

positive

acids.

an

a cDNA library

and one open

of

the

plaques

inserts

nucleotides

bases

and

outlined

the

synthesized

screened

of

AND BIOPHYSICAL

is not

it

molecular

electrophoresis of

using

16,785

& Osborn

(15.5

chromatography

(17.3

kDa)

that

the

kDa but

is

molecular

18,785

Da.

the

Da agrees

Weber

appears 12.8

reported

value of

to be 16,785

kDa) (Fig.

mass Profilins

6 kD

Relative

kD

mobility

K av

Fig.2 Determination of the molecular weight of Tetrahymena The molecular weight was estimated by mobility of profilin. electrophoresis according to the method of Weber i% Osborn (16)(A) of Tetand by gel filtration chromatography (B). The positions rahymena profilin are indicated by arrowheads. The electrophoretic pattern (A, left) is also shown. M; marker, P; Tetrahymena profilin. 546

of

Vol.

175,

No.

BIOCHEMICAL

2, 1991

AND

A

BIOPHYSICAL

kb

B

RESEARCH

COMMUNICATIONS

kb

-23.6

-96 -66 -43 -22

_=1 : -a2

EHB

Fig.3 Southern and Northern blot analyses. (A) Southern blot Z'etrahymena genomic DNA (10 pg) digested by EcoRI (43ne analysis; (lane B) was hybridized with PEl, Hind111 (lane H), or BglII (B) Northern labeled full length cDNA of Te-trahymena profilin. blot analysis; total RNA (15 pg) was hybridized with the same

probe

used in (A).

previously

reported

are

profilin

rahymena

seems

To determine in

the

band

in

the

(Fig.

1B).

sesses

result

a single

Northern

recognized vivo

(Fig.

3B).

Next,

we examined

other

are

highly the

are

Tetrahymena ic

profilins,

lowest

also

latter

from

are

profilin but

that

among lower

has

among less

but

3A),

is

shown

among

homologies is

profilins

547

only

of

cDNA pos-

0.73

Thus,

it

in

profilin

profilins

lower

eukaryotic

while

the

other

(Table

with

lower

Zl-29%, (Table

was

transcribed

that each

kb

is proved

mammalian

themselves, with

the

macronucleus.

Tetrahymena

that

in

coincided

pyriformis

actively

and

conserved identity

which

in the

a

bands

maps of

3B).

between

shows higher

and

band

(Fig.

themselves

eukaryotic

two

a single

been

conserved

DNA,

gene

probe

When

as a probe,

Tetrahymena

of profilin

gene

was used

restriction

homologies

the

so Tet-

was performed.

(Fig.

the

analysis,

It

conserved

profilins and

not

profilins.

analysis

a pseudogene

the

and

gene is

blot

recognized

type

that

weight,

as a single-type

profilin

32P-labeled

by the

exists

shows

blot

molecular

BglII-digested

deduced

This

only In

or

DNA were

patterns

in

gene

Tetrahymena

EcoRI-

HindIII-digested with

the

Southern

cDNA for

single

kDa

to be exceptional.

whether

macronucleus,

full-length

12-15

I).

which The

former I).

eukaryotis

the

sequence

Vol.

BIOCHEMICAL

175, No. 2, 1991

Table

I.

Comparison

of

*Hu Human Bovine

identities

Bo

between

Cl

An

RESEARCH COMMUNICATIONS

various

AcI

profilins

AcII

PhA

PhP (%

95 21 21

Clypesster Anthocidaris Acanthamoeba I Acanthamoeba II Physarum A Physarum P Saccharomyces Tetrahymena

Amino (191,

AND BIOPHYSICAL

acid

22 22

23-24

24-25

23 23 20 25 20

sequences

were

Clypeaster

identity)

04 31-34

23 23 20 25 20 compared

31-34 36 33 35 32 22

38 36 36 31 23 between

egg (20), Anthocidaris Physarum (A, P)(23),

11)(21, (I, Tetrahymena

82-85 50-52 52-53

54 51 41 26

40 26-27 bovine

66 50 28

(17,

18),

42 29

Bovine Human

human

LIAQTKDASGTGHS . . . . TNLVGTGAV . . . . TNLVGTGAV

I II

Saccharomyces Tetrahymena

.HDGNV.&S.K...NL........ [email protected]........ .RAGDAwTSG...GL........ .KTDGTI@ASNVGLTTLYNNYQIDV

M

Bovine Human

Clypeaster

Anthocidaris Acanthamoeba Acanthamoeba Physarwn A Physarum P Saccharomyces Tetrahymena

I II

..VNITPAEVGILVGKDRSSFFVNGLTL ..VNITPAEVGVLVGKDRSSFYVNGLTL ., .KLQGTEGANIAKCFKSKDF.SAFMA . . .KLEGQEGPNIARCFKSKDF.TPFMS . . .AVTPAQG*TLAGAFNNA...D*IRA . . .AVSPANGAALANAFKDA...TAIRS . . . SLKAGEGAKIVNGFKDS...ASVLS . . . TLKAGEGQAIAALFKTP...ANVFA . ..SLQPNEIGEIVQGFDNP...AGLQS EGQKANVNETANLLAAMNNNGV.PTDPL

Bovine Human Clypeaster Anthocidaris Acanthamoeba Acanthamoeba Physarum A Physarum P Saccharomycea Tetrahymena FiP.4 profilins. Fen.%,

as to yet are I, from

AVP.GKTF........ TAGHANAL........

Clypeaster Anthocidaris Physarum A Physarum P

21

egg (20), Acanthamoeba Saccharomyces (6), and

22), (this paper) profilins. Identities are expressed percentages. In comparing Acanthamoeba profilin I, minimum maximum identities are shown, because amino acids are not determined at five positions (21). *The abbreviations used bovine, human, Clypeas ter egg, Anthocidaris egg, Acanthamoeba Acanthamoeba II, Physarum A, Physarum P, and Saccharomyces the left.

Acanthamoeba Acanthamoeba

SC

Optimal Optimal al (25)

PTFNITVTMTAKTLVLLMGK........EG PTFNVTVTKTDKTLVLLMGK........EG ITLQASKTAIVIAHCP........EG ITLQSSKTAIVIGHAP........EG ITVKTSK.*ILVGVYN........EK ITVKTSK.AILIGVYN........EKI VLVKTGQ.SVLIGHYN........ET ATVITGQ.CILIGYYN........EK VCVRTKQ.TVIIAHYP........PT ACIAITNQALVIGTFDITKKQQNGVAQ alignment of alignments between bovine

amino were (17,

IMNQKYYTVKYDADSQV V V G G I Q I Q V

RSQY RSQY SLGM SLGM GQGF GQGF ENGY ENGY GVQY QAGY

acid sequences of various performed by the method of et 18), human (19)) Clypeaster II) (21, 22), egg (20), Acanthamoeba (I, egg (20), Anthocidaris Physarum (A, P) (23), Saccharomyces (6), and Tetrahymena (this paper) profilins. In Acanthamoeba profilin I, five positions shown by asterisks are T or Q, P or A, G or S, S or A, and S or A Gaps are indicated by dots. Amino acids from the N-terminal. conserved in more than 9 species are shaded. For the boxed region and brakets, see the text.

548

Vol.

175,

No.

analysis

clearly

profilin

is

result

is

capable

BIOCHEMICAL

2, 1991

diverged

with

fact

that

inhibiting

the

of

that

of

such

low

homology

sus

common

eukaryotic (Fig.

region

has

lins

same

may

also

to actin framework be

will

this

as all

other

responsible

for polyproline

Further

experiments

be required

to

be well

conserved.

this

site

in

the

region

N-terminal (Fig.

In This

profi-

chemically shares it

4 brackets),

(ex. actin)

mutagenized

4).

C-terminal

is

and/or

using

test

the

N-

consen-

(Fig. in

properties

to

the

possessed

profilins the

actin

of Acanthamoeba

Because

is

profilins,

octapeptide

region

profilin

profilins to

The

(8).

be an actin-binding

(24).

phosphatidylinositol, lins.

to

profilins.

actin

other

other

known

Tetrahymena

Tetrahymena

profilin

of is

suggested in

with

the

region)

Lys-115

cross-linked

those

of

Tetrahymena

muscle

Tetrahymena

to

COMMUNICATIONS

other

of

rabbit

profilins,

been

because

the

of

4 boxed

of

polymerization

than

regions

sequences

the

RESEARCH

structure

compatible

spite

region

primary those

and C-terminal

lower

the

than

degree

In

that

BIOPHYSICAL

much more

of

a greater

shows

AND

binding

common

recombinant

to

with profi-

profilins

possibility.

ACKNOWLEDGMENTS We thank Dr. Takashi help in amino acid analysis Dr. Shin Sugiyama for his

Takagi of Tohoku University for his and computer analysis. We also thank critical reading of this manuscript. REFERENCES

Carlsson, L., Nystrom, & Lindberg, U. (1977) 2. Blikstad, I., Sundkvist, Biochem. 105, 425-433 3. Mabuchi, I., & Hosoya, 4. Reichstein, E., & Korn, 6179 5. Ozaki, K., Sugino, H., s.(1983) J. Biochem. 6. Oechsner, V., Magdolen, Acids Res. 15, 9078 7. Hirono, M., Endoh, H., (1987) J. Mol. Biol. 8. Edamatsu, M., Hirono,

L.E.,

1.

Biophys.

Res.

9. Cupples, sci.

10.

Hirono,

M.,

Proc.

Natl.

Biol.

I., 115,

g, Eriksson,

Markey, 465-483 S. (1980)

F., Eur.

H. (1982) Biomed. Res. 3, 465-476 E.D. (1979) J. Biol. Chem. 254, Hasegawa, 93,

V.,

T.,

Takahashi,

170,

S.,

J. 6174-

& Hatano,

295-298

& Bandlow,

W. (1987)

Okada, N., Numata, 194, 181-192 M., & Watanabe, Y.

& Pearlman,

83,

Sundkvist, Mol.

I.,

Commun.

C.G., U.S.

J.

O.,

Nucleic

& Watanabe,

(1990)

Biochem.

957-962

R.E.

(1986)

Proc.

Nat].

Acad,

5160-5164

Kumagai, Acad.

Y., Sci.

Numata, U.S. 86. 549

O., & Watanabe, 75-79

Y.

(1989)

Y.

Vol.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

175, No. 2, 1991

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hirono, M., Tanaka, R., & Watanabe, Y. (1990) J. Biochem. 107, 32-36 Watanabe, Y., Hirono, M., Takemasa, T., & Numata,O. (1990) Jpn. J. Protozool. 23. l-11 Gubler, U., & Hoffman, B.J. (1983) Gene(Amst.) 25, 263-279 Takemasa, T., Takagi, T., Kobayashi, T., Konishi, K., & Watanabe, Y. (1990) J. Biol. Chem. 265, 2514-2517 Sanger, F., Nicklen, S., & Coulson, A.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 Weber, K., & Osborn, M. (1969) J. Biol. Chem. 244, 4406 Nystrom, L.-E., Lindberg, U., Kendrick-Jones, J., & Jakes, R. (1979) FEBS Lett. 101, 161-165 Ampe, C., Markey, F., Lindberg, U., & Vandekerckhove, J. (1988) FEBS Lett. 228, 17-21 Kwiatkowski, D.J., & Bruns, G.A.P. (1988) J. Biol, Chem. 263, 5910-5915 Takagi, T., Mabuchi, I., Hosoya, H., Furuhashi, K., & Hatano, S. (1990) Eur. J. Biochem. 192, 777-781 Ampe, C., Vandekerckhove, J., Brenner, S.L., Tobackman, L., & Korn, E.D. (1985) J. Biol. Chem. 260, 834-840 Ampe, C., Sato, M., Pollard, T.D., & Vandekerckhove, J., (1988) Eur. J. Biochem. 170, 597-601 Binette, F., Benard, M., Laroche, A., Pierron, G., Lemieux, G & Pallotta, D. (1990) DNA Cell Biol. 9, 323-334 Vandekerckhove, J., Kaiser, D.A., & Pollard, T.D. (1989) J. Cell Biol. 109, 619-626 Feng, D.F., Johnson M.S., et Doolittle, R.F. (1985) J. Mol. Biol. 21, 112-125

550