Studies of odd bases in yeast mitochondrial tRNA: III. Characterization of the tRNA methylases associated with the mitochondria

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEIRCH COMMUNICATIONS

STUDIES OF ODD BASES IN YEAST MITOCHONDRIAL tPNA III.CHARACl-ERIZATION OF THE tRNA METHYLASES ASSOCIATED WITH THE MITCCHONDRIA C. Schneller,

J.M.

Schneller

and A.J.C.

Stahl

Laboratoire de Biochimie, Faculte de Pharmacie, 3, rue de l'Argonne, 67083 Strasbourg (France). Received

March

1,1976

Whereas mlG, m2G, m:G, m7G, T, mlA, m5C and Cm methylase activities in total cell enzyme of Saccharomyces cerevisiae using under++ with or without Mg , coii tRNA and E. coli B tRNA in reaction m G and T methylases occurred in mitochondria. Mitochondrial by their homologous enzymes ; and cytoplasmic ! z RNA cannot be methylated only mitochondrial tRNA can be met r: ylat f d in a heterologous re 1 ction by2total cell enzyme with formation of T, m C, m A and low amounts of m G and m2G. SUMMARY

:

were found rnnF;a;;drn"G

INTRODUCTION : Study presence Till

of four

now only

reported

(3).

methylases ferent

of the odd bases

distinct

the These

present

methylating

occurrence

bases

of yeast

results

prompted

in mit.

enzymatic

activities

revealed

E. coli

B- or undermethylated

E. coli

whether

it

was possible

present

methylated

homologous

or heterologous

MATERIALS

AND METHODS :

mit.

mit.

us to cha#acterize extracts.

tRNA showed

This

in mit.

the different report

and total

tRNA as substrate.

to overmethylate

tRNA with

in yeast

the

: T (1,2), m2G, m;G and miG (2). 2 2 m G and m2G methylases have been

the yeast

deals cell

the dif-

extracts

We studied

functional

with

tRNA

mit.-

using also or cyt.

enzymes.

1) Preparation of mit. tRNA from Saccharomyces cerevisiae p' 1~8-8~ protoplasts was as in ref. 4. Cyt. tRNA was either total cell tRNA purchased from BoehTinger-Mannheim or cyt. tRNA from mit. DNA less mutant ILE-EC/H 71 or from p cells ILE-EC, prepared as in (5). E. coli B tPNA was purchased from Boehringer. Undermethylated tRNA was e$$facted according to Rammler et starved for methionine during al (6) from E. coli K 12 58/161 Met- B - RC were chromatographed on the stationary phase (7). All the tPNAg preparHd, enzyme was prepared from highly purified Sephadex GlOO. The mitochond$ial mitochondria isolated from p protoplasts and the total cell enzyme was extracted as earlier described (4,5,8). medium contained Tris-HCl pH 8 10 poles, 2) The 100 ~1 methylation 2-mercaptof$hanol 0.5 pole, glutathione KC1 3 poles, MgC12 1.5 Foles, 0.3 pmole, S-adenosyl L methionine (SAM) [ Cl CH 46 Ci/mole (CEA) 3 nmoles, we 700 to 900 pg enzymatic proteins and 4 to 5 pg t R&L In some reactions are referred to as added to th$a medium 3 pmoles EDTA : these reactions a two hours incubation at + 37OC an aliquot is applied "without Mg "- After Abbreviation

: mit.

: mitochondrial

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

; cyt. 1003

: cytoplasmic.

BIOCHEMICAL

Vol. 70, No. 3,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on a Whatmann 3 MM paper disk, which is further washed 3 times with cold trichloracetic acid 5%, 2 times with ethanol 95%, dried and counted for radioactivity by liquid scintillation. In the conditions used, a plateau of methylation was reached. For kinetic measurements 200 pg undermethylated E. coli tBNA were methylated for 5 to 40 minutes by 500 pg enzymatic proteins. 3) The analysis of the methylated bases formed with the different enzymes, was performed submitting 1 ml of the above mentioned methylation medium, at the end of the reactions, to phenolic extractions ; the modified tBNA was then purified by Sephadex ~25 chromatography, dialysed and dried under vacuum. It was however more convenient to elute the modified tBNA from the disk filters with triethylamine bicarbonate 0.1 M pH 9.7, after removing the scintillator with toluene and ethanol 95%, and drying. The elution was repeated twice and the IZNA was recovered by drying under vacuum. 4) Two dimensional thin layer chromatography on cellulose plates (Schleicher and SchUll G1440) of methylated tBNA formic acid hydrolysates (9) was accordi f g to2Munn et 31 (19). Tlje bages wFre iaentified by cochromatoand graphy of m G, m G, m G, m G, m A, m A, m A, m C, m hX, (Cyclochemicals Sigma), and T formed i y formic acid hydrolysis of ribothymidine (Cyclochemicals). The position of the different bases was verified by their alkaline and acidic spectra ; they were essentially similar to those reported in ref. 10. The radioactive spots were detected using one month long autoradiofor radioactivity by liquid scingraphs ; they were then scraped and counted tillation. Less than 4% radioactive material was present at the origin. RESULTS : 1) Overall

yield

undermethylated

of E. coli

E. coli

B tBNA by respectively Total

tBNA methylation

(Table

tBEA can be 7 and 4 times mit.

and total

Ia,b)

shows that

more methylated

than

E. coli ++ of Mg .

cell

enzymes in the presence ++n , when compared to reactions Mg

cell

enzyme in reactions "without ++ the presence of Mq , methylates more E. coli B tBNA, whereas mit. gives "without Mg++,a a very poor methylation if significant (Table Measurement tIWA

of methylase

in the presence

methylating

activity

compared

(5 pmoles

to total

cell

2) Methylase methylated cell

enzyme

indicates

with that

in the presence with

total

enzyme whereas poorly

those

cell well

with

undermethylated

mit.

extracts

contain

per

mn and per

half

enzyme Ia). E. coli of

mg of proteins)

(11 pmoles/mn/mg). revealed

with

E. coli

undermethylated besides

of ~g++,

m7 G, mlA and m5C methylases

revealed very

formed

shown)

that

E. coli

activities

T, m2G, m;G, mlG methylases,

revealed i.e.

(not

CH3 incorporated

extracts

activities

bases

Mg++ i.e.

activities ++ reveals

of Mg

in

which

appear II).

"without

found

: Analysis

tFWA in presence revealed

are activities

(Table

enzyme in presence revealed

best

there

tEEA

in reaction

of the of total

in the presence

of

not or poorly "without Mg++ II

Furthermore the methylases ++ of w are found in the mit. ++,, Ug are not (m7G methylase)

or

(m5C, mlA methylases) (Table II). It must be noticed that m2G and m2G methylases in the two with E. coli B tBNA we found essentially enzymatic extracts in the presence of Mgu2 , and mlA and m5C methylases with ++,I the total cell enzyme "without Mg (not shown). This explains also why total

1004

Vol. 70, No. 3,1976

BIOCHEMICAL

AND BIOPHYSICAL

Table I : Overall yields of various tRNA+r$ethylation mitochondrial enzymes with or without Mg . Results methyl incorporated per nmole tRNA.

Total with

a. E.coli

B tRNA

b. E.coli undermethylated tRNA

Mg

++

enzyme without

Mitochondrial Mg

++

Mg

without

Mg

4

1060

400

525

90

95

14

0

0

4

0

0

Table II : Base analysis of E. coli undermethylated dr&l tRNA methylated with total or mitochondrial are given in percentage of recovered MS - Results

E.coli

undermethylated

enzyme ++ Mg

or

enzyme

75


without

with

++

340

d. Cytoplasmic tRNA

with

in presence of total are given in pmoles

240

c. Mitochondrial tRNA

Total ++ Mg

RESEARCH COMMUNICATIONS

tRNA and yeast mitochonmethylases with or without radioactivity.

Mitochondrial tRNA

tRNA

Mitochondrial enzyme ++ -I-+ with Mg without Mg

Total

enzyme ++ With Mg

m7G

0

3

0

0

0

1 mG

5

1

6

1.5

0

2 mG

2

1

6.5

1

3

2 mG 2

13

5

8.5

3

1.5

5 mC

Cl

mA1 T

1.5 77

27

0

<1

7

17

0

41

38

45

79

92

48.5

1005

-I-+

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1 : RPC 5 chromatograptty (5) of yeast total yeast methylases with [ H]CH~ SAM.

cell

enzyme gives

compared results (not cell

increased

to the reaction using

the

with

nucleosides

Mg++

of E. coli

(Table

in mit.

Ia).

chromatography

methylase shown) ; furthermore'Cm enzyme as already reported (12),

detected

"without

and heterologous

RPC 5 chromatography

could

(11) total be

(Table

tRNA with

mit.

of methylated

(not

mit.

d) with

enzymes.

species

This

was cyt.

However

mit.

in the presence

the methyl

counts

were

tRNA species

are methylated

tRNA (experiment

performed

several peaks (figure 5 T, m C and mlA besides low

or

of yeast

shown).

enzyme specially that

IC,

of

assosince

with

The methylated bases formed 2 2 formation of m G and m2G 1).

II).

DISCUSSION

:

1) The main result

m;G and T methylases

of this in yeast

l- the lack of methylation w++ll in presence of mit. cell

in

: no significant

reactions

tRNATrp

c3H] CH3 SAM) yielded

.-.

reactions

of some purified

tRNA could be methylated with total cell ++ We verified by gel electrophoresis Mg ciated to the peak of tRNA. Several mit.

essentially

be revealed

no O'-methylases

methylation

confirmed

(Table

these

to Rogg et al.

could

whereas

by lack of methylation Ala tRNA : tRNAPhe, tRNA , tRNAASP

were

we confirmed

according

by

Mg ++ II when

B tRNA "without

Finally

activity

could be obtained in homologous ++,I . Mg we could not methylate cyt.

methylation

tRNA methylated

enzyme.

3) Homologous

also

methylation

mitochondrial

enzyme

enzyme.

;

report

is

the finding

mitochondria.

of normal enzyme,

This

E. coli

while

result

of active is

mlG, m2G,

strengthened

B tRNA in reactions

m5C and mlA are formed

by :

"without by the total

2- the formation of m5C and mlA in mit. tRNA with total cell ++ has opposite effects in the reactions of cyt. mlA and

Note that Mg

1006

Vol. 70, No. 3, 1976

m5C methylases rences

BIOCHEMICAL

with

E . coli

m5C and mlA sites not

need Mg

mitochondria (14),

thow

it

is

are

mitochondria

not known

and its

in

low

used conditions

where

enzyme.

et al

tained

(3)

the

as cyt.

essentially

expected

since

occur

This

may be an indication

the

same enzymes,probably

would

also

in this

heterologous

This

mit.

actually

in progress

contrast

not

find

a

nuclear than is

for

coded

ACKNOWLEDGEMENTS : we thank G. fied yeast tRNA. This work was the Scientifique et Technique Recherche Scientifique and the 1'Energie Atomique.

(3).

rather

shows that methylation

the

mit.

tRNA as

of mit.

can be somewhat

puzzling

existence

because

methylases

nuclear

however mit.

would

of

be

in mit.

formation

of T

enzyme does not

of an active

of distinct

the mit. are

of the bases

(1,2),

the presence

they

methylate

enzyme.

tRNA. The low formation

Analysis

sequence

to know whether

con-

G25 and DEAE-

m2G and m;G methylases

1T per

the

and if

in mit.

of

which

we could

However

of

methylases,

An inhibitor

by Sephadex that

in

analysis

mit.

extracts,

reactions

and mit.

despite

with

we

of incompletely modified sites and may would then be too low for detection.

but

cyt.

reaction

are distinct

mit.

in mit. detected

yeast

m5C and mlA formation

not present

tRNA in vitro

may be an indication

counterparts

in

of

enzyme since

agreement

our mit.

in vivo.

possible.

that less

explain

more with

methylated are

present

are well

Studying crude

in homologous

enzyme also,

a little

in full

in their

m2G and m;G may be due to methylation

methylate

that

by cyt.

and was not purified

This

enzyme is

well

tRNA yielded

mlA

organisms

we could

m2G and rnz G methylases.

of methylation

mit.

(13),

methylases

III’A

are

tRNA 10 times

the bases

with

be emphasized

tRNA (2).

present

membranes

cell

must

m5C and mlA methylases in mit.

tRNA are fully

tRNA by total

and mit.

in higher

may be due to evolution

contamination

the results

present

E. coli

3) Absence

It

of m5C and

cyt.

chromatography.

undermethylated

diffe-

methylable

chloroplastic

mitochondria

of a low

have been

mitochondrial

report

mitochondria

systems.

result

found

might

cellulose

from

and chloroplasts

of a mlA methylase

the HeLa cells

Finally

bases

mitochondria

latter

in yeast

activities

be the

the methylated

This

mitochondria.

enzyme may well

Smolar

in the

absence

in yeast

methylation

if

and in plant

biosynthesis

2) The very

may result ++ need Mg so that

tRNA would

The presence

report

mLA methylase

cell

(13)

different.

to Klagsburn's

tRNA.

RESEARCH COMMUNICATIONS

may fit to the enzyme, whereas these sites on E . coli tRNA ++ . However a mlA methylase has been characterized in the

the mitochondrial

total

: mit.

of HeLa cells

methylases

well

B tRNA or mit.

in tRNA conformations

would

AND BIOPHYSICAL

T methylase.

enzymes. and their

Work is cytoplasmic

coded.

Dirheimer and coworkers for their gift of puripartly supported by the Delegation a la Recher(no 72-7-0179), the Centre National de la Departement de Biologie du Commissariat a

1007

Vol. 70, No. 3, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES : 1. Schneller, J.M., Faye, G., Kujawa, C. and Stahl, A.J.C. (1975) Nucleic Acids Res., 2, 831-838. 2. Martin, R., Schneller, J.M., Stahl, A. and Dirheimer, G. (1976) Biochem. Res. Commun. This issue. Biophys. 3. %olar, N. and Svensson, I. (1974) Nucleic Acids Res., L, 707-718. 4. Schneller, J.M., Martin, R., Stahl, A. and Dirheimer, G. (1975) Biochem. Biophys. Res. Commun., 2, 1046-1053. 5. Accoceberry, B., Schneller, J.M. and Stahl, A.J.C. (1973) Biochimie, 55, 291-296. 6. Rammler, D.H., Okabayashi, T. and Delk, A. (1975) Biochemistry, 4, 1994-2002. 7. Mandel, L.R. and Borek, E. (1961) Biochem. Biophys. Res. Commun., 4, 14-18. 8. Schneller, J.M., Accoceberry, B. and Stahl, A.J.C. (1975) FEBS Lett., 53, 44-48. 9. Munns, T.W., Podratz, K.C. and Katsman, P.A. (1974) Biochemistry, 13, 44094416. 10. Munns, T.W., Podratz, K.C. and Katsman, P.A. (1973) J. of Chromatography, 16, 401-406. 11. Rogg, H., Brambilla, R., Keith, G. and Staehelin, M. (1976) Nucleic Acids Res., 3, 285-295. 12. Svensson, I. and Patel, V.J. (1975) Nucleic Acids Res., 2, 603-611. 13. Klagsbrun, M. (1973) J. Biol. Chem., 248, 2606-2611. 14. Dubois, E., Dirheimer, G. and weil, J. (1974) Biochim. Biophys. Acta, 2, 332-341.

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