The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases
Animal and fungal cells (in contrast to prokaryotes) contain two distinct sets of related aminoacyl-tRNA synthetases (aaRSs) encoded by nuclear genes and functioning in cytosol and mitochondria. The structural differences between mitochondrial and cytoplasmic enzymes may reflect the functional adapt...
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2000
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| Zitieren: | The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases / L.L. Sidorik, T.A. Rybkinska, N.G. Bakhiya, N.V. Rodnin, V.V. Filonenko, N.S. Entelis, I.A. Tarassov, R.P. Martin, G.Kh. Matsuka // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 363-368. — Бібліогр.: 26 назв. — англ. |
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irk-123456789-1528332019-07-05T19:10:55Z The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases Sidorik, L.L. Rybkinska, T.A. Bakhiya, N.G. Rodnin, N.V. Filonenko, V.V. Entelis, N.S. Tarassov, I.A. Martin, R.P. Matsuka, G.Kh. Структура и функции биополимеров Animal and fungal cells (in contrast to prokaryotes) contain two distinct sets of related aminoacyl-tRNA synthetases (aaRSs) encoded by nuclear genes and functioning in cytosol and mitochondria. The structural differences between mitochondrial and cytoplasmic enzymes may reflect the functional adaptation to fulfil mitochondrial processes in addition to protein synthesis. Mitochondrial import of nuclearencoded tRNAs has been described in yeast, plants and protozoans but it has not been observed in mammalian cells. Ifs established that mitochondrial lysyl-tRNA synthetase (MSK) plays a prominent role in the transport of tRNA into yeast mitochondria for complementation o f mitochondrial tRNAs genes mutations. We tried to identify MSK homologues in mammalian cells with the help of monospecific antibodies against pre-MSK by ELISA and Western-blot analysis. We have identified cross-reactive proteins in mitochondrial and cytoplasmic fractions of mammalian cell lysates. These data, together with the results of cross-aminoacylation on mitochondrial and cytoplasmic tRNAs, suggest the presence of common antigenic determinants in the mitochondrial and cytoplasmic lysyl-tRNA synthetases from higher animals. Клітини еукаріот на відміну від прокаріот містять дві різні групи аміноацил-тРНК синтетаз, які кодуються ядерним геномом та функціонують в цитозолі і мітохондріях. Структурні відмінності між ферментами мітохондрій і цитоплазми можуть бути відображенням функціональної адаптації до процесів, які відбуваються в мітохондріях, крім участі в біосинтезі білка. Імпорт цитозольних тРНК у мітохондрії описано для дріжджів, рослин і найпростіиіих, однак він не спостерігався в клітинах ссавців. Виявлено, що мітохондріальна лізил-тРНК синтетаза (MSK) відіграє провідну роль у транспорті тРНК у мітохондрії дріжджів для комплементації мутацій мітохондріальних генів тРНК За допомогою моноспецифічних антитіл проти npe-MSK ми зробили спробу ідентифікувати гомологи MSK у клітинах ссавців методами ELISA і Вестерн-блотинга. В цитоплазматичних і мітохондріальних фракціях лізатів клітин ссавців нам вдалося виявити білки, які мають імунологічний перехрест з MSK Разом з результатами перехресного аміноацилювання ці дані дають підставу для припущення щодо наявності спільних антигенних детермінант у мітохондріальних і цитоплазматичних лізил-тРНК синтетаз ссавців. Клетки эукариот (в отличие от клеток прокариот) содержат две различные группы аминоацил-тРНК синтетаз, кодируемых ядерным геномом и функционирующих в ц и то золе и митохондриях. Структурные отличия между ферментами митохондрий и цитоплазмы могут быть отражением функциональной адаптации к процессам, происходящим в митохондриях помимо участия в биосинтезе белка Импорт цитозольных тРНК в митохондрии описан у дрожжей, растений и простейших, однако не наблюдался в клетках млекопитающих. Установлено, что митохондриальная лизил-тРНК синтетаза (MSK) играет ведущую роль в транспорте тРНК в митохондрии дрожжей для комплементации мутаций митохондриальных генов тРНК. С помощью моноспецифических антител против npe-MSK мы попытались идентифицировать гомологи MSK в клетках млекопитающих методами ELISA и Вестерн-блотинга. В цитоплазматических и митохондриальных фракциях лизатов клеток млекопитающих нам удалось обнаружить белки, имеющие иммунологический перекрест с MSK В совокупности с результатами перекрестного аминоацилирования эти данные дают основание предположить наличие общих антигенных детерминант у митохондриальных и цитоплазматических лизил-тРНК синтетаз высших животных. 2000 Article The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases / L.L. Sidorik, T.A. Rybkinska, N.G. Bakhiya, N.V. Rodnin, V.V. Filonenko, N.S. Entelis, I.A. Tarassov, R.P. Martin, G.Kh. Matsuka // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 363-368. — Бібліогр.: 26 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.00057E http://dspace.nbuv.gov.ua/handle/123456789/152833 577.1 en Биополимеры и клетка Інститут молекулярної біології і генетики НАН України |
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Структура и функции биополимеров Структура и функции биополимеров |
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Структура и функции биополимеров Структура и функции биополимеров Sidorik, L.L. Rybkinska, T.A. Bakhiya, N.G. Rodnin, N.V. Filonenko, V.V. Entelis, N.S. Tarassov, I.A. Martin, R.P. Matsuka, G.Kh. The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases Биополимеры и клетка |
| description |
Animal and fungal cells (in contrast to prokaryotes) contain two distinct sets of related aminoacyl-tRNA synthetases (aaRSs) encoded by nuclear genes and functioning in cytosol and mitochondria. The structural differences between mitochondrial and cytoplasmic enzymes may reflect the functional adaptation to fulfil mitochondrial processes in addition to protein synthesis. Mitochondrial import of nuclearencoded tRNAs has been described in yeast, plants and protozoans but it has not been observed in mammalian cells. Ifs established that mitochondrial lysyl-tRNA synthetase (MSK) plays a prominent role in the transport of tRNA into yeast mitochondria for complementation o f mitochondrial tRNAs genes mutations. We tried to identify MSK homologues in mammalian cells with the help of monospecific antibodies against pre-MSK by ELISA and Western-blot analysis. We have identified cross-reactive proteins in mitochondrial and cytoplasmic fractions of mammalian cell lysates. These data, together with the results of cross-aminoacylation on mitochondrial and cytoplasmic tRNAs, suggest the presence of common antigenic determinants in the mitochondrial and cytoplasmic lysyl-tRNA synthetases from higher animals. |
| format |
Article |
| author |
Sidorik, L.L. Rybkinska, T.A. Bakhiya, N.G. Rodnin, N.V. Filonenko, V.V. Entelis, N.S. Tarassov, I.A. Martin, R.P. Matsuka, G.Kh. |
| author_facet |
Sidorik, L.L. Rybkinska, T.A. Bakhiya, N.G. Rodnin, N.V. Filonenko, V.V. Entelis, N.S. Tarassov, I.A. Martin, R.P. Matsuka, G.Kh. |
| author_sort |
Sidorik, L.L. |
| title |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases |
| title_short |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases |
| title_full |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases |
| title_fullStr |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases |
| title_full_unstemmed |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases |
| title_sort |
immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-trna synthetases |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2000 |
| topic_facet |
Структура и функции биополимеров |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152833 |
| citation_txt |
The immunochemical cross-reactivity between cytoplasmic and mitochondrial mammalian lysyl-tRNA synthetases / L.L. Sidorik, T.A. Rybkinska, N.G. Bakhiya, N.V. Rodnin, V.V. Filonenko, N.S. Entelis, I.A. Tarassov, R.P. Martin, G.Kh. Matsuka // Биополимеры и клетка. — 2000. — Т. 16, № 5. — С. 363-368. — Бібліогр.: 26 назв. — англ. |
| series |
Биополимеры и клетка |
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ISSN 0233-7657. Биополимеры и клетка. 2000. Т. 16. № 5
The immunochemical cross-reactivity between
cytoplasmic and mitochondrial mammalian
lysyl-tRNA synthetases
L. L. Sidorik, T. A. Rybkinska, N. G. Bakhiya, N. V- Rodnin, V. V. Filonenko,
N. S. Entelis1, I. A. Tarassov1, R. P. Martin1, G. Kh. Matsuka
Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine
Acad. Zabolotnoho vul., 150, Kyiv, 03143, Ukraine
!UPR 9005, IBMC CNRS
Rue Rene Descart, 15, Strasbourg, France
Animal and fungal cells (in contrast to prokaryotes) contain two distinct sets of related aminoacyl-tRNA
synthetases (aaRSs) encoded by nuclear genes and functioning in cytosol and mitochondria. The structural
differences between mitochondrial and cytoplasmic enzymes may reflect the functional adaptation to fulfil
mitochondrial processes in addition to protein synthesis. Mitochondrial import of nuclear-encoded tRNAs
has been described in yeast, plants and protozoans but it has not been observed in mammalian cells. Its
established that mitochondrial lysyl-tRNA synthetase (MSK) plays a prominent role in the transport of
tRNA into yeast mitochondria for complementation of mitochondrial tRNAs genes mutations. We tried to
identify MSK homologues in mammalian cells with the help of monospecific antibodies against pre-MSK
by ELISA and Western-blot analysis. We have identified cross-reactive proteins in mitochondrial and
cytoplasmic fractions of mammalian cell lysates. These data, together with the results of cross-
aminoacylation on mitochondrial and cytoplasmic tRNAs, suggest the presence of common antigenic
determinants in the mitochondrial and cytoplasmic lysyl-tRNA synthetases from higher animals.
Introduction. Aminoacyl-tRNA synthetases (aaRSs)
(EC 6.1.1) — are a heterogeneous family of enzymes
differing in size and subunit s tructure but catalyzing
the same reaction of the selective a t tachment of amino
acids to their cognate tRNAs. T h e animal and fungal
cells (in contrast to prokaryotes) contain two different
sets of related aaRSs encoded by nuclear genes, one
set localized in the cytosol and functioning in the
cytoplasmic protein synthesis while the second lo
calized in mitochondria and functioning in the or
ganella translation. In addit ion, plant cells posses the
third set of aaRSs involved in the chloroplastic protein
synthesis [1 , 2 ] .
T h e molecular and catalytic properties of cyto
plasmic enzymes have been extensively studied over
© L. h. SIDORIK, T. A. RYBKINSKA, N. G. BAKHIYA,
N. V. RODNIN, V. V. FILONENKO, N. S. ENTELIS,
I. A. TARASSOV, R. P. MARTIN, G. Kh. MATSUKA, 2000
the past 20 years . However, the information about
mitochondrial aaRSs remained scarce so far. Studies
of the organelle-specific synthetases a re important for
several reasons . T h e information about sequences of
these enzymes contr ibute to bet ter unders tand ing of
the evolution of this enzyme family. On the other
hand , the structural differences between the mito
chondrial and cytoplasmic enzymes reflect the func
tional adaptat ions to fulfil mitochondrial processes in
addition to the protein synthesis . For example, in
fungi, certain mitochondrial aaRSs a re involved in
either the splicing of mitochondrial RNAs [1 , 2 ] , or
the import of tRNA from the cytoplasm [3 ].
In the yeast Saccharomyces cerevisiae, the se
lective import into mitochondria of one of the two
cytoplasmic lysine isoacceptors, t R N A L y s C U U , requi
res the integrity of the mitochondrial pre-protein
import machinery as well as soluble cytosolic factors
[4] . One of the essential import factors has been
identified as the precursor of the mitochondrial lysyl-
363
SIDORIK L L. ET AL.
tRNA synthetase (pre-MSK). Fur thermore , it has
been shown that pre-MSK can selectively bind to the
aminoacylated form of t R N A L y s C U U (and to im
portable mutant tRNAs) and is, therefore, likely to
act as a carrier for t ransport of the tRNA across the
mitochondrial membrane [5 ] .
T h e mi tochondr ia l import of nuc lear -coded
tRNAs appears to be a widely spread process among
eukaryotes since it has been described not only in
yeast but also in plants and protozoans [6, 7 ] .
However, this process is likely not to occur in
mammalian cells. Establishing artificial system of
tRNA mitochondrial import in human cells could be
of high biomedical interest because several human
mitochondrial neurodegenerative and muscular dise
ases (such as MERRF and MELAS syndromes and
several cardiomyopathies) are associated with point
mutations in mitochondrial tRNA genes [8 ]. Indeed,
theoretically such defects should be cured by exp
ressing functional tRNAs in the nucleus and impor
ting them from the cytoplasm to mitochondria.
Because the mitochondrial lysyl-tRNA synthetase
plays a prominent role in the transport of tRNA into
yeast mitochondria, we tried to identify MSK homo-
logues in mammalian cells with the help of mono
specific polyclonal antibodies directed against the
yeast pre-MSK by ELISA and Western-blot analysis.
We have identified cross-reactive proteins in mito
chondrial and cytoplasmic fractions of mammalian
cells lysates possessing an electrophoretic mobility
similar to that of yeast pre-MSK. These data , toge
ther with the results of cross-aminoacylation studies
on mitochondrial and cytoplasmic tRNAs, suggest the
presence of common antigenic determinants in mito
chondrial (mt) and cytoplasmic (ct) lysyl-tRNA syn
thetases (LysRSs) from the mammals and allow us to
suppose that mammalian mitochondrial and cyto
plasmic lysyl-tRNA synthetases could be encoded by
the same gene.
Materials and Methods. Protein G-Sepharose was
from «Sigma-Aldrich» (USA); anti-rabbit IgG labelled
by horse-radish peroxidase from «DAKO» (Den
mark) ; unfractionated yeast cytoplasmic and Escheri
chia coli tRNAs were from «Boehringer-Mannheim»
(Germany); the immunoblotting detection ECL sys
tem and [ 1 4 C]-Lys from «Amersham» (USA); all
other chemicals were from «Мегск» (Germany) , «Sig-
ma» (USA) and «Віо-Rad» (USA).
The high molecular weight complex of cyto
plasmic aaRSs was purified from bovine and rabbit
liver by the immunoprecipitation method on the
affinity column with coupled monoclonal antibodies
against rabbit cytoplasmic GluRS accordingly to the
described method [9] .
Recombinant pre-MSK was obtained using the
Pichya pastoris expression system («Invitrogen») and
purified on DEAE-cellulose and Mono-Q Sepharose.
Isolation of mammalian mitochondria and prepa
ration of a mitochondrial crude extract Mitochondria
from rabbit liver were obtained as described in [10]
with some modifications. Rabbit liver was briefly
homogenized in buffer A: 25 mM T r i s / H C l , pH 7.5,
2 mM EDTA, 1.5 mM CaCl 2 , 250 mM sucrose. Nuclei
and cell debris were removed by two 5 min sequential
centrifugation at low speed (1000 x g for 5 min) and
mitochondria were collected by high-speed centri
fugation (10000 xg for 10 min) and washed three
times with 250 mM sucrose in Т Е buffer (25 mM
T r i s / H C l , pH 7.5, 2 mM EDTA) . T h e mitochondrial
fraction in 250 mM sucrose and Т Е was layered on a
sucrose step-gradient consisting 50 %, 36 % and
20 % sucrose in buffer В (50 mM T r i s / H C l , pH 7.5,
2 mM EDTA, 1 g/1 bovine serum albumin) and
centrifuged at 25000 rpm (1 h, rotor Beckman SW27).
Intact mitochondria collected from the 36 % sucrose
layer were diluted in 4 volumes 0.5 M sorbitol and
centrifuged at 22000 x g for 15 min.
To obtain a mitochondrial crude extract, pelleted
mitochondria were suspended in buffer С (25 mM
potassium phosphate, pH 7.5, 10 mM MgCl 2 , 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 %
glycerol) and disrupted by sonication. Membranous
debris were removed by centrifugation at 10000 x g,
50 min.
Protein concentration was estimated by the Brad
ford method [11 ].
Mitochondrial tRNA was purified from bovine
liver mitochondria according to the method [12] .
Preparation of antibodies against pre-MSK. The
partially purified protein (50 jug) was electrophorized
in 12 % PAAG-SDS gel [13] and transferred onto a
nitrocellulose filter. T h e colored band with apparent
molecular weight consistent with that of pre-MSK was
cut, dissolved in DMSO and used for immunization.
Rabbit was immunized by 30—40 jug of pre-MSK
extracted in DMSO with complete Freund ' s adjuvant
(1:1, v /v ) . A booster of 30 jug of antigen in incomplete
Freund ' s adjuvant was administered twice at 3 weeks
intervals. The serum was collected, and immuno
globulins were precipitated with ammonium sulfate
buffer at 50 % of saturat ion, and then purified by
three chromatographic steps on DEAE-cellulose, on
Protein G-Sepharose (according to the Firm protocol)
and on an affinity column with coupled antigen [14]
to obtain high-affinity and specific anti-pre-MSK
antibodies.
The specificity of antibodies fractions obtained at
each purification step was controlled by the ELISA
364
method and their purity was estimated by 12 %
PAGE-SDS electrophoresis.
Western-blot analysis. Lysates (cytoplasmic and
mitochondrial) of cells and organs were electro-
phorized in 12 % PAAG-SDS gel and transferred
onto a nitrocellulose membrane . Washing and blocka
ge of the membrane was done in TBS-T-M buffer
(TBS buffer with 0Л % Tween-20 and 5 % dry milk)
during at 1 h at 25 ° С Incubation with antibodies in
TBS-T-M was during 2 h at 25 ° С After extensive
washing with TBS-T-M, the filters were incubated
with anti-rabbit IgG labelled by peroxidase in TBS-
T-M during 2 h at 25 ° С After extensive washing
with TBS-T, the fluorogene ECL was added to the
filters (according to the firm protocol) and after
10 min of incubation filters were autographied on an
X-ray film («Kodak») for visualization of the signals.
Aminoacylation assay. Lysyl- tRNA synthetase
aminoacylation test was performed in 0.1 ml of the
mixture containing 100 mM T r i s / H C l , pH 7.5,
30 mM KC1, 10 mM MgCl 2 , 2 mM A T P , 60 / Ш
[ 3 H]-Lysine (60—80 Ci /mmol , «Amersham»), with
saturating amounts of relevant unfractionated tRNA
and limiting amounts of enzyme. A unit of activity
corresponds to 1 nmol aminoacyl-tRNA formed at
37 ° С
The influence of antibodies on the aminoacy-
lating activity of the mitochondrial and cytoplasmic
lysyl-tRNA synthetases was detected according to the
method [14] .
Results and Discussion. A problem of obtaining
antibodies of high specificity and affinity is connected
with the homogeneity of antigen, the scheme of
immunization and the methods of antibodies purifica
tion. The purification of majority of recombinant
antigens is problematic without use of specific affine
ligands. In our case the absence of such a ligand for
the antigen (pre-MSK), low level of the recombinant
protein secretion by producent and disability to purify
the antigen to homogeneity put forward a problem of
working out an effective method of immunization and
following antibodies purification to produce antibodies
of high titer and specificity.
Our scheme of immunization allowed us to obtain
the antisera with titer more than 1:10000. T h e affinity
of antibodies increased after each purification stage
accompanied by simultaneous nonspecificity decre
asing. Fig. 1 and Fig. 2 04, B) represent the
immunoreactivity of the antibodies after different
stages of purification. It is obvious that even after
protein G-Sepharose chromatography (Fig. 1) the
affinity of antibodies is not enough for the antigen
detection in cell and tissue lysates. Only after their
purification on the affine column with coupled antigen
THE CROSS-REACTIVITY BETWEEN LYSYL-tRNA SYNTHETASES
1 2 3 4
Fig. 1. Immunoreactivity of anti-pre-MSK antibodies after pu
rification on DEAE-cellulose and Protein G-Sepharose columns,
determined by Western-blot analysis: / — yeast pre-MSK; 2 —
ctLysRS (high molecular weight complex from bovine liver); 3 —
lysate of mitochondria from bovine liver; 4 — post-mitochondrial
supernatant (SI00) from bovine liver; The position of protein
molecular weight standard indicated by arrows
1 2 3 1 2
Fig. 2. Immunoreactivity of monospecific anti-pre-MSK antibodies
(after immune-affine purification) determined by Western-blot ana
lysis: (Л) 1 — lysate of mitochondria from rabbit liver; 2 —
postmitochondrial supernatant (S100) of rabbit liver; 3 — yeast
pre-MSK; (В) 1 — lysate of mitochondria from human placenta;
2 — posmitochondrial supernatant (SI00) from human placenta
(pre-MSK) the affinity was considered satisfactory
(Fig. 2 ) .
The studies of antibodies immunoreactivity with
cell and tissue lysates have shown that mentioned
antibodies recognized the polypeptide band with elec-
trophoretic mobility corresponding to that of LysRS
subunit in the majority of the lysates investigated
365
SIDORIK L. L. ET AL.
kDa
45
67
Fig. 3. Immunoreactivity of monospecific
anti-pre-MSK antibodies detected by Wes
tern-blot analysis in mitochondrial and cyto
plasmic (SI00) lysates from different cells
and mammalian organs: 1 — bovine serum
25 albumin (control of non-specificity); 2 —
high molecular weight complex from bovine
liver; 3 — mitochondrial lysate from bovine
liver; 4 — cytoplasmic lysate (S100) from
rabbit liver; 5 — lysate of yeast mitochon
dria; 6 — cytoplasmic (S100) lysate of ye
ast; 7 — pre-MSK
(Fig. 3) . T h e occurence of several low molecular
weight peptides in yeast lysate (Fig. 3 , lane 6) is a
result of high level of endogeneous proteolysis in
yeast.
The study of he antibodies influence on the
enzymat ic activity of mammal ian mtLysRS and
ctLysRS in the reaction of aminoacylation did not
demonstrate any effect on this activity. One could
suggest that anti-pre-MSK monospecific antibodies
were directed against non-catalytic domains of both
enzymes investigated (data not shown).
The data of the cross-aminoacylation experi
ments (Fig. 4) have shown unusual properties of the
mammalian mitochondrial LysRS — a high level of
aminoacylation of the cytoplasmic tRNA by the mito
chondrial enzyme comparing with that for the mito
chondrial tRNAs. We have found that mtLysRS
aminoacylated the cytoplasmic tRNAs with the same
efficiency as LysRS from cytoplasmic high molecular
weight complex (ctLysRS). On the o ther hand ,
mtLysRS aminoacylated the mitochondrial tRNAs but
did not aminoacylate E. coli tRNAs in fact. In
contrast yeast mtLysRS is known to aminoacylate E.
coli tRNA with the same efficiency as yeast mito
chondrial tRNA but practically is not able to amino
acylate the cytoplasmic tRNA from yeast [15] . T h e
data published recently about expression and charac
terization of the human mitochondrial PheRS [16]
and LeuRS [17] have shown that these enzymes
expressed in functionally active monomelic form are
able to aminoacylate E. coli tRNA but less effectively
than the yeast cytoplasmic aaRSs. Whether the dis
tinct properties of mtLysRSs from higher eukaryotes
revealed are specific or common features of other
mammalian mitochondrial aaRSs — is to be eluci
dated.
Most eukaryotic cells possess at least two com-
partmentally distinct sets of aaRSs, one located in the
cytoplasm, and the other — in mitochondria (plants
have the third set of chloroplast-specific synthetases) .
In some instances the same gene can code for both
the mitochondrial and cytoplasmic aaRSs [18, 19] .
More commonly, however, the functionally equivalent
homologues are encoded by different genes. This is
the case for the two lysyl- tRNA synthetases (KRS
and MSK) of yeast. T h e gene KRS1, described by
Mirande and Waller [20] , has been shown to code for
the cytoplasmic LysRS. T h e MSK1 gene, reported by
Gatti and Tsagoloff [15] , codes for the homologous
yeast mitochondrial synthetase . However, concerning
the LysRSs from higher eukaryotes , our knowledge is
restricted to the information on the cytoplasmic
synthetases functioning in the high molecular weight
complex.
Based on the theory of Wong [21 ] that the
genetic code may have evolved «hand- in-hand» with
biosynthetic pathways for new amino acids and the
hypothesis about mitochondria as prokaryotic endo-
symbiont incorporated into ancient eukaryotic cells
[22—25], our data on the immunochemical cross-
reactivity between the cytoplasmic and mitochondrial
mammalian lysyl-tRNA synthetases taken together
with those on the cross-aminoacylation (Fig. 4) allow
us to suppose that the mitochondrial and cytoplasmic
366
THE CROSS-REACTIVITY BETWEEN LYSYL-tRNA SYNTHETASES
Fig. 4. Aminoacylation of tRNA by cytoplasmic LysRS (o) and
mitochondrial LysRS (o): The time courses of aminoacylation of
cytoplasmic tRNA by LysRS from high molecular weight complex of
rabbit liver (/) and LysRS from rabbit mitochondria (2); of
mitochondrial tRNA (3) and E. coli tRNA (4) by LysRS from rabbit
mitochondria
lysyl-tRNA synthetases of mammals could be enco
ded by the same gene. Our suggestion has been
approved by recently published data concerning the
unusual alternative splicing of the human lysyl- tRNA
synthetase gene which encodes for both the cyto
plasmic and mitochondrial enzymes [26 ].
Fur ther investigation of the mammalian mito
chondrial LysRS is important not only for solving
many questions in the protein biosynthesis, molecular
import into organelles, molecular evolution of aaRSs,
but has a practical value as many severe pathologies
are connected with mitochondrial disfunctions, espe
cially with mutations in t R N A L y s gene and defects in
the protein importing machine, with a leading role of
KRS and preMSK.
This work was supported by grant INTAS 96-
1515.
Л. Л. Сидорик, T. О. Рибкінська, H. Г. Бахія, M. В. Роднін,
В. В. Філоненко, И. С. Ентеліс, І. О. Тарасов, Р. П. Мартан,
Г. X. Мацука
Перехресна імунореактивність між цитоплазматичними
і мітохондріальними лізил-тРНК синтетазами ссавців
Резюме
Клітини еукаріот на відміну від прокаріот містять дві різні
групи аміноацил-тРНК синтетаз, які кодуються ядерним
геномом та функціонують в цитозолі і мітохондріях. Струк
турні відмінності між ферментами мітохондрій і цитоплаз
ми можуть бути відображенням функціональної адаптації до
процесів, які відбуваються в мітохондріях, крім участі в
біосинтезі білка. Імпорт цитозольних тРНК у мітохондрії
описано для дріжджів, рослин і найпростіиіих, однак він не
спостерігався в клітинах ссавців. Виявлено, що мітохон-
дріальна лізил-тРНК синтетаза (MSK) відіграє провідну роль
у транспорті тРНК у мітохондрії дріжджів для комплемен
тації мутацій мітохондріальних генів тРНК За допомогою
моноспецифічних антитіл проти npe-MSK ми зробили спробу
ідентифікувати гомологи MSK у клітинах ссавців методами
ELISA і Вестерн-блотинга. В цитоплазматичних і міто
хондріальних фракціях лізатів клітин ссавців нам вдалося
виявити білки, які мають імунологічний перехрест з MSK
Разом з результатами перехресного аміноацилювання ці дані
дають підставу для припущення щодо наявності спільних
антигенних детермінант у мітохондріальних і цитоплазма
тичних лізил-тРНК синтетаз ссавців.
Л. Л. Сидорик, Т. А. Рибкинска, Н. Г. Бахия, Н. В. Роднин,
В. В. Филоненко, Н. С. Энтелис, И. А. Тарасов, Р. Л. Мартан,
Г. X. Мацука
Перекрестная иммунореактивность между
цитоплазматическими и митохондриальными
лизил-тРНК синтетазами животных
Резюме
Клетки эукариот (в отличие от клеток прокариот) содержат
две различные группы аминоацил-тРНК синтетаз, кодируе
мых ядерным геномом и функционирующих в ц и то золе и
митохондриях. Структурные отличия между ферментами
митохондрий и цитоплазмы могут быть отражением функци
ональной адаптации к процессам, происходящим в митохонд
риях помимо участия в биосинтезе белка Импорт цитозоль-
ных тРНК в митохондрии описан у дрожжей, растений и
простейших, однако не наблюдался в клетках млекопитаю
щих. Установлено, что митохондриальная лизил-тРНК син
тетаза (MSK) играет ведущую роль в транспорте тРНК в
митохондрии дрожжей для комплементации мутаций мито-
хондриальных генов тРНК. С помощью моноспецифических
антител против npe-MSK мы попытались идентифицировать
гомологи MSK в клетках млекопитающих методами ELISA и
Вестерн-блотинга. В цитоплазматических и митохондриаль-
ных фракциях лизатов клеток млекопитающих нам удалось
обнаружить белки, имеющие иммунологический перекрест с
MSK В совокупности с результатами перекрестного аминоа-
цилирования эти данные дают основание предположить нали
чие общих антигенных детерминант у митохондриальных и
цитоплазматических лизил-тРНК синтетаз высших живо
тных.
REFERENCES
1. Akins R. S., Lambowitz A. M. A Protein required for splicing
group I introns in Neurospora mitochondria is mitochondrial
367
SIDORIK L. L. ET AL.
tyrosyl-tRNA synthetase or a derivative thereof / / Cell.—
1987.—50, N 1.—P. 331—345.
2. Labouesse J., Herbert C. Dujardin G., Slominski P. P.
Three suppressor mutations which cure a mitochondrial RNA
maturase deficiency occur at the same codon in the open
reading frame of the nuclear NAM2 gene / / EMBO J.—
1987.—6, N 2.—P. 713—721.
3. Entelis N. S., Krasheninnikov I. A , Martin R. P., Tarassov /.
A Mitochondrial import of a yeast cytoplasmic tRNALys:
possible roles of aminoacylation and modified nucleosides in
subcellular partitioning / / FEBS Lett.—1996.—384.—P. 38—
42.
4. Tarassov /., Entelis N, Martin R. P. An Intact protein
translocating machinery is required for mitochondrial import of
a yeast cytoplasmic tRNA / / J. Мої. Biol.—1995.—245.—P.
315—328.
5. Entelis N. S.t Kieffer S., Kolesnikova O. A , Martin R. P.
Tarassov I. A. Structural requiremets of tRNA L y s for its import
into yeast mitochondriaad / / Proc. Nat. Acad. Sci. USA.—
I998.—95, N 6.—P. 2838—2843.
6. Dietrich A , Weil J. #., Marechal-Drouard L. Nuclear-en
coded transfer RNAs in plant mitochondria / / Annu. Rev. Cell
Biol.—1992.—8.—P. 115—131.
7. Schneider A , Martin J., Agabian N. A nuclear encoded tRNA
of Trypanosoma brucei is imported into mitochondria / / Мої.
Cell. Biol.—1994.—14.—P. 2317—2322.
8. Luft R. The development of mitochondrial medicine / / Proc.
Nat. Acad. Sci. USA.—1994.—91.—P. 8731—8738.
9. Filonenko V. V., Deutcher M. Evidenve for similar structural
organization of the multienzyme aminoacyl-tRNA synthetase
complex in vivo and in vitro II J. Biol. Chem.—1994.—269.—
P. 17375—17378.
10. Ausenda C , Chromyn A. Purification of mitochondrial DNA
from human cell cultures and placenta / / Meth. Enzymol.—
1996.—264.—P. 122—128.
11. Bradford M. M. A rapid and sensitive method for quantitation
of microgram quantities of protein utilizing the principle of dye
binding / / Anal. Biochem.—1976.—72.—P. 248—254.
12. Brungraber E. A. A simplified procedure for the preparation of
^soluble* RNA from rat liver / / Biochem, Biophys. Res.
communs.—1962.—8.—P. 1—3.
13. Laemmli V. K. Cleavage of structural proteins during in the
assembly of the bacteriophage T4 / / Nature.—1970.—227, N
5.—P. 680—685.
14. Sidorik L L., Gudzera O. /., Dragovoz V. A , Tukalo M. A ,
Beresten S. F. Immuno-chemical non-cross-reactivity between
eukaryotic and prokaryotic seryl-tRNA synthetases / / FEBS
Lett—1991.-292, N 1, 2 . - P . 7 6 - 7 8 .
15. Gatti D. L., Tsagoloff A. Structure and evolution of a group of
related aminoacyl-tRNA synthetases / / J. Мої. Biol.—1991.—
218.—P. 557—568.
16. Bullard J. M., Cai У.-С, Demeler B.t Spremulli L. L.
Expression and characterization of a human mitochondrial
phenylalanyl-tRNA synthetase / / J. Мої. Biol.—1999.—
288.—P. 567—577.
17. Bullard J. M.f Cay Y. C, Spremulli L. L. Expression and
characterization of the human mitochondrial leucyl-tRNA syn
thetase / / Biochim. et biophys. acta.—2000.—1490, N 3.—P.
245—258.
18. Tzagoloff A , Gatti D.t Gampel A Mitochondrial aminoacyl-
tRNA synthetases / / Progr. Nucl. Acid Res. Мої. Biol.—
1990.—39.—P. 129—158.
19. Natsoulis G., Higler F.t Fink G. R. The HTS1 gene encodes
both the cytoplasmic and mitochondrial histidine tRNA syn
thetases of S. cerevisiae II Cell.—1986.—46.—P. 235—243.
20. Mirande M., Waller J.-P. The yeast lysyl-tRNA synthetase
gene / / J. Biol. Chem.—1988.—263.—P. 18433—18451.
21. Wong J. T.-F. Role of minimization of chemical distances
between amino acids in the evolution of the genetic code / /
Proc. Nat. Acad. Sci. USA.—1980.—77.—P. 1083—1086.
22. Schwartz R. M.t Dayhoff M. 0.y Schwartz R. M., Dayhoff M.
Origins of prokaryotes, eukaryotes, mitochondria and chlo-
roplasts / / Science.—1978.—199.—P. 395—403.
23. Borst P., Grivell L. A. The mitochondrial genome of yeast / /
Cell.—1978.—15.—P. 705—723.
24. Nagel G. M., Doolittle R. F. Evolution and relatedness in two
aminoacyl-tRNA synthetase families / / Proc. Nat. Acad. Sci.
USA.—1991.—88.—P. 8121—8125.
25. Lipman R. S. A., Hou Y.-M. Aminoacylation of tRNA in the
evolution of an aminoacyl-tRNA synthetase / / Proc. Nat. Acad.
Sci. USA.—1998.—95.—P. 13495—13500.
26. Tolkunova E.t Park #., Xia J., King M. P., Davidson E. The
human lysyl-tRNA synthetase gene encodes both the cytoplas
mic and mitochondrial enzymes by means of an unusual
alternative splicing of the primary transcript / / J. Biol.
Chem.—2000, August 21 (preprint).
УДК 577.1
Received 10.06.99
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