Ellropcan Juurnal or Cell 8iolol)' 55. 114 III (19911
0
WiiH nstharllit he Ve rlagsgcscllsdlal'l mbH . SllIuga n
Prosomes (proteasomes) of higher plants
Martin Schliephacke 2 ), Anette Kremp, Hans-Peter Schmid 3 ) , Kurt K6hler, Ulrich KuH I )
Biologisthts Institut d t r Univc:rsitlit. Stllugart/ Dundtutpublik
Ocut5~hlalld
Re~cived November 7. 1990
Accepled February 24. 1991
Prosomes - prolrasome.f - muf/ica/afy/ic pro/ease - serological refationship - glycoproleins
From different planlliuues such as lobacco (Nko lians ruslica). poo
lalo (Solanum luberosum). and mung bun (Phaseolus radi.IlUS).
ring- or cylinder-shapW particles called prosomes "rere isolaled by
eilher sucrose gradient cenlrirugalion or fast prOlein liquid chromalography (FPLC). These particles have a diameler of t2 10 14 nm
and a lenglh of 16 to 18 nm. They migrale undl'r eondilions of nondenalUrlng gel electrophoresis as one distinct band. Sedimenlallon
coefficient and buoyanl density in (51S0. of Ihe plant prosomes
.... rre determined by analytical ullracentrifugation to be approxima lely 238 and l.23g/em J , re!ip«tively. The lolal molecular mass
was estimated by gel fillralion 10 be 650 kOa. Planl prosomH are
composed of 12 to 15 protelM with molecular muses in tbe range of
14 to 35 kOa with isorleclrie points of pH 5 to 7 as revealed by twodimensional gel electrophoresis. The proldn palterns or prosomes
from the three differenl plant species are nry similar. Polyclonal
antisera against potato prosomes resclfd in WHlern blots .... ith prosomal proleins of alllhrH pllllt sp«ies. They also bind to some prosomal proleins of animal SpeciH. Aotisera against aaimal prosome!i
ruet with some proltins of plant prosomH. As shown by lectin bioIlin~. plant prosomes .rt glycosylaled carrying glucosyl- or mannosyl. and N-.cetylgalael05aminyl residues. Prosomal preparations
contain non-stoichiometric amounts of small RNA of about 80 kOa.
These fe!iulls suggest that planl prosome!i lire slructurally and funcliooally homologous to prosornes of other ellkaryolic cells.
IntrMuctinn
Proso mes are small particles with sedimentation coeffi·
cients of 19 to 225 in the cytoplasm and in nuclei of various animal tissues with very similar morphology and biochemica l properti es (1 1. 22, 33, 38, 40). Th ese ring-shaped
or cylindrical pa rticl es have a diameler o f 10 to 14 nm and
a length of 16 to 19 nm. Although various functions have
been proposed for these particles. so far only an involvement in the posuranscriptional control of gene expression
Of Prof. Or. Ulrich Kut!, Biologisches InSlilUI der Universit4t, pfaf·
fe nwaldriog 57. 0 -7000 Stullgan SO/ Federal Republic or Gennany.
: , Present address: I05lilul fur Klin ische Genetik der Universillit,
Wilhelmstr. 27. D-7400 Tiihinge n/ Federa l Republic of Germany.
\, Present address: Biochimie. Univcrsite de Clermont-Ferrand Ill ,
24 Avenue de Landai s, F·63170 Aubiere/ France.
114
has been demonstrated (21. 29. 35, 37]. Beca use o f this
function the particle was nam ed " prosome" (short ve rsion
for "programosome". (371). Indepe ndently. an intracellular, non-lysosomal proteinase was described ("multi catalytic proteinase" (8, 46]; "high molecular weight proteinase" 141 , 42]), which strikingly resembles the prosome in
structure and biochemical properties. Lately it was shown
that both types of panicles share antigenic determinants 14.
D). and the new name "proteasome" was proposed. Quite
likely, proteasome and prosome designate the same particle ; we prefer to use the latter name in the following investigation.
Prosomes of animal tiss ues are resistant to I O~ of the detergent sodi um-N- Iauroylsarcosi nate and consist of a set of
slightly acidic polypeptides with molecular masses of
about 20 to 35 kDa. They are often reported to be associated with small RNAs of about 80 nucleotides (2. 10,291The protein subunit pattern and the number of the associated RNAs show species-specific differences (3, 291. and
also deve lopmental changes in the protein subunit pattern
are reported (1 6).
Prosomes were also found in higher plants and in yeast
13.251, and one may consider them 10 be ubiquitous in eukaryotic cells. Whereas the isolation of prosomes from
Escherichia col i was unsuccessful (3), recently prosomelike parti cles were detected in archaebacteria (9). In this
paper we prese nt comparative biochemical and im munological data of prosomes isolated from three different species of higher plants (tobacco. potato and mung bean).
Materials and methods
Cullure of plant.t
a j Tobacco (Nicotiana rustiea). Plants were grown in pots in a
green-house at temperatures of 2S ce (day. 12 h) and 20 ce (night).
Leaves or 6to 8-weelr:-old planlS o r slems and pelals or 10 10 12week-old plan ts were cut and frozen al - 20 ce.
b) Potato (Solanum tuberosum va r. ~ Aula"). POla lo tubers we re
kept in darkness al 18°C and a l a relati ve air moiSlure of about
40%. The germinating sprouts were removed when they reached
aboul 20 cm and then kepI frozen a t _ 20°(,
c) Mung bean (Phaseol us radiatus). Seedlings we re grow n in the
dark at 25 °e for 3 days and Ihen slored at _20°(,
Purification by sucrose gradient centn!ugation
The homogenization of the plant material, preparation of the postribosomal pellet and purification of the particles by sucrose gradient centrifugation was done as described (2 5).
Purification by fast protein liquid chromatography (FPLC)
The postribosomal pellet was resuspcnded in TBK 100 (20 mM
Tris-HCI, pH 7.4, 100 mM KCI, 3 mM MgCI:, 7 mM 2.mercapto·
ethanol) and aliquots of about 20 A280 units applied to an anion
exchange column of a FPlC system (Mono Q. Phannacia. FreiburglFRG) at a salt concentration of 240 mM KCI. The chromatography was perfonned at a now rate of I ml / min. and a program
comprising a linear salt gradient from 240 mM to 600 mM KCI in
FPLC buffer (20 mM Tris-HCI, pH 7.4, 2 mM MgCh. 7 mM 2-mercaptoethanol) was used. The elution of bound malerialtook place
within an elution volume of 13 ml and the absorption at 280 nm
was measured during the run, The prosomal fractions of several
runs were pooled and concentrated by anion exchange chromato'
graphy using a salt step from 360 mM to 560 mM KCI in FPLC
buffer. Subsequenliy, aliquots of 200 111 were subjected 10 a gel fil tration column of a FPLC system (Supcrose 6B: Phannacia. Freiburg/ FRG) equilibrated with 560 mM KCl in FPlC buffer. The
chromatography was performed at a now rate of 0.5 ml /min. and
the absorption at 280 nm was measured during the run. Fractions
of I ml were collected and precipitated with 2.5 vol. ethanol.
Determination of buoyant densities
Buoyant densities of puriricd prosomes were detennined byanalytical ultracentrifugation. Prosomal fractions in FPlC buffer were
brought to a density of 1. 19 to 1.22 g/ cml with CS l S0 4 and centrifuged in a Centriscan 75 (48 h. 45000 rpm, 20 °C; MSE, Crawley/
UK). After reaching the equilibrium, the absorbance at 280 nrn
was measured along the sample cells and recorded. Bouyant densities were calcu lated as described (19. 27. 45).
Dl'terminalion of sedimentation coefficients
Sedimentation coefficients of plant prosornes were determined by
analytical ultracentrifugation. Prosomes were centrifuged through
5% (w/ w) sucrose in FPLC buffer using Vinograd cells (Centriscan
75, six-place analytical rOlOr. 40000 rpm, 20°C: MSE). During
centrifugation the absorbance at 280 nm was measured along the
sample cells and recorded. Sedimentation coefficients were calculated as described [32J.
Gel electrophoresis of prottins
Denaturing polyacrylamide gel electrophoresis (SOS·PAGE) of
proteins was performed in 12. 5% polyacrylamide (PA) gels with 5%
PA stacking gels (26]. Molecular weight markers (lKB·Pharmacia,
FreiburglFRG) were phosphorylase b (94 kOa). albumin (67 kDa).
ovalbumin (43 kDa), carboanhydrase (30 kDa). trypsin inhibitor
(20 kDa) and lactalbumin (14.4 kDa). Non-equilibriurn electrofocusing (34J was performed using ampholines (lKB-Phannacia) in
the range of p H 3.5 to 10. The gels for the rirst dirnension were run
for 4 h at 400 V. For the second dimension 12.5% PA gels (see
above) were used.
Non-denaturing gel electrophoresis (7] was carried out using 2%
PA/05% agarose geb in Tris-borate buffer, pH 8.3. Prosomal fractions were concentrated by ultracentrifugation. resuspended in
sample buffer (IOmM Tris-HCI. pH 7.4, 20mM EDTA. 10mM
NaCI. I mM 2-mercaptoethanol. 20'10 (w/ w) sucrose) and afte r
short centrifugation (12000 rpm, I min) to remove insoluble material. used for electrophoresis. After 20 min pre-electrophoresis at
200 V and 4°C. the samples were applied to the gel, and electro-
phoresis took place for 2 h at 200 V. The gels were stained with
Coomassie Brilliant Blue.
Gel electrophoresis of RNA
Ethanol-precipitated material from prosomal fractions was resuspended in digestion buffer (IOrnM Tris-HCI, pH 7.5, 100mM
NaCI. 2 mM EDTA, 0.5% Na-N-Iauroylsarcosinate) and digesled
for 2 h at 37 °C with proteinase K (10 mglml, Boehringer. Mannheim/ FRG). RNA was extracted with chloroform / phenol [28],
precipitated with 2.5 vol. ethanol/O.S vol. 3 1\1 sodium acetate/ acetic acid, pH 5.5, and analyzed in 11 % PA gels containi ng 7 M urea.
SO mM Tris-bOTllte buffer, pH 8.3. and I mM EOTA (l4J. Molecular
weight markers we re phe-tRNA, TMV-RNA, 5S-, 18S-. and 28SrRNA. The gels werc silver-stained 139].
Digestion of RNA
The postpolysomal peliet was resuspended in Tris buffer (10 mM
Tris-HCI, pH 7.5. IS mM NaCI) and incubated witn 670).lg/ ml
RNase A in this buffer for 5 min. After addition of dete rgent buffer (20 mM Tris- HCI, pH 7.4, 1% Na-N-Iauroylsarcosinate. 7 mM
2·mercaptoethanol). the postpolysomal pellet was centrifuged in
sucrose gradients as described (25].
£l~ctron
microscopy
Drops of purified prosomal fraction s were adsorbed onto freshly
glow-discha rged and carbon-coated cop~r grids. The grids were
rinsed with water and the particles stained with 2% uranyl acetate.
They were then rinsed again and air-dried. The micrographs were
taken with a EM 10 electron microscope (Zciss, Oberkochen/
FRG) at 80 kV.
Immun oblolfing
An an tiserum agains t polato prosomes was raised in rabbit using
prosomal particles isolated and concentrated by FPLC. For immunization, about 1.2 rng of prosomes. mixed with Freund's complete
adjuva nts (Oifco. Detroit, MIIUSA). were injected. Afler two and
three weeks. the rabbit was boostered with about 1.0 and 0.7 mg of
prosomes. respectively, each mixed with Freund's incomplete adjuvanls (Oifco).
Prosomal proteins were separated by SOS-PAGE and electrophoretically transfe rrcd to nitrocellulose (44]. After tr:lnsfer, the remaining binding sites on the nitrocellulose sheets were blocked
with 2% gelatine/ 0.5'1o Tween 20 in TBS (20 mM Tris-HCI. pH 7.5.
500 mM NaCI). After washing with TTBS (20 mM Tris·HCI, pH
7.5,500 mM NaCI, 0.05% Tween 20). the nitrocellulose sheets were
incubated for 3 h at 37 "C with antisera (rabbit anti ·potato-prosome, rabbit anti-calf-prosome) diluted I : 100 in TTBS. After re·
peated washing in TTBS, the nitrocellulose sheets were incubated
at 3JOC with pcroxidase-Iabeled swine anti-rabbit antibodies (Oakopatts, Hamburg/ FRG) diluted 1, 200 in TTBS for I h. After
washing in TTBS and TBS.the sheets were assayed for peroxidase
activity by incubation in peroxidase substrate solution I (SO ml
100 mM Tris·HCI. pH 7.6, 10 ml 3 mg/ ml 4-Cl-I -naphthol. 50111
300/, H I 0 1) .
uetin blotting
After separation on SDS-PAGE, prosomal proteins were transferred electrophoretically from the gel to nitrocellulose [44]. After
transfer. the remaining binding sites were blocked by incubation
for 2 min in 2% Tween 20 in T8S. After washing in TBS and lectin
buffer (10 mM Tris· HCI. pH 7.2, 1 M NaCl, I mM CaCI 1 • I mM
MgCl 1 • 1 mM MnCl~, 10 mM NaN•• 0.05% Tween 20).thc nitrocel·
lulose sheets were incubated overnight with biotinylated lectlns
(2I1g/ ml : Kem-En-Tec. Hellerup/ Denmark) in lectin buffer. The
11 5
following biotinylated lectins were used : Bandei raea simplicifolia
agglutin ins I and 11 ( RS I and BS 11 ). Concanavalin A (Con A)
from Ca mH'alia ensiformis, and agglutinins from Dolichos binorus
(DRA), Lens culinaris (LC H), Limulus polyphemus (LPA, Limulin
Il l), I'haseolus vulga ris (I' HA), Arachis hypogaea _ peanut (I'NA).
l'isum sativum ( PSA), Glycine max =soybean (S OA), Solanum tuberosum (STA). Ulex europaeus (UEA), Vicia faba (VFA). Triticu m vulgaris = wheat germ (WGA). After incubation with the lee·
tins, the nit rocellulose sheets were washed in lecti n buffer and incubated for 3 h with horseradish peroxidase-conjugatcd biotin y1:lIcd strepla vidin (Amersham, Braunschweig/ FRG ) dil uted 1, 400
to I: 1000 in lectin buffcr. After washing in lectin buffer and TUS.
the nitrocellul ose sheets we re assayed for peroxidase activity by
incubation in peroxidase substrate solution 11 ( 100 ml 50 mM Na·
acetate/ acetic acid, p H 5.5. 4 ml 10/. )-amino-9-ethylcarbazole in
ace tone. 50 ~I 300 . H:0 1 ) . For competition experiments with Con
A. VFA. and DBA. the corresponding sugar residues methyl-n-Dmannoside, methyl-(l-o -gl ucoside, and N-acetyl- D-galactosamine
(0.5 M: Sigma. Oeisenhofen/ FRG) were added 10 the reaction
mixtures and treated as described above.
Results and discussio n
of prosomes. Quantities of pure proso mes can be obta ined
wi th non-green plant tiss ues (etiolated mung bean seedlings and potato s prouts or tobacco stem s and petals),
whereas preparations de ri ved from green lear tiss ue te nd
to be hea vily contaminated with ribu lose- I,5.bis phosphatecarboxylase/ -oxygenase
( rubisco,
14 kDa
and
55 kDa s ubunits); this protein is mos t abundant in green
leaves and ca nno t be easily separated rrom the prosomal
particles (Fig. I ),
Analysis or the protei n composition or prosomes o r the
th ree species re vealed a characteristic set of protei ns in the
molecu lar mass range or 25 to 35 kDa (Fig, I). When prosomes or dirrere nt tissues or mung bean and tobacco were
analyzed, no difrerences in the protein paHern rrom the
different kinds o r tissues or the sa me s pecies were detected
(Fig. I). Obviously, there is no tissue s pecificity or the protein paHern.
Electro n mi croscopic examination of the prosomal rractions revealed identica l structures fo r the prosomes or all
three plants (tobacco, potato, and mung bean), having the
sa me characte ristic reature as prosomes or animal ti ssues.
As s hown in Figure 2, two types or structures are vis ible in
Prosomes can be pre pared rrom plant tiss ues by two diITer·
ent me thods. In itia lly. prosomes were purified by s ucrose
g radien t cen trirugation made up with 1% sodium-N-Iau ro ylsa rcosinate, since proso mes are known to be resistent
to this s trong detergent 1371. Alternative ly. ras t protein liquid chromalOgraphy ( FPlC) was chosen ror the isolation
m
,
b
,
d
•
g
94 - __
67
30 -
t44Fig. I. SOS-polyacrylamide gel electrophoresis of prosomal fractions from different plant tissues. Prosomal fractions of different
plant tissues were anal )'zed by one-dimensionat {1 7. 5~o) polyacrylamide gel electrophoresis. All ti ssue fractions revealed a characteristic set of proteins in the range of 25 to 35 kDa. The two fractions
from leaf tissue in addi tion sho wed a contamination with proteins
of about 55 kOa (rubisco). - /..ones 0 to (.: tobacco : blossoms (1011/.'
(1), stems (Ialle b), It'aves (fall(' r): lan e d : potato sprouts: IIIII I'S I' to
g : mung-bt'an seedlings : stems (fonl' r). rOOIS (/0111.' fJ. cotyledons
(hlll(' g): /OllC Ill : motecul:lr weight marker protei ns. All prol ei n ~
( · ooma ~~ie - s tained . The prosomal fra ct ions were isolltted by FI'LC
{Iall/'f a. h. (I) and sucrose gradie nt centrifugluion, respectively.
116
Fig. 2. Electron micrographs of prosomal fracti ons from differem plant species. Prosomal fra ctions of tobacco (a l. potato ( b)
and mung bea n (c, d) were exam ined by elect ron microscopy after
staining with 2". uranyl ace tatc. Two types of structures are \'isible: ring-shaped particles of t2 to t4 nm diameter and cyli ndrical
particles of the same diameter. but the y probabl y represent different perspecth'es of the same particle. Digestion of mung-bean par!icles by RNase A prior to examination did nOl alter the shape of
the particles (d J. - Bar lOO nm.
th e va ri ous prosoma l preparations: One type is ringshaped with a diameter of 12 to 14 nm. the OIher one is
cylindrical with the sa me diameter and a length of 16 to
18 nm. These two structures probably represent side views
l!Od th e end·on views of the sa me cylindri cal panicle [5].
To verify the homogenous character of th e prosomal preparations. we ana lyzed them funher by electrophoresis un·
der non ·denaturing co nditions. Since only one distinct
band could be detected, we assume that on ly one type of
particle was present. Further. when sedimentat ion coefficients of purified plant prosomes were determined by ana-
+
67
-
•3
-
30
-
m
• -
20.1-
•
14.4 -
67
-
30
-
., -
20.1 -
b
14,4 -
Fig. 3. Analysis of protei n and RNA components. Two·dimen·
sional PAG E of prosomal proteins. Prosomal rractions were anaIyzed by tWo-dimensional PAGE. revealing similar patterns of 12
10 15 slightly acidic protei ns. - a. Tobacco. - b. Potato- c.
Mung bean. - m: Molecular weight marker proteins. All proteins
Coomassie·stained.
Iytical ultracen trifugation. aga in a si ngle peak was visible
as shown for tobacco prosomes in Figure 3h.
The fact that plant proso mes migrated as one band under condit ions of non ·denaturin g electrophoresis. has also
been reponed for prosomes/ proteasomes of oth er spl'l-it's
(3. 8. 41) and suggests the presence of one distinct panicle.
This was further supponed by analytical ultracentrifugation ana lysis by us and by others 142), where only onc peak
of sedimenting panicles was detected.
The sedimentation coefficients of prosomes from th e
three different plant species were between 22S and 23S.
This is slightly higher than the coefficient s of animal prosomes, reported to be between 19S and 22S (3. 22. 37 ).
Prosomes/ proteaso mes are particles of approximately
650 kDa total molecular mass, but the values reponed in
the literature vary, which is likely to be due to the different
eval uati on methods used. Our data suggest that prosomes
of higher plants seem to be somewhat larger complexes
than the prosomes/ proteasomes of most animal species .
Thi s assumption is consistent with results of a compa rat ive
study, in which th e sizes of prosomes from several eukaryotic speci es were determined : the largest dimensions were
fou nd for prosomes from wheat germ , th e only plant tissue
tested (3).
Analysis of the protein constituents of pl ant prosomes
by two-dimensional gel electroph oresis revealed a complex
pattern of 12 to 15 slightly acidic proteins with isoelectric
point s of pH 5 to 7 (Fig. J). This co rrespond s with th e protei n pattern of animal prosomes (3, 29). The prosoma l protein patterns of tobacco and potato are nearly identi cal.
Ten out of the twelve proteins of the two Solanaceae have
the same electrophoretic mobililies. The protein pattern of
proso mes from mung bean is somewhat different but
shares several subu nits wi th the ot her plant prosomes. as
far as molecular weight and isoelectric point are concerned. These simil arities could be supported by immu no·
logical studi es.
A rabbit antiserum against prosoma l particles of pOl<tto
sprouts recognized all proteins of the homologous potato
prosome in Western blot analysis. When tested on proso·
mal proteins of the other two species. tobacco and mung
bean, the antibody reacted with all proteins with th e ex ·
ception of the smallest ones. p24 and p25 (Fig. 4b). Th is
antiserum cross-reacted also with prosomes from animal
tissues. It recognized on ly the 29 kDa protein of prosomal
proteins from cal f liver and two proteins (25 kDa and
29 kDa) of proso mes from mouse spleen cells (Fig. 4b). On
th e othe r hand , an antiserum against ca lf liver prosomes.
which recognized at least 4 proteins of the homologou s
ca lf liver proso me. reacted with only one prosoma l protein
from mou se. tobacco, potato, and mung bean in the molec·
ular mass range of 25 to 26 kDa (Fig. 4c). Previously. im munological relationship between proteins from prosomes/ proteasomes of different animal species was re·
poned in several studi es (13,15), and even immunological
relationsh ips between proteins of prosomes/ proteasomes
from Xenopus and yeast (231. and from rat and archaebacteria (91 are repo rted. From these data a rather hi gh evolu ·
tionary conse rvation of some componen ts of th e prosome
may be concluded .
11 7
.,
m
67
"
2
-
3
,
5
•
2
2
5
3
,
5
!
1-
,.
••
•I
I
.
~-=.--~
-=.~ -~
a
,
~
30
20.1-....
3
"
-
'
b
c
Fig. 4. Cross reacti ons of antisera against po tato and calf live r
prosomes wit h prosoma l proteins of different species. Prosoma l
protei ns of dirrerent species separated on 12.5~. polyacrylamide
gels were transferred 10 nitrocell ulose sheets and incubated with
rabbi t a ntiserum against POI:1I0 or calf liver prosomes. Binding
reactions were visualized by incubation with peroxidase·labeled
swine anti -rabbi t antibodies after peroxidase/ suhslTale reaction.a. Polyacrylamide gels ( 12.5%) of prosomal fra ctions of mung
bean (lam· I), potato (/(l/Ie 2).lohacco (lane J). mouse erylhrohlasls
(lan e 4). ca lf liver (lane 5). ~ m : Molecular weight marker proteins.
Coomassie-stainl:d. - b. Incubation after Western blotting with an
antiserum raised against prosomal proteins of potato. The other
two plant species showed positive signals for all proteins except in
the range of 24 to 25 kDa. Cross reaction with calf liver and mouse
proteins was obtained with a protein of about 29 kDa and an additional protein of about 25 kDa for mouse. - c. Incubation after
Western blotting with an antiserum raised agai nst calf liver prosomal proteins. This antiserum reoognized protei ns in the range of 25
to 26 kDa in alt plant species.
However, the question arises whether or not prOleins of
plant prosomes are glycosylated, as it had been reported
for anima l prosomes previously [43). To verify this, prosomal proteins from potato a nd mung bea n were transferred to nitrocellul ose and incubated with various biotinylated lectins. As shown in Figures 5a and 5c, the lectins
Con A. VFA, and DBA bind to some prosomal proteins.
regularly to p27, indicating the presence of glucosyl- or
mannosyl-, and N-acetylgalactosaminyl residues. The specificity of these reactions was demonstrated in competition
experiments, with th e corresponding I-methylsaccharides.
Incubation with Ihese sugars resulted in reduced lectin
binding to the respective prosomal proteins (Fig. 5b).
These findings were not unexpected since prosomal proteins of an im al cells are modified by phosph orylation.
ADP-ribosylalion. and glycosylation (30). Glu cosyl- or
mannosyl residues have also been found in prosomal proteins in mouse [43). So far N-acetylglucosamine a nd Nacetylneumminic acid were identified in mouse prosomes
(43), while. accord ing to our investigations. N-acetylga lactosamine is likel y to be prese nt in plant prosomes. One
may argue that suga r residues could be impo rtant for th e
stabilit y of the particle against self-digestion or proteolysis
by other proteases 120). It was al so proposed that glycosylat ion of cytoplasmic proteins might be essential for the
formation and stabilizatio n of multiprotein complexes or
could direct particles to specific intracellular targets [17.
181.
lt has been reported th;1I proso mes of animal cell s or
rather the 19S fractions of Ih e postribosomal supern ata nts
contain RNA. Th ese RNA as well as purified prosomes
rcact with vi ral mRNA and may thus be of fun ctional importance for the cell 121]. From prosomal fraction s of all
three plant species RNA could be purified ei ther by FPLC
o r by sucrose gradient cen trifugation followed by CS 2 S04 gradient centrifugation. When examined by PAGE, a small
RNA band of approximately 80 nucleotides was detected
(Fig. 6). From FPLC-isolated prosomes, the yield of RNA
was lower than from prosom es purified through sucrose
gradients. The amount of RNA in prosoma l fractions va ried somewhat. but stoich iometric amou nts of RNA could
not be detected. This could be con firmed by density determinations. The buoyant densities in CS ZS04 of proso mes
isolated from tobacco and potato were 1.232 g/ cm l and
1.229 g/ cml, respective ly. Preparati ve ultracentrifugation
in a Cs zS04 -gradient of prosomes isolated from mung
bean revealed a similar low value of 1.24 g/ cm). RNAs of
abo ut 70 to 80 nucleotides have also been detected in prosomes of anima l tissues wi th buoyant densiti es of 1.29 and
1.3 1 g/ cml in CS~S04' which allows to estimate a RNA
content of 12 to 15% (2, 37, 38]. But in other studies. no
R NA or o nl y non-stoichio metric amounts of RNA could
be detected 14, 6, 22, 23, 42). The buoyant density and absorption spectra (not shown) rather indi cate that prosomes
in hi gher plants are comprised exclusively of proteins.
After incubatio n with RNase, no RNA cou ld be detected
in purified prosomal fractions fro m mung bean (Fig. 4b),
whi ch is in co ntrast 10 findings in anima l prosomes 11 01.
The morphology of th e plant particles is not affected by
the RN ase treatment (Fig. 2d). It seems as if RNA is nOI a
11 8
a
bed
m
6400 nt
3400 nt
1800 nt
-
..
-
120 nl
74 01
Fig. 6. Gel electrophoresis of RNA. RNA extracled from prosomal fractions was submitted 10 11% polyacrylamide urea gels. The
preparations of all plants showed an RNA of about 80 nucleotides
(nl). When digested by RNase A prior to RNA isolation. the prosomal fraction of mung bean revealed no presenc(' of RNA. _
Lane a: tobacco : lane b: POtaIO; lalle c: mung bean: la/ll' d : mung
bean after digestion by RNase A: lalle m: molecu lar weighl marker
RNA. All RNAs sil ver-stained.
Fig. S. Analysis of glycoproteins. Glycoprotcins were delermined
after separalion of prosomal proteins by 12.5". polyacrylamide gel
electrophoresis and Western blotting, followed by incubation with
different biolinylated leellns. The leelins were visualized by incu bation with streptavidin·eonjugaled peroxidase and subsequent
peroxidase/s ubstrale reaction. - a. Prosomal proteins of potato
were incubated wilh different biotinylated lectins (abbrevialions
see Materials and methods). Positive reactions, mainly with a protein of about 27 !tDOl, were obtained with Con A and VFA (both
specific for Cl-D-glucosyl and Cl-D-mannosyl residues) as well as
wilh DBA (specific for N-acetyl-(l-D·galactosaminyl-residues).UlIIe a : PAGE-separated proteins, Coomassie·stained: lalll' b;
moleCular weight marker proteins; lan e K ; sITepl3vidin control. b. The speeificity of the rc:.ctions is shown by competition experiments. Western blotted prosomal proteins of potalo were incubated with the biotinylated leelins Con A. VFA and DBA and with
the corresponding I·methylglycosides. - Lalle a ; without glyco·
side: lan c b ; methyl,u-D-glucoside: lane c: methyl-n-D-mannoside:
lane d : N-acctyl-o- D-galactosamine: lane K: streptavidin cOnlrol;
l(lllt m ; molecular weight marker protei ns. Amido black -stained.
Concentration or haptens: 0.5 M. - c. Prosomal proteins of mung·
bean scedlings revealed a positi\·e reaction with the biolinylated
ge nuine structural component of plant prosomes but rath er
associates in a nonspecific way.
In summary, our data indi cate that plant prosomes are
identica l wi th prosomes/proteaso mes of animal tissues.
supporting the idea of ubiquity of these p.lrticl es. Thus. a
basic cellular function can be envisaged [35]. It has been
s hown that prosomes play a role in the posttranscriptional
reg ul ation of gene expression, since proso mes were initially found to cosediment with repressed messenger ribonucleoprotein particles in extracts o f animal ti ssues [I. 29,
371. An interesting inhibitvry effect of prosomes and/o r
RNA of prosomal fra ct io ns from mouse on the expression
of viral mRNA has been reported. This inhibition is
thought to be due to an association of proso mal RNA with
the S'-regions o f viral mRNA, thus preventing initiation of
translation (211. Surprisingly, in preli minary experiments
an inhibi to ry effect of purifi ed plant prosomes (which d o
not contain stoich iometri c amounts of RNA) on the translation of toba cco mosa ic virus RNA was also observed,
whereas the translation of globin-m RNA was not affected
(24). Possibly, th e inhibition is not likely to be due to the
lectins VFA and DBA in the range of 25 to 26 kDa. - Lane K :
strepta\'idin control; lant m: PAG E-se parated proteins. Coomas·
sie-stained.
119
action of RNA. One may rather speculate that it is a result
of a proteolyti c activity of the prosome, affecting some viral-specific messenger binding proteins, since a multi catalytic proteinase act ivity is present in prosomes/ protcasomes 14, 8, 9, 12, 13,23, 31,35, 41]. As was found in o ur
laboratory, purified mung bean prosomes were able to hydrol yze methylcasein (36J, a proteolytic activity which is
known from animal proteasomes [4, 8, 13).
By the physical. biochemical and immunological data
presented in this paper, and by functi onal aspects (36], the
plant prosomes are demonstrated to be particles homologous to th e proteasomes (multicatalytic protease) described from animals. yeast and prokaryotes. Therefore,
the need of evaluati on of the basic physiological role of
these enzyme complexes in eu karyot ic and prokaryotic
cells still increases.
A cknowledgements. We thank Dr. G. Adam for his help in raising
antibodies against potalo proso mes, Dr. L KOhn, W. Tomek, S.
GroBkopf, and R. Vallon fo r providing antisera and animal prosomes, K. Maurer for the non-denaturing gel e l ectrophoresi~ , M.
Flachmann and Dr. H. Ungibauer-Kremp for hetp in electron microscopy and analytical ultracentrifugation, Prof. K. Scherrer for
di scussions, and U. Vallon for help in preparing the manuscript. This work was su pported by a grant from the Deutsche Forschungsgemeinschaft (Ku 31017 ).
Rererences
[1 1 Akhayat, 0 ., M.- F. G rossi de Sa, A. A. Infante: Sea urchin prosome : Cha racterization and changes during develo pment. Proc.
Natl. Acad. Sci. USA 84, 1595 - 1599 (1987).
[21 Arrigo. A.- P., l -L DarHx, E. W. Khandijan. M. Simon, P.-F.
Spa hr : Characteriutio n of the prosome fro m Drosophila and its
similarity 10 the cytoplasmatic structures formed by the low molecular weight heat-shock protcins. EMBO J. 4 ,399- 406 (1985).
[31 Arrigo, A.-P., M. Simon, J.-P. Darlix, P.- F. Spahr : A 20S particle ubiquitous from yeaslto human. 1 Mol. Evol. 25 , 141 - 150
(1987).
(41 Amgo, A.-P., K. Ta naka, A. L Goldberg, W. J. Welch : Identit y
of the 19S "prosome~ particle wilh the large mullifunctional protease complex of mam malian cells (the proteasome). Nature 331,
192- 194 (1988).
(51 Baumeister, W., B. Dahlmann, R. Hegerl, F. Kopp, L. Ku ehn,
G. pfelrer: Electron microscopy and image analysis of the multi catalytie proteinase. FEBS UII. 241 , 239- 245 (1988).
16} Castano, 1 G., R. Ombers, J. G. Koster. J. A. Tobian, M. Zasloff: Eukaryotic pre-tRNA 5'processing nuclease : Copuri fi calion
with a complex cylindrical particle. Cell 46 , 377 -387 ( 1986).
(71 Oahlberg. A. E., C. W. Dingman, A. C. Peacock. : Electrophoretic characterization of bacterial palyribosomes in agarose-acrylamide composite gels. J. Mol. Bioi. 41. 139- 147 ( 1969).
(81 Dahlmann, B., L Kuehn, M. Rutschmann, H. Reinauer: Punlication a nd characterization of a multicatal ylic high-molecularma ss proteinase from ra t skeletal muscle. Diochem. J. 228, 161 - 170
( 1985).
[9] Dahlmann, B., F. Kopp, L. KUehn, B. Niedel, G. pfeifet, R.
Hegerl, W. Baumeister : The multicatalytic proteinasc (prosomc) is
ubiquitous rrom eukaryotes to a rchaebacteria. FE 8 S Lell. 251 ,
125- 13 1 ( 1989).
1101 Di neva, 8 ., W. Tomek, K. KOh ler, H., P. Schmid : Evidence for
a highly nuclease resistant RNA fragm ent in pr050mes. Mol. BioI.
Rep, 13,207- 21 1 ( 1989).
120
[Ill Domae. N" F. R. Hannon, R. K. Busch, W. Spahn, C. S. Subrahmanyam, H . Busch : Donut-shaped " miniparticles~ in nuclei of
hu man and rat cells. Lifc Sci. 30 , 469 _477 (1982).
1121 Driscoll, J., A. L Goldberg : Skeletal muscle prOleaso me can
degrade pro teins in an ATP dependent process thal does not require ubiquilin. Proc. Na tl. Acad. Sci. USA 86, 787-791 ( 1989).
[13] Falkenburg. P.- E., C. Haass, P.-M. K10etzel , B. Niedel, F,
Kopp, L Kue hn, B. Dahlmann : Drosophila small cytoplasmic 19S
ribon uclcoprotein is homologous to the multicatalytic proteinase.
Nat ure 331,1 90- 192 (1988).
114) Grierson, D.: Gel electrophoresis of RNA. In: D. Rickwood.
B. D. Hames (eds.): Gel electrophoresis of nucleic acids: a practical app roach. IRL Press. Oxford, Washington 1982.
(1 51 Grossi de Sa, M.,F., C. Ma rti ns de Sa, F. Harper, M. Olink coux, M . Huesca, K. Scberrer : The association of prosomn with
some of the intermediate filament networks of the animal cell. J.
Cell BioI. 107,151 7- 1530 ( 1988).
116) Haass, Co. P.-M, K1oetzd : The Drosophila proteasome undergoes changes in its subunit pallern during development. Ex p. Cell
Res. 180 , 243- 252 (1989).
[1 71 Hart,G. W., G . D. Holt, R. S. Haltiwanger: Nuclear a nd cytoplasmic glycosylation : no vel saccharide linkages in unexpected
places. Trends Biochem. Sci. t3, 380-384 (1988).
[18] Ha rt, G. W., R. S. Halliwange r, G. D. Holt, W. G. Kelly: Glycosylation in the nucleus and cytoplas m. An nu. Rev. Biochem. 58.
841 -874 (1989).
[191 Hennig, W.: Ultrazentrifugation und ihre Anwendung in der
Untersuchu ng dcs Eukaryoten-Genoms. Instrument und ForsChung 3175 , 3- 22 (1 975).
[20] Holzer, H., P. C. Heinrich : Control of proteolysis. Annu. Rev,
Biochem. 49 . 63-9 1 (1980).
[21) Horsch, A., C. Martins de Sa, B. Dineva, E. Spindler, H.- P.
Schmid: Prosomes discriminate between mRNA of Adeno virus-infected and uninfected HeLa cells. FEBS Lell. 246 , 13 1- 136
(1989).
[22) Kle insch midt, J. A., B. Hllgie, C. Grund. W. W. Franke: The
22S cylinder particles of Xeno pus laevis. L Biochemical and electro n microscopic cha ractcrization. Eur. J. Cell Bioi. 32 , 143- 156
(1983).
[23) Kleinschmidt, J. A., C. Escher. D. H. Wolf: Proteinase yscE of
yeas t shows ho mology with the 20S cylinder particles of Xenopus
laevis. FEBS Lell. 239 ,35-40 (1988).
[24} Kremp, A. : Isolierung und Charaklerisierung vo n 23S- Partiketn (Prosomen) aus Nicotiana ruslica L und Solanu m tuberosum
L. Dissertation, Universitlit Stuttgart (1989).
(25) Krcmp, A., M. Schliephacke, U. Kull, H.-P. Schmid : Prosomes exist in plant cells too. Exp. Cell Res. 166 ,553-557 (1986).
[26) Laemmli, U. K. : Cleavage of stru ctural proteins during the assembly of the head of bacteriophage T4. Nature 227 . 680-685
( 1970).
[271 Ludlum, O. B., R. C. Warner : Equi librium centrifugation in
cesium sulfale solutions. J. BioI. Chem. 240,296 1- 2965 (1965).
(281 Man ialis, T., E. F. Fritsch. 1 Sambrook: Molecular Cloning.
A laboratory man ual. Cold Spring Harbor laboratory. Cold
Spring Harbor, NY, 1992.
[29) Martins de Sa, C , M.-F. Grossi de Sa, O. Akhayat, F. Broders,
K. Scherrer, A. HOT"Sch, H.- P. Schmid : Prosomes : Ubiquity and
inter-species st ructural variation. J. Mol. BioI. 187, 479- 493
( 1986).
(301 Martins de Sa, C .• E, Rollet, M.-F. G rossi de Sa, R. M. Tanguay, M. Belpamme, K. Scherrer: Prosomes and heat-shock complexes in Drosophila me1anogaster cells. Mol. Cell. BioI. 9, 2672268 1 ( 1989).
[31) Matthews, W., K. Ta naka, J. Driscoll, A. Ichihara, A. L. Goldbe rg : Involvement of the proteasome in various degrada ti ve processes in mammalian cells. Proc. Natl. Acad. Sci. USA 86. 2597260 1 ( 1989).
1321 MSE Tcchn ical Publication No. 73 (S upplemcnt) : Basic the-
(40) Shelton, E., E. L Kuff, E. S. Maxwell . 1. T. Ha.rringto n : Cyto·
ory and application of analytical ulrraccntrifugation.
13l} Narayan, K. S., D. E. Rounds: Minute ring-shaped panicles
in cultured cells of malignant origin. Nature New BioI. 243, 146150 ( 1973).
134) O·Fatrell. P. z.. H. M. Goodman. P. H. O'Fan-ell ; High resolution two-dimensional electrophoresis of basic as well as acidic
proteins. Cell 12, 113J- 1142 (1977).
PS) ScherTer, K. ; Prosornes, subcomple:le$ of untranslatcd mRNP.
Mol. Bioi. Rep. 14. 1-9 (1990).
136) Schliephacke, M.; lsolierung und Charaktcrisierung von 22SPanikel n (" Prosomen~) aus Tabak (N icotiana ruslica) und Mungbohne (Phaseol us radiatus). Dissertation, Universit:i.1 Stuugan
(1989).
137) Schmid, H. P., O. Akha yat, C. Manins de Sa, F. Puvion, K.
Scherrer: The Prosome: an ubiquitous morphologicall y distinct
RNP particle associa ted with repressed mRNPs and containing
specific scRNA and a characteristic set of proteins. EM80 J. 3 ,
29-34 ( 1984).
(38J Sch uldt, c., P.· M. Kloetzel ; Analysis of cytoplasmi c 195 ringtype pa rticles in Drosophila which conlain hsp 23 a t normal
growth lemper.alure. Dev. BioI. 110 ,65 - 74 ( \985).
139] Schumacher, J. : Horizontale Elektrophoresc in ultradun nen
Polyacrylamidgelen zu r AuOOsung vo n RNAs und DNA· Fragmen ten. LKD Sonderdruck RE 040 ( 1985).
plasmic p:uticles and :1minoacyl transferase I activity. J. Cell Bioi.
45 , 1- 8 (1970).
(41) Tanaka, K., K. li, A. Ichiha.ra, L. Waxrna n, A. L. Goldberg : A
high m olecular weight protease in the cytosol of ral liver. 1. Pun ·
I1cation, e nzymological properties, and tissue distribution. J. BioI.
Chem. 261 , 15 197· 15203 (1986).
(42J Tanaka, K., T. Yoshimura, A. Ichihara, K. Kameyama, T. Ta-
bgi : A high molecular weight prolease in the cytosol of rat liVe r.
11. Propcnies of the purified e nzyme. J. BioI. Chem. 261 , 1520415207 ( 1986).
1431 Tomelt, W., G. Adarn, H.· P. Schmi d : Prosomes, small cytoplasmic RNP pa nicles. conlain glycoproleins. FEBS Lelt. 239 ,
155- 158 (1988).
(44) Towbin, H., T. Staehelin, J. Gordon : Electrophoretic transfer
.of proteins from polyacrylamide gels to nitrocellulose sheets: pro·
cedure and some applications. Proc. Natl. Acad. Sci. USA 76 ,
4350-'U 54 (1979).
]451 Vinograd, J.: Sedimentation equilibrium in a buoyant density
gradient. Methods Enzymol. 6, 854-870 (1963).
(46) Wilk, S., M. Orlowski : Evidence thal pituitary cation·sensitive
neutral endopeptidase is a multi calalytic protease C"omple)! . J.
Neurochem. 40, 842- 849 ( 1983).
121