(Received for publication, September 26, 1994; and in revised form, November 23, 1994)
From the
The ubiquitin-dependent pathway for protein degradation has been
found to play a major role in controlling protein turnover in the cell.
Ubiquitin is one of the most conserved proteins yet identified, and up
until now it has been thought to be present only in eukaryotes and
archaebacteria. This is the first report on the detection and
purification of ubiquitin from a eubacterium, the cyanobacterium Anabaena variabilis. The purification procedure included a
heat denaturing step, fractionated ammonium sulfate precipitation, two
gel filtration runs (Sephadex G-50 and Superose 12), and a final
hydroxylapatite chromatography. Comparisons with bovine ubiquitin
showed a high similarity with respect to antigenicity to anti-ubiquitin
(bovine), molecular mass (M = 6,000),
isoelectric point (pI 6.5), and NH
-terminal sequence. The
existence of ubiquitin in A. variabilis was confirmed by
Southern hybridization. In in vitro experiments both
cyanobacterial and bovine ubiquitin were covalently attached to several
target proteins from A. variabilis, respectively. Data are
presented which suggest ubiquitination of dinitrogenase reductase, the
Fe-protein subunit of nitrogenase. Our findings imply that
ubiquitination equivalent to the eukaryotic system is instrumental in
this organism.
Intracellular protein degradation performs an array of essential functions in the physiology, biochemistry, and development of all organisms. Most important is its role in cellular housekeeping(1, 2) . Abnormal proteins continually arise by mutations, biosynthetic errors, spontaneous denaturation, and free radical or temperature-induced damage. To avoid toxic levels in the cell a fast removal of these aberrant proteins is mandatory. Both eukaryotes and prokaryotes have proteolytic machineries that specifically degrade abnormal proteins(3, 4) .
A proteolytic pathway that plays a major role in the selective removal of abnormal proteins requires the attachment of ubiquitin to a target protein prior to degradation(1, 5, 6) . Ubiquitin is a small protein (76 amino acid residues) with a tightly packed globular structure(7) . Its conjugation to target proteins is an ATP-dependent multistep process catalyzed by the enzymes E1, E2, and E3(6) . The coupling of ubiquitin to acceptor proteins involves a preliminary ATP-dependent step catalyzed by the ubiquitin-activating (E1) enzyme. The activated ubiquitin is transferred to a further cysteine in a ubiquitin-conjugating enzyme (E2), which attaches ubiquitin to its target. Some reactions require additional E3-proteins (ubiquitin ligases), which serve as substrate recognition proteins(8) . Once modified with multiubiquitin chains proteins are degraded by an ATP-dependent, multicomponent proteinase complex, the so-called 26-S proteasome, with a concominant recycling of free ubiquitin(9) . Alternatively, ubiquitinated proteins are rescued by isopeptidases, which cleave the isopeptide bond between ubiquitin and the target protein(10) .
Ubiquitin is present in all eukaryotic cells. Its amino acid sequence is of unparalleled high conservation. There is little variance in all animal species examined including insects, fish, amphibians, birds, and humans. Only two amino acids differ between the lower eukaryote Trypanosoma cruzi and the consensus established for higher eukaryotes; three residues vary between the yeast and plant protein (11) . At the moment ubiquitin and its conjugating system are thought to be present only in eukaryotes and archaebacteria(1, 2, 12, 13, 14) .
Extending our studies on nitrogen fixation by cyanobacteria(15, 16) , we have begun to investigate whether ubiquitin may be present in these organisms. Cyanobacteria are a diverse group of Gram-negative photosynthetic prokaryotes, often with the ability to fix atmospheric nitrogen. Biological nitrogen fixation is catalyzed by an extremely oxygen-sensitive mechanism. In filamentous cyanobacteria such as Anabaena, nitrogen fixation is restricted to differentiated cells termed heterocysts, with special features providing adequate protection of nitrogenase(17) . However, prolonged oxygen treatment or activated oxygen species leads to proteolytic degradation of nitrogenase itself (for review see (18) ). Although many structural nif genes are coregulated by oxygen, repression in Anabaena 7120 requires a concentration of oxygen greater than that needed to destroy nitrogenase(19) . This in turn implicates the existence of a permanent and controlled turnover of the newly formed nitrogenase components.
In this paper, we first describe the purification of ubiquitin from a eubacterium, namely the cyanobacterium Anabaena variabilis. Initial biochemical characterizations showed remarkable similarity to the mammalian form. Moreover, added to cell-free extracts from A. variabilis heterocysts both the bacterial and the bovine ubiquitin became conjugated with several target proteins.
In a first attempt to detect ubiquitin in A. variabilis crude extracts were analyzed with anti-ubiquitin (bovine). Western blotting of both heterocyst and vegetative Anabaena cell extracts has revealed the presence of high molecular weight proteins representing ubiquitin conjugates formed in vivo. However, even under the homogenization conditions described which preclude COOH-terminal proteolysis of ubiquitin (possibly leading to a loss of antigenicity; 23) only trace amounts of unbound ubiquitin could be detected (not shown). A previous report on the detection of ubiquitin in crude extracts from Escherichia coli(24) has been never confirmed. On the other hand, there are very recent speculations that parts of the ubiquitin system may occur in eubacteria(25) . Accordingly, we considered purification of the cyanobacterial protein and proof of its functionality as important. Of course we have been aware of the risk of contamination by eukaryotic ubiquitin. However, the strain used is an axenic one. Photoautotrophic growth conditions implicate the absence of any organic carbon source. The columns used for purification had been cleaned prior to the addition of the cyanobacterial samples.
Previous studies have shown
that ubiquitin, regardless of the source, is remarkably heat-stable.
Therefore, by heating the crude extract to 86 °C, it was possible
to denature and precipitate the majority of proteins, leaving ubiquitin
in solution. After concentrating the supernatant by fractionated
ammonium sulfate precipitation (45-90%), a gel filtration run on
Sephadex G-50 was carried out. Besides desalting, this column served to
separate ubiquitin from proteins of high molecular weight. Subsequently
the concentrated ubiquitin-containing fractions were subjected to a
second gel filtration on Superose 12 preparation grade. The
cyanobacterial protein eluted at a position identical to bovine
ubiquitin (M = 10,000). The final
purification was by hydroxylapatite chromatography (Fig. 1).
Ubiquitin prepared by this protocol was more than 90% homogeneous as
determined by SDS-PAGE. The final yield was very low. 50 g of cell
paste yielded 450 ng of free homogeneous ubiquitin. In eukaryotes
ubiquitin is a fairly abundant protein, in the range of some mg/kg of
tissue(23, 26) . Our modest yield was caused by the
low ratio of free to conjugated ubiquitin, which might be improved by
extraction procedures other than that reported. As described, cells
were broken by two cycles through a French pressure cell. This
procedure was carried out at room temperature and required more than 30
min. During this period the main part of free ubiquitin was possibly
conjugated to damaged target proteins. Furthermore, it has been shown
with tissues from various organisms that there is a dramatic decrease
of unbound ubiquitin in response to stress
conditions(27, 28) . We did not address this question
for A. variabilis. An effective method of increasing the pool
of free ubiquitin in eukaryotic cells is based on ATP depletion.
Corresponding experiments with Anabaena cells were hampered by
the well known and very fast adaption of cyanobacteria to starvation,
which is accompanied by a fundamental reorganization of the
cells(18) . A comparison of different homogenization procedures
of Nostoc commune UTEX 584 cells with respect to the yield of
unbound ubiquitin is shown in Fig. 2. In a crude extract treated
as described for Anabaena there was no detectable free
ubiquitin (lane 1). A small amount of cell paste (0.25 g)
homogenized in a mortar under liquid nitrogen immediately subjected to
SDS-PAGE resulted in a substantial decrease of conjugates and in an
easily detectable amount of free ubiquitin (lane 2).
Up-scaling of this procedure is not possible at the moment.
Figure 1:
Last step of purification of ubiquitin
from A. variabilis by hydroxylapatite chromatography. Protein
is plotted as absorbance at 280 nm at the left axis. The insets show the relevant sections of a silver-stained SDS-PAGE
(a 16.5% Tricine gel with a 10% spacer) and an immunoblot developed
with anti-ubiquitin (bovine), respectively. Bovine ubiquitin (U(bov)) served as standard (20 ng for silver staining, 5 ng
for the Western blot). The purified protein migrated in a position
identical to bovine ubiquitin (M = 5,500)
and showed antigenicity comparable to the ubiquitin
antibody.
Figure 2: Immunoblot analysis of extracts from N. commune UTEX 584 showing ubiquitinated proteins and the presence of unbound ubiquitin. Cultivation of the axenic strain was carried out in the absence of an organic carbon source and nitrogen(47) . After separation (16.5% Tricine gel) the detection was performed with the ubiquitin antibody. Lane 1 shows a sample homogenized as described for Anabaena. Lane 2 represents a sample (0.25 g) broken with a mortar under liquid nitrogen. Protein content in the samples was 20 µg. The arrow indicates the position of free ubiquitin.
Initial
biochemical analysis of the Anabaena ubiquitin indicated that
it was very similar to the mammalian form. On SDS-PAGE, the
cyanobacterial protein migrated slightly above the bovine ubiquitin (M 5,500), with an apparent M
of 6,000 (Fig. 1, upper inset). Both of the
proteins showed antigenicity comparable to that of the antibody raised
against ubiquitin (lower inset). Isoelectric focusing
indicated an isoelectric point (pI) at pH 6.5 (Fig. 3A). This is somewhat different from the reported
pI of 6.7 of the plant and mammalian proteins(5, 23) .
Since in our experiment bovine ubiquitin also focused at pH 6.5, we
attribute this difference to our gel system or the marker proteins
used. Fig. 3B compares the NH
-terminal
sequence of the cyanobacterial and the mammalian ubiquitin. As expected
there was complete identity. A striking feature of ubiquitin is the
exceptional conservation of its primary structure. Analyses of the
amino acid sequence of ubiquitin from a variety of organisms indicate
that within the 76 residues only 7 are variant(11) . Still,
there might be differences between the Anabaena protein and
the eukaryotic ubiquitin. A slightly different size was observed with
various gel systems (Tricine gels and conventional SDS-PAGE). There was
also a reproducible difference in the elution from hydroxylapatite
(elution was effective at 30 mM phosphate compared with 15
mM for the bovine protein). These findings suggest high
similarity but not complete identity between the two proteins. This
fact excludes any contamination of our preparation by eukaryotic
ubiquitin. As a further proof we carried out hybridizations of digested
DNA from Anabaena with two different oligonucleotides
corresponding to two regions of the mammalian protein (Fig. 4A). The cross-reaction, i.e. the
detection of identical bands by both oligonucleotides, provides strong
evidence for the existence of at least one ubiquitin gene in Anabaena. Fig. 4B shows the absence of
corresponding sequences in E. coli and Brevibacterium
linens. Although we have carried out only some initial genetic
work it seems clear that the cyanobacterial system does not match the
eukaryotic one, which consists of a variety of ubiquitin genes usually
resulting in Southern hybridization with a much more complex band
pattern (see (11) ).
Figure 3:
Panel A, determination of the isoelectric
point. 10 ng of bovine ubiquitin (U(bov)) and 5 ng of the
purified protein (U(Av)) were subjected to isoelectric
focusing. The precast gel (pH 3-9, Serva) was run for 4 h at a
constant current of 3.6 mA, reaching a final voltage of 3,000 V. After
blotting, detection was with ubiquitin antibodies. Panel B,
NH-terminal amino acid sequence homology between the
purified cyanobacterial protein (U(Av)) and bovine ubiquitin (U(bov)). The primary sequence of the purified protein was
determined as described.
Figure 4: Panel A, Southern hybridization of digoxygenin-labeled oligonucleotides with genomic DNA from A. variabilis. 10 µg of DNA was digested with EcoRV (lanes 1 and 1`) or HindIII (lanes 2 and 2`) and hybridized under high stringency conditions to oligonucleotides I (lanes 1 and 2) or II (lanes 1` and 2`). Lane M shows a digoxygenin-labeled molecular size standard. Corresponding bands are marked with open triangles. Panel B, dot-blot with 3 µg of DNA from B. linens DSM 20426 (spot 1), E. coli (spot 2), A. variabilis (spot 3), and calf thymus (spot 4). Spots C serve as controls (no DNA). Detection was as described for immunoblotting.
The functionality of cyanobacterial ubiquitin is shown in Fig. 5. Although the conjugation to target proteins was somewhat less compared with the experiment with bovine ubiquitin, the pattern of conjugates was identical (lanes 1 and 3). The lower activity may reflect nonproteolytic modifications during extraction or purification or inherent structural differences between the two ubiquitins. A similar difference in activity probably caused by attachment of quinones has been reported for oat ubiquitin(23) . Considering the conjugation of the mammalian form by our cyanobacterial system it is likely that the cyanobacterial ubiquitin is susceptible to an unknown damage during extraction.
Figure 5:
Ubiquitination in extracts of heterocysts
using either A. variabilis or bovine ubiquitin. The reaction
mixtures (130 µg of cellular protein, 100 µM ATP, 2
mM MgCl, 1 mM dithiothreitol, 50 mM Tris/HCl, pH 7.8, plus 0.1 µg of ubiquitin either from A.
variabilis (U(Av)) or calf (U(bov)) were
incubated aerobically for 30 min at 24 °C in presence of protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 µM antipain, and 100 µM leupeptin). All samples (5
µg of protein) were separated on the same gel (12.5%) and analyzed
after blotting with anti-ubiquitin. Co, aliquots treated
identically without the addition of ubiquitin. The arrowhead at the right indicates the position of free
ubiquitin.
It has been shown that conjugation of ubiquitin to
protein substrates requires ATP (for review see (6) ).
Ubiquitin is activated by formation of an acylphosphoanhydride bond
between ATP and the COOH-terminal Gly-76 carboxyl group. Subsequently,
a high energy thioester linkage is formed with an unique cysteine
residue of the activating enzyme E1. To show this
ATP-dependent activation and conjugation in Anabaena extracts,
we carried out experiments using an ATP-generating and an ATP-depleting
system, respectively. Because we have isolated only small amounts of Anabaena ubiquitin these assays were done with bovine
ubiquitin. The results are presented by Fig. 6. Even in the
absence of added ATP or an ATP-generating enzyme substantial
conjugating activity could be observed (panel A). A similar
phenomenon has been reported for extracts from green plant
tissues(29, 30) . The authors attributed this activity
to high endogenous levels of ATP (about 50 µM), which in
turn correspond to the reported K of the
activating enzymes E1 from oat (29) or
reticulocytes(31) . In fact, the Anabaena extracts
used contained about 20 µM ATP. After incubation in the
presence of 200 µM ATP, creatine phosphate, and
phosphocreatine kinase, an increase of high molecular weight conjugates
occurred (panel B). Neither desalting nor the presence of an
ATP-depleting system (hexokinase) allowed a complete inhibition of
ubiquitin conjugation. Nevertheless, as shown in panel C, the
decrease of ubiquitinated proteins was significant. Therefore we
conclude that the ubiquitin-activating system in Anabaena is
essentially like the eukaryotic one. Most recently sequence comparisons
of a newly discovered human ubiquitin-activating enzyme (32) showed significant identity (although not within the E1 portion) with an open reading frame from Anabaena 7120 obviously involved in nitrogen fixation(33) .
Figure 6: ATP-dependent formation of ubiquitin conjugates. Bovine ubiquitin was added to crude extracts as described for Fig. 3. Panel A, no addition of ATP. Panel B, the assay contained 200 µM ATP, 5 mM creatine phosphate, and 1 unit of phosphocreatine kinase (Sigma). Panel C, the assay contained 10 mM deoxyglucose and 1 unit of hexokinase (Sigma). Before the addition of ubiquitin the assays were preincubated for 5 min. After separating the extracts by SDS-PAGE and subsequent immunoblotting with anti-ubiquitin, the blots were scanned densitometrically. The positions of the molecular weight markers are indicated.
One might
speculate on the function of ubiquitin in cyanobacteria. In eukaryotes
regulation of ubiquitin conjugation is presumably performed through
various events. Substrates of the N-end rule pathway are recognized and
degraded depending on the identity of their NH-terminal
residues(34) . Denaturation of proteins, e.g. by heat
stress or synthesis of misfolded proteins, may lead to exposure of
other recognition sites for the conjugating enzymes E2 or E3. Other signals are phosphorylation of cyclins(35) ,
light-induced conversion of the plant phytochrome into the far red
light-absorbing form(36) , or the binding of the papilloma
virus E6 protein to the tumor suppressor p53(14) .
Many filamentous cyanobacteria differentiate heterocysts with
biochemical and structural features providing adequate protection of
nitrogen fixation against oxygen. However, it is well documented that
prolonged oxygen treatment can destroy the integrity of the Fe-S and
Mo-Fe cofactors (for review see (18) ). Harsh in vivo conditions with respect to oxygen tension or C-starvation
irreversibly affect and degrade cyanobacterial nitrogenase subunits
itself and probably many of the associated
components(18, 37) . Therefore a possible
participation of ubiquitin in the degradation of dinitrogenase
reductase (Fe-protein) was examined (data on the oxygen-induced
degradation of nitrogenase will be published elsewhere). Fig. 7presents Western blots of cell extracts developed against
anti-Fe-protein (15) and anti-ubiquitin antibodies. Because of
the crude extracts used a number of heavily stained bands (see also Fig. 5) had to be accepted on the ubiquitin blot. However, with
protease inhibitors present (lanes 2 and 5; arrowheads 3 and 4), and even more pronounced after
the addition of bovine ubiquitin (lanes 3 and 6; arrowheads 1 and 2), a ladder of protein species (M 60,000-100,000) cross-reacting with both
antibodies became apparent. After complete degradation of the
Fe-protein (lanes 1 and 4) only one stable product of M
43,000, which was recognized by both of the
antibodies, escaped proteolysis (arrowhead 5). Especially the
appearance of additional cross-reacting products after incubation with
ubiquitin (arrowheads 1 and 2) suggest ubiquitination
of the Fe-protein of nitrogenase.
Figure 7: Appearance of ubiquitinated bands of the cyanobacterial Fe-protein. Western blot analysis was performed with either Fe-protein (left panel) or ubiquitin antibodies (right panel). Aerobic incubation of extracts was for 45 min with 100 µM ATP present as described for Fig. 5. As indicated at the bottom, samples applied to lanes 2, 3, 5, and 6 contained protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 µM antipain, and 100 µM leupeptin). Prior to incubation, 0.5 µg of ubiquitin (from bovine red blood cells) was added to samples 3 and 6. For SDS-PAGE a 12.5% gel was used. This gel system did not separate the modified from unmodified forms of Fe-protein(15) , which is indicated by an arrow. Arrowheads 1-5 point to the bands recognized by both the Fe-protein and the ubiquitin antibodies, respectively. U, free ubiquitin.
In addition, the ubiquitin system may be necessary during dehydration-induced stresses in cyanobacterial cells. Many cyanobacteria colonize extreme habitats and have an extraordinarily desiccation tolerance. Terrestrial cyanobacteria such as N. commune are subjected to multiple and rapid cycles of wetting and drying with oxidation- and radiation-induced stresses(38) . These events are accompanied by a massive turnover of proteins. Cellular housekeeping may represent one aspect of the cyanobacterial ubiquitin system. During initiation of nitrogen fixation and heterocyst differentiation about 50% of the abundant proteins are subject to degradation, among them ribulose-1,5-bisphosphate carboxylase (which has been identified as a target for ubiquitination; 29) and phycobiliproteins(39) . The cyanobacterial ubiquitin system probably plays a major role in this fundamental reorganization of the differentiating cell.
Our identification of ubiquitin in heterocystous cyanobacteria of the Anabaena and Nostoc type continues a series of recent publications on eukaryotic features unique to these prokaryotic organisms. Anabaena species possesses both self-splicing group I introns (40) and eukaryotic RNA-binding proteins of the ribonucleoprotein family responsible for the excision of introns(41) . Considering their multicellular organization, the discovery of eukaryotic-type protein kinases in Anabaena species (42) and of a eukaryotic tyrosine/serine phosphatase in Nostoc species (43) was not unexpected. In contrast to the simple prokaryotic systems these enzymes are thought to be instrumental in intercellular signal transduction and cell differentiation. Furthermore, both Nostoc and Anabaena species possess calmodulin (44, 45) , which mediates the regulation of a variety of cellular processes in plants and animals. Like ubiquitin, calmodulin is highly conserved and has been thought to exist only in eukaryotes. Interestingly it has been shown that in lower protists like Trypanosoma species a genomic and transcriptional linkage of the calmodulin and the ubiquitin systems exists(46) . In this context the presence of a protozoon-like myoglobin in Nostoc species should be mentioned(47) . These unexpected eukaryotic features in cyanobacteria may raise some questions whether there are common prokaryotic origins of the corresponding genes. It has been suggested that during evolution many genes have migrated from the endosymbiont genome to the eukaryotic nucleus (e.g.(41) ).