(Received for publication, June 30, 1995; and in revised form, September 27, 1995)
From the
A 645-kDa proteasome was purified from Methanosarcina
thermophila which had chymotrypsin-like and
peptidylglutamyl-peptide hydrolase activities and contained
(24-kDa) and
(22-kDa) subunits. Processing of both subunits was
suggested by comparison of N-terminal sequences with the sequences
deduced from the
- and
-encoding genes (psmA and psmB). Alignment of deduced sequences for the
and
subunits revealed high similarity; however, the N-terminal sequence of
the
subunit contained an additional 24 amino acids that were not
present in the
subunit. The
and
subunits had high
sequence identity with
- and
-type subunits of proteasomes
from eucaryotic organisms and the distantly related archaeon Thermoplasma acidophilum. The psmB gene was
transcribed in vivo as a monocistronic message from a
consensus archaeal promoter. The results suggest that proteasomes are
more widespread in the Archaea than previously proposed.
Southern blotting experiments suggested the presence of ubiquitin-like
sequences in M. thermophila.
Proteasomes are prevalent in eucaryotes (Eucarya domain) where they have at least three distinct endopeptidase activities which include hydrolysis of peptide bonds on the carboxyl side of hydrophobic, basic, and acidic amino acid residues (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities, respectively)(1) . It is proposed that the 20 S eucaryotic proteasome is the ``catalytic core'' in a larger 26 S complex that degrades proteins labeled with ubiquitin in an ATP-dependent process(1) .
A 20 S proteasome from the
archaeon Thermoplasma acidophilum has been extensively
characterized. The thermoplasma proteasome has and
(25.8-
and 22.3-kDa) subunits with significant identity to the sequences of
all described eucaryotic 20 S proteasomes(2, 3) . The
quaternary structure is also highly conserved with the eucaryotic
proteasome(4) . The fully assembled thermoplasma proteasome is
a barrel-like structure of four stacked rings(5) . Each of the
two inner rings are comprised of 7
subunits, and each outer ring
contains 7
subunits(5) . Assembly of the proteasome
proceeds by formation of the
ring which is proposed to
``chaperon'' assembly of the
subunit ring(6) .
The integrity of the N-terminal region of the
subunit is
necessary for proper assembly of rings. The
subunits are
synthesized with a propeptide that is processed during assembly.
Recently, the crystal structure of the thermoplasma proteasome was
published(5) , and it is proposed that this proteasome is a
novel threonine protease(7) .
Attempts to demonstrate eucaryotic-like proteasomes from procaryotes (Bacteria and Archaea domains) other than thermoplasma have been unsuccessful(8, 9, 10) . The results have led to the suggestion that thermoplasma, an atypical member of the Archaea, is the only procaryote containing proteasomes(10) . Recently, an enzyme with a quaternary structure similar to the 20 S proteasome was isolated from the eubacterium Rhodococcus; however, the primary structure of the eubacterial enzyme has low identity to the archaeal and eucaryotic proteasomes(11) . Thus, questions remain regarding the distribution of proteasomes in the Bacteria and Archaea domains (procaryotes), as well as the origin and evolution of proteasomes from the Eucarya and Archaea domains(10) .
Here we provide evidence for an enzyme highly identical to the eucaryotic-like 20 S proteasome in a representative of methanogenic microbes, the largest group known for the Archaea. These results suggest that proteasomes are more widespread among the Archaea than previously proposed. Although proteolysis is almost certain to be of fundamental importance for methanogenic microbes, the process has not been investigated and the isolation of proteolytic enzymes has not been reported. The discovery of proteasomes in methanogenic microbes marks an entry for investigating the physiology, biochemistry, and molecular biology of protein turnover in this major group of strict anaerobes.
Chymotrypsin- or
trypsin-like peptide hydrolyzing activities were assayed by
fluorimetric measurement (13) of the release of
7-amino-4-methylcoumarin from Suc()-Ala-Ala-Phe-AMC or
Pro-Phe-Arg-AMC at 42 °C for 60 min. The assay mixture (0.3 ml)
contained protein and 20 µM fluorigenic substrate (Sigma)
in 20 mM Tris-HCl (pH 7.2) containing 0.4% dimethyl sulfoxide.
Peptidylglutamyl-peptide hydrolase activity of the purified proteasome
was assayed by colorimetric measurement of the release of
-naphthylamine (14) from Cbz-Leu-Leu-Glu-
-NA (Sigma)
at 42 °C for 60 min. The assay mixture (0.2 ml) contained 5 µg
of protein and 300 µM substrate in 20 mM Tris-HCl
(pH 7.2) containing 3% dimethyl sulfoxide. Protein concentration was
determined by the bicinchoninic acid method (15) using bovine
serum albumin as the standard.
N-terminal sequencing was as follows. Subunits of the purified proteasome were separated by SDS-PAGE (16) using 10% polyacrylamide. The subunits were electroblotted onto a polyvinylidene difluoride (Immobilon-P) membrane (Millipore) and sequenced by automated Edman degradation at the University of Florida-Interdisciplinary Center for Biotechnology Research protein chemistry core facility.
Figure 1:
Location of the psmB gene
relative to the cdh operon in the M. thermophila chromosome. The cdh operon of M. thermophila encodes subunits of the CO dehydrogenase enzyme complex. The cdhE gene encodes the -subunit of CO dehydrogenase and is
the most distal gene in the cdh operon. cdhE` and orf5` are truncated. Abbreviations: Bs, BstEII; S, SmaI; B, BamHI; RI, EcoRI; H, HindIII.
The DNA sequence of both strands of the psmB and psmA genes was determined according to the dideoxy chain termination method(17) . The nucleotide sequence of the M. thermophila psmA and psmB genes have been submitted to the GenBank data base and assigned the accession numbers U30483 and U22157.
Figure 2:
Multiple amino acid sequence alignment of M. thermophila PsmB with selected -type proteasomal
subunits. Periods represent gaps introduced for alignment.
Highlighted amino acid residues are identical or functionally similar
to PsmB. The N-terminal protein sequence of PsmB is represented above
the deduced amino acid sequence, where X is an uncertain
assignment. Abbreviations: MT
, M. thermophila PsmB; TA
,
-subunit of the T. acidophilum proteasome (51% identity)(2) ; CC1, Gallus
domesticus proteasome C1 chain (33% identity)(35) ; HR10, Homo sapiens proteasome-related RING10 (33%
identity)(36) .
Figure 6: Primer extension analysis of the 5`-end of psmB-specific mRNA. Upper, the products of extension reactions using total RNA from methanol (lane 1) and trimethylamine grown (lane 2) M. thermophila cells and DNA sequencing reactions (lanes A, T, G, and C) in which the same oligonucleotide primer was used. Lower, the double underlined DNA sequences indicate the boxA and boxB consensus archaeal promoter. The putative transcriptional start site is indicated by a down arrow and is numbered relative to the translational start site. Single underlined sequences indicate a potential ribosome binding site upstream of the initiator codon. The N-terminal deduced amino acid sequence of psmB is shown in single letter code beginning with the first base of each codon.
A proteasome was purified from M. thermophila which contained 24- () and 22-kDa (
) subunits (see
below). Analysis of the
-subunit revealed two N-terminal sequences
that were identical except for the length (Fig. 3). A
GEM-11 clone bank containing M. thermophila genomic DNA was
screened with a degenerate oligonucleotide probe based on the
N-terminal sequence. Southern blot analysis of the DNA from a
hybridizing phage isolate identified a 980-bp EcoRV-XmnI fragment which hybridized to the probe.
Sequence analysis of the fragment identified an open reading frame (psmA) with a putative translational start corresponding to
the longer of the two
subunit N-terminal sequences (Fig. 7). The psmA gene encoded a putative 246-amino
acid protein (PsmA) with a calculated anhydrous molecular mass of
27,139 Da. PsmA was highly identical (up to 53%) to
-type
proteasome subunits from phylogenetically diverse organisms (Fig. 3). The DNA sequence downstream of the only consensus
ribosome binding site contained three potential translational start
sites, two of which corresponded to the N termini determined for the
purified
subunit and a third located two codons upstream of psmA (Fig. 7). Two of the potential start sites are
separated from the consensus ribosome binding site by 7 and 13 bases
which is typical for archaeal genes(40) . Although a
translational start site corresponding to the shorter of the two N
termini (25 bases from the consensus ribosome binding site) cannot be
ruled out, it is more probable that PsmA is processed to yield the
shorter N-terminal sequence.
Figure 3:
Multiple sequence alignment of M.
thermophila PsmA with selected -type proteasomal subunits. Periods represent gaps introduced for alignment. Highlighted
amino acid residues are identical or functionally similar to PsmA. The
N-terminal protein sequences of PsmA are represented above the deduced
amino acid sequence, where X is an uncertain assignment.
Abbreviations: MT
, M. thermophila PsmA; TA
,
-subunit of the T. acidophilum proteasome (53% identity)(3) ; H
, H.
sapiens proteasome
chain (41% identity)(38) ; AT
or AT-27, Arabidopsis thaliana
-type proteasome (44%
identity)(39) .
Figure 7: Primer extension analysis of the 5` end of psmA-specific mRNA. Upper, the products of extension reactions using total RNA from trimethylamine grown (extreme left lane) M. thermophila and DNA sequencing reactions (lanes A, T, G, and C) in which the same oligonucleotide primer was used. Lower, the bases complementary to the 5` ends of the mRNA are indicated by down arrows and are numbered relative to the translational start site. A potential ribosome binding site is underlined. Predicted stem loop structures are indicated by arrows below the DNA sequence. The N-terminal deduced amino acid sequence of psmA is shown in single letter code beginning with the first base of each codon.
Alignment of deduced sequences for the
and
subunits revealed high similarity (46%); however, the
N-terminal sequence of the
subunit contained an additional 24
amino acids that were not present in the
subunit (Fig. 4).
When the CLUSTAL program was used to generate a dendrogram relating all
proteasome subunit sequences currently available, PsmA and PsmB
clustered with the
- and
-type subunit group, respectively,
in the recently proposed classification scheme (10) and were
closest relatives to the thermoplasma proteasome subunits (Fig. 5). No established proteinase motif was identified in
either PsmA or PsmB using PROFILESCAN(41) , a result which is
in agreement with all proteasome subunit sequences to date.
Figure 4:
Alignment of the deduced sequences of the M. thermophila (PsmA) and
(PsmB) subunits. Periods represent gaps introduced for maximum alignment.
Functionally similar amino acids are boxed in black.
Figure 5:
Dendrogram relating currently available
proteasome subunit sequences to the (PsmA) and
(PsmB)
subunits from M. thermophila.At, Arabidopsis
thaliana; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Gd, Gallus
domesticus; Hs, H. sapiens; Mm, Mus musculus; Mt, M. thermophila; Rn, Rattus norvegicus; Rr, Rattus rattus; Sc, Saccharomyces
cerevisiae; Sp, Schizosaccharomyces pombe; Ta, Thermoplasma acidophilum; Xl, Xenopus laevis.
Primer extension analysis of total RNA isolated from trimethylamine-grown cells identified two products using a psmA-specific oligonucleotide. The 5` ends of the mRNA mapped to the G bases located -117 and -48 relative to the predicted translational start site (Fig. 7). No sequences resembling the consensus boxA were at the expected 16-23-bp upstream of either of the two G bases. Potential stem-loop structures were detected upstream of both 5` mRNA ends. Although transcriptional start sites cannot be ruled out, the results are more consistent with mRNA processing at the G bases.
Figure 8: SDS-PAGE of the proteasome purified from M. thermophila. Electrophoresis was performed as described under ``Materials and Methods'' using 12% polyacrylamide. Four µg of protein was loaded. The molecular weight standards were: bovine plasma albumin, 66-kDa; ovalbumin, 45-kDa; bovine carbonic anhydrase, 29-kDa; and bovine trypsinogen, 24-kDa (Sigma). Protein was stained with Coomassie Blue R-250.
Figure 9: Southern hybridization analysis of M. thermophila genomic DNA using a probe specific for yeast ubiquitin genes. M. thermophila genomic DNA was digested with EcoRI or PstI restriction endonuclease as indicated.
The results document a proteasome in M. thermophila representing anaerobic methanogenic microbes, the largest known
group in the Archaea. Therefore, proteasomes outside the Eucarya are not restricted to the atypical archaeon
thermoplasma as previously suggested(10) . T. acidophilum is a thermoacidophile that can grow aerobically utilizing glucose
as an energy source and is phylogenetically distant from the strictly
anaerobic methanosarcina (42) which obtain energy for growth by
converting simple one- and two-carbon substrates to methane at neutral
pH. Thus, proteasomes occur in physiologically and phylogenetically
diverse Archaea. The results lessen the probability that the
thermoplasma proteasome originated by horizontal gene transfer from the Eucarya(10) and supports the proposal that eucaryotic
proteasomes evolved from an archaeal predecessor(2) . Multiple
DNA sequence alignment of the T. acidophilum and M.
thermophila and
proteasomal subunit genes revealed
high identity (46-60%) among all four genes with the highest
identity between
subunit genes (not shown). This result suggests
that the gene encoding the
subunit is more closely related to an
ancestral proteasome gene from which both the
and
subunit
genes derived.
Thermoplasma and methanosarcina are classified to the Euryarchaeota kingdom(42) . Presently, there is no evidence for an archaeal proteasome in microbes from the Crenarchaeota kingdom(10) ; however, additional surveys are necessary before it can be concluded that the archaeal proteasome is unique to the Euryarchaeota.
The availability of and
subunit sequences
for a second proteasome from an archaeon that is physiologically and
phylogenetically distant from thermoplasma will allow comparisons to
guide site-directed mutagenesis experiments for identification of amino
acids involved in assembly, the catalytic mechanism, substrate
targeting, regulation, and thermostability of archaeal and eucaryal
proteasomes. These experiments will be especially productive because
the crystal structure is known for the thermoplasma
proteasome(5) . Proteasomal
subunits from the Eucarya and thermoplasma (3) display putative NLS and cNLS motifs
exemplified by
sdKKvR
and
EEgEElkapE
(Fig. 3) for thermoplasma
(where upper case letters conform to consensus sequences). The cognate
methanosarcina sequences (
vdKRit
and
EgkfdagtlE
) (Fig. 3) have low
identity with the putative thermoplasma NLS and cNLS motifs, suggesting
these sequences are not strictly conserved in the archaeal proteasome.
It is postulated that either a target for the NLS and cNLS exist in
thermoplasma or that these sequence motifs existed prior to evolution
of the corresponding eucaryotic receptor(3) . Two-dimensional
PAGE reveals multiple forms of the thermoplasma
subunit
indicating it is modified which alters the pI(43) . The
thermoplasma
subunit contains the sequence
KKGST
(Fig. 3) which has identity to
cAMP/cGMP-dependent phosphorylation sites, where serine is
phosphorylated(3) . The thermoplasma
subunit also
contains a putative tyrosine autophosphorylation site (
LVKRVADQMQQYTQYGGVRPY
) (Fig. 3),
where the underlined tyrosine is phosphorylated. The cognate
methanosarcina sequences
KRGTT
and
ISKKICDHKQTYTQYGGVRPY
(Fig. 3) have
high functional similarity, suggesting they may be phosphorylation
sites as predicted for the thermoplasma and eucaryal
subunits(3) . The N-terminal sequence deduced for the
methanosarcina
subunit is extended relative to the
subunit (Fig. 4), as is the case for thermoplasma(2) . Deletion
of this N-terminal extension in the thermoplasma
subunit prevents
assembly of the
ring, demonstrating the importance of N-terminal
sequences for assembly of an active proteasome(6) . The
N-terminal sequences may also be involved in recognition of substrates
because the
rings are located on the ends of the barrel-like
proteasome where the substrate is thought to enter(6) . Unlike
the proteasome purified from thermoplasma(3) , N-terminal
sequencing of the methanosarcina
subunit was not blocked. The
sequencing revealed two N termini resulting from either dual
translational start sites or processing. The significance is unknown;
however, it is conceivable that subunits with different N termini may
assemble into proteasomes with different substrate specificities.
The proteasome from M. thermophila is the first proteolytic enzyme isolated from methanogenic microbes for which there is considerable biochemical and physiological understanding(44) . The discovery of proteasomes in M. thermophila will aid in understanding fundamental properties of the enzyme, including physiological roles in methanogenic microbes and other members of the Archaea. The native molecular mass and subunit composition of the methanosarcina proteasome was comparable to the thermoplasma and eucaryal 20 S proteasome; however, the results do not rule out the existence of a larger complex similar to the eucaryal 26 S complex which degrades proteins labeled with ubiquitin in an ATP-dependent process. Recently, ubiquitin has been identified in T. acidophilum(45) . The thermoplasma proteasome degrades partially unfolded and ubiquitin-associated proteins(46) , suggesting the possibility of ubiquitin-dependent proteolysis in this microbe. The hybridization of a M. thermophila genomic fragment to a yeast ubiquitin gene probe suggests that homologous sequences are present in this methanogenic microbe. Several physiological roles can be envisioned for ubiquitin-dependent proteolytic pathways in M. thermophila. The methanosarcina are the most versatile methanogenic microbes, having the ability to utilize several growth substrates and regulate the synthesis of catabolic enzymes in response to the growth substrate. Two-dimensional PAGE reveals more than 100 mutually exclusive peptides present in acetate- and methanol-grown M. thermophila(47) , including subunits of the CO dehydrogenase enzyme complex which comprise approximately 10% of the cellular protein during growth on acetate(48) . Proteolytic pathways for rapid turnover of specific enzymes would be advantageous when cells switch from one substrate to another. The rapid turnover of specific proteins could also be important during periods of stress such as heat shock or exposure to oxygen. All of these potential physiological roles of the archaeal proteasome pose interesting questions for further investigation.