 |
INTRODUCTION |
Modification of proteins by ubiquitin is involved in many cellular
processes including cell cycle progression, signal transduction, ligand-inducible endocytosis of cell-surface proteins, mating type
switching in yeast, and elimination of damaged proteins (reviewed in
Refs. 1 and 2). Attachment of polyubiquitin chain(s) to a protein
frequently serves to target the modified protein for proteolysis by the
26 S proteasome. The ubiquitin molecules in these chains are most
often linked to one another by isopeptide bonds between the C terminus
of one ubiquitin and the 
-amino group of lysine 48 of the next
ubiquitin.
Protein ubiquitination is reversible. Deubiquitination is catalyzed by
specialized thiol proteases, called deubiquitinating or
DUB1 enzymes, which hydrolyze
the amide bond between the C-terminal Gly of ubiquitin and primary
amino groups of the substrate protein (3). One of the two known classes
of DUB enzymes is the ubiquitin-specific processing protease or UBP
class. All members of the UBP family contain two short consensus
sequences, the Cys and His boxes, which are likely to help form the
active site (4, 5). Several additional short sequences are also
moderately well conserved (3). The DUB family is large. For example, 17 genes encode DUB enzymes in the yeast Saccharomyces
cerevisiae, of which 16 are in the UBP class (2). However, little
is known about their physiological functions.
One role of DUBs is the regulation of protein degradation by the 26 S
proteasome. Mammalian 26 S proteasomes contain a DUB enzyme that
specifically removes ubiquitin molecules from the distal ends of
polyubiquitin chains attached to protein substrates (6). It may
function to rescue inappropriately ubiquitinated proteins from
degradation or may facilitate movement of substrates within the
protease complex. Two UBP enzymes from yeast have been shown to have
general roles in proteasome-mediated proteolysis in vivo.
The Doa4 enzyme appears to cleave ubiquitin chains from proteins
already committed to degradation by the proteasome (5). On the other
hand, Ubp14 has recently been found to be the major yeast DUB that
disassembles free ubiquitin chains (7). Both Doa4 and Ubp14 may
function by preventing competition between products generated (at least
in part) by the proteasome (ubiquitinated protein remnants and
unanchored ubiquitin chains, respectively) and polyubiquitinated
protein substrates of the protease.
Other DUBs may have more specialized functions. The one demonstrated
example of a DUB enzyme that is crucial for the development of a
multicellular eukaryote is the Drosophila fat facets UBP enzyme (5). In addition to having a maternal effect lethal phenotype,
mutant fat facets (faf) flies have a specific defect in eye
development characterized by extranumerary photoreceptor cells.
Interestingly, partial loss-of-function alleles of a proteasome subunit gene suppress the faf developmental defect,
suggesting that the Faf protein normally functions to antagonize the
degradation of one or more key regulators of eye development (8).
The simple eukaryote D. discoideum is an excellent model
system because its growth is independent of development. As the
vegetative amoebae starve, they enter an initial stage of development
wherein they stop dividing and begin secreting the cell density sensing factor, CMF (9-13). When there is a high concentration of starving cells, as indicated by high levels of CMF, the cells enter another stage of development where relayed pulses of cAMP cause an increase, from a basal level, of the transcription of genes encoding proteins such as the cAMP receptor, cAR1, and a phosphodiesterase (PDE) that
causes extracellular cAMP levels to return to a base-line level in the
interval between the pulses of cAMP (see Refs. 14-17 for review). The
pulses of cAMP also function as a chemoattractant that causes the cells
to aggregate (reviewed in Ref. 18). The aggregate, which is held
together by adhesion proteins such as gp80, subsequently develops into
a fruiting body consisting of a mass of spore cells supported on a
column of stalk cells (see Refs. 19-24 for review). The entire
developmental process takes only 24 h. In
Dictyostelium, ubiquitin genes have been identified, and
ubiquitin mRNA species were shown to be developmentally regulated, suggesting a role for ubiquitin in development (25, 26). In addition, a ubiquitin-conjugating enzyme, UBC1, has been
implicated in Dictyostelium development (27).
Here we demonstrate that the D. discoideum UbpA protein is a
deubiquitinating enzyme, that it is a functional homolog of yeast Ubp14
and shares the highly restricted substrate specificity found previously
for Ubp14 (7) and mammalian isopeptidase T (1, 28, 29), and that the
UbpA enzyme is crucial for development. We used a random mutagenesis
protocol (30) and isolated ubpA as a mutant with defective
aggregation. Mutants lacking UbpA grow and enter the CMF secretion
stage of development normally, but these cells fail to reach the stage
where pulses of cAMP normally increase the transcription of genes
required for further development, such as those encoding cAR1, PDE, and
gp80. It is intriguing that such a specific developmental defect
results from the loss of a DUB enzyme with a very general but
nonessential function in ubiquitin-dependent proteolysis.
These findings suggest that there is a central role for the
ubiquitin-proteasome pathway in setting the proper expression level of
specific regulatory factors controlling progression between stages of
Dictyostelium development.
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EXPERIMENTAL PROCEDURES |
Growth and Manipulation of Dictyostelium, Yeast, and Escherichia
coli Cells--
D. discoideum Ax4 cells were grown as
described previously (31). The uracil-lacking strain DH1 (a gift of
Peter Devreotes, Johns Hopkins University. Baltimore), a
pyr5-6 derivative of Ax4, was grown in HL5 medium (32)
supplemented with 20 µg/ml uracil. Cells were developed on filters
(31) or starved in shaking cultures as described (13). Shaking cultures
that were pulsed with cAMP received pulses to 30 nM every 6 min between 1 and 6 h of starvation. Low cell-density assays for
prestalk and prespore cells were carried out as described by Clay
et al. (33). Time lapse videomicroscopy, motility assays,
and trypan blue exclusion were done as described in Yuen et
al. (13).
The yeast strains used in this work were MHY501 (MAT
his-
200 leu2-3, 112 ura3-52 lys2-801 trp1-1) and MHY840
(MAT his-200 leu2-3, 112 ura3-52 lys2-801 trp1-1
ubp14-1::HIS3i). Yeast-rich and minimal media were
prepared as described, and standard yeast genetic methods were used
(34). E. coli strains used were JM101 and SURE (Stratagene,
La Jolla, CA), and standard procedures for recombinant DNA work were
used (35).
Disruption, Cloning, and Sequencing of ubpA--
Restriction
enzyme-mediated integration (REMI) was carried out following Kuspa and
Loomis (30) by electroporating EcoRI-linearized DIV2 (an
insertion vector carrying the Dictyostelium pyr5-6 gene, see
Fig. 1A) into DH1 (pyr5-6
) cells
along with the restriction enzyme MunI and selecting for uracil prototrophs. In a screen of 230 transformants, 6 had defective developmental morphology. Two of these, M7 and M11, showed defective aggregation. Plasmids pM7E and pM7N, which contained DNA flanking the
DIV2 insertion site in the M7 transformant, were prepared from M7
genomic DNA that had been cut with EcoRI and
NsiI, respectively. A 250-bp
PvuII-EcoRI fragment from pM7E, which contains
the genomic MunI-EcoRI region that flanks the
insertion site plus 46 bp from the vector, was used as a probe to
screen a 
gt11 library of Dictyostelium cDNA
(CLONTECH, Palo Alto, CA) following Jain et
al. (31). The cDNA inserts of positive clones were amplified
by 35 cycles of PCR (1 min at 94 °C, 1 min at 40 °C, 4 min at
74 °C) using Pfu enzyme (Stratagene, La Jolla, CA) and
gt11 forward and reverse primer (New England Biolabs, Beverly, MA).
PCR products were purified using the Wizard PCR kit (Promega, Madison,
WI) and desalted/concentrated using a Microcon-100 microconcentrator
(Amicon, Beverly, MA). DNA sequencing was performed using an Applied
Biosystems sequencer at the Microbiology Department core sequencing
facility, University of Texas Medical School, Houston.
The largest cDNA clone was 3 kb in length and contained an open
reading frame from one end through to a poly(dA) region at the other
end. RACE-PCR was used to obtain additional sequence at the 5'-end (10,
36). First strand cDNA synthesis from 5 µg of total RNA was
carried out using the gene-specific antisense primer
5'-GATTCTGCATTCCAACTGACG-3' with the Life Technologies, Inc.
SuperScript preamplification system following the manufacturer's protocol. Primers and dNTPs were removed by using a Microcon-100 microconcentrator. A poly(dC) tail was added to the cDNA using terminal deoxynucleotidyltransferase (U. S. Biochemical Corp.). The
dC-tailed cDNA was amplified by PCR, carried out for 30 cycles (1 min at 94 °C, 1 min at 48 °C, 2 min at 72 °C), with the
5'-RACE Anchor Primer (Life Technologies, Inc.) and a nested
gene-specific primer, 5'-ACTTTATCTCTTCATCC-3', using the GeneAmp kit
(Perkin-Elmer). Product (0.1 µl) from this PCR was used as template
for nested amplification, carried out for 17 cycles (1 min at 94 °C,
1 min at 49 °C, 1 min at 72 °C), with the Universal Amplification
Primer (Life Technologies, Inc.) and the antisense primer,
5'-CCACCATTCTCTATAGTTGG-3'.
The 5'-RACE fragment extended the cDNA sequence an additional 135 bp, which included the first 4 bp of the open reading frame. The
cDNA sequence matched the sequence obtained from the genomic clones
except for the 5'-end of the cDNA (exon 1; Fig.
1A), which was not present on the genomic fragment of pM7E,
suggesting that a deletion occurred during the cloning of this plasmid.
This was confirmed by PCR of genomic DNA from wild-type cells using a
primer specific for a region upstream of the presumptive deletion and a
gene-specific (exon 2) antisense primer. The sequence of the genomic fragment obtained by PCR matched the cDNA and 5'-RACE sequence. Plasmid pM7E thus contains a deletion of 350 bp including exon 1.
Construction of Plasmids for the Expression of UbpA and
Ubp14--
For ubpA cloning, the ubpA coding
sequence was amplified by PCR from a gt11 cDNA clone that
contained the entire ubpA open reading frame. PCR was
carried out for 30 cycles (45 s at 95 °C, 45 s at 45 °C, 3 min at 72 °C) using a AmpliTaq (Perkin Elmer, Branchburh,
NJ)/Pfu enzyme (Stratagene) mix (37) and the primers 5'-CGGATCCAATGGAATTATTCCCAGAATTAAAAAATATTAAAGTACC-3' and
5'-GCTCTAGAAATTAAAATTTAATTTAGTTGTC-3', which correspond to the 5'- and
3'-ends of the ubpA coding region, respectively. The PCR
product was digested with BamHI and XbaI (restriction sites which were built into the 5'- and 3'-primers) and
subcloned into BamHI, XbaI-cut pYES2 (Invitrogen,
Carlsbad, CA), a yeast expression vector, to generate pYEU1. The
ubpA insert of pYEU1 was removed by digestion with
BamHI, blunt-ended using Klenow, and digested with
XbaI, and then subcloned into similarly cut pDXA-3H, a
Dictyostelium expression vector (38). For expression in
E. coli, the complete ubpA coding sequence,
contained on a gt11 cDNA clone, was amplified by 30 PCR cycles
(45 s at 95 °C, 45 s at 55 °C, 3 min at 74 °C) using
PrimeZyme (Biometra, Tampa, FL) and the primers
5'-CGGGATCCTAATGGAATTATTCCCAGAATTAAAAAATATTAAAGTACC-3' and
5'-CGGGGTACCTAAATTAAAATTTAATTTAGTTGTC-3'. The PCR product was
blunt-ended using Klenow, digested with BamHI, and cloned into pGEX-3 (Amersham Pharmacia Biotech) that was cut with
EcoRI, blunt-ended using Klenow, and digested with
BamHI to generate pGEX-ubpA. To express the yeast
ubp14 in Dictyostelium, the ubp14 cDNA obtained by digesting pGEX-UBP14 (7) with XhoI,
blunt-ended using Klenow, followed by digesting with BamHI,
was cloned into similarly cut pDXA-3H.
Determination of Isopeptidase Activity in E. coli
Extracts--
To induce expression of GST-Ubp14 and GST-UbpA, JM101
bacterial cells transformed with pGEX-UBP14 and pGEX-ubpA plasmid DNAs, respectively, were grown to an A600 of 0.7 in LB + 100 µg/ml ampicillin at 30 °C. After addition of 1 mM isopropyl-1-thio-b-D-galactopyranoside and
incubation at 30 °C for 3 h, cells (100 ml) were collected by
centrifugation and resuspended in 3 ml of phosphate-buffered saline
(PBS). The suspension was sonicated with a microtip attachment until
clarified; 1/20 volume of 10% Triton X-100 was added, and the
suspension was gently mixed by inversions of the tube. Cell debris was
removed by centrifugation at 14,000 × g for 10 min, and the extracts were adjusted to 0.5 mM EDTA, 10 µg/ml
aprotinin, 5 µg/ml pepstatin A. Expression of fusion proteins was
checked by PAGE and Coomassie Blue staining. Isopeptidase activity was assayed in 50 mM Tris-HCl, pH 7.3, 2 mM
dithiothreitol in a total volume of 20 µl following Amerik (7).
Reaction mixtures contained 2 µl of bacterial extracts and 0.5 µg
of Ub-Ub or Ub-UbGG. After 1 h at 37 °C, aliquots were
removed and analyzed by anti-ubiquitin immunoblotting.
RNA Analysis--
RNA isolation, electrophoresis, and transfer
to Duralon UV membrane (Stratagene) were performed as described (31). A
250-bp PvuII-EcoRI fragment (described above) and
a 400-bp BspDI fragment from genomic DNA clones pM7E and
pM7N, respectively, were used to prepare ubpA-specific DNA
probes labeled with [32P]dCTP by random hexamer-primed
DNA synthesis (Amersham Pharmacia Biotech). Additional probes were
prepared using the inserts of the following genomic or cDNA clones.
Jakob Franke (Columbia University, New York) provided plasmid pDE-0.9,
containing the 0.9-kb BstBI-EcoRI fragment of the
cyclic nucleotide phosphodiesterase cDNA (39). Chi-Hung Siu
(University of Toronto, Toronto, Canada) provided the gp80 clone which
carried a 1.0-kb EcoRI cDNA insert (40). Alan Kimmel
(National Institutes of Health, Bethesda) provided the cAR1 clone which
carried a 1.3-kb EcoRI cDNA insert. Hybridization was
performed at 60 °C in 0.125 M
Na2HPO4, pH 7.2, 0.25 M NaCl, 5%
SDS, 1 mM EDTA, 10% PEG (Mr 8,000).
Blots were washed with 50 mM
Na2HPO4, pH 7.2, 0.5% SDS twice at room
temperature for 30 min each and then at 60 °C for 30 min. Molecular
size standards were from the Life Technologies, Inc., 0.24-9.5-kb RNA
ladder.
Antibody Production and Purification--
An anti-UbpA
polyclonal serum was prepared using a bacterially expressed UbpA fusion
protein. The primer 5'-CGGGATCCACCAACAGAAAAATCACG-3' and the antisense
primer 5'-CGGGATCCAAGATGGACGAG-3' were used in a PCR to amplify
cDNA encoding a 438-residue segment of UbpA (residues 13-450; Fig.
1). PCR was carried out with Pfu enzyme for 35 cycles (1 min
at 94 °C, 1 min at 45 °C, 4 min at 74 °C). The primers added
BamHI sites at the ends of the amplified region. Following
digestion with BamHI, the 1.3-kb PCR product was cloned in
frame into the BamHI site of the expression vector pET15b
(Novagen, Madison, WI). After the construct was verified by DNA
sequencing, protein was expressed in the bacterial strain
BL21(DE3)pLysS (Novagen) and purified on His-Bind resin (Novagen)
following the manufacturer's protocol. The recombinant protein was
isolated further by 15% SDS-PAGE followed by electroelution of the
excised protein-containing gel slice and lyophilization (33). A rabbit
was then immunized by Cocalico Biologicals, Inc. (Reamstown, PA) with
30 µg of fusion protein in complete Freund's adjuvant injected into
the popliteal lymph nodes followed 30 days later with 150 µg in
incomplete Freund's adjuvant injected subcutaneously. Serum was
collected 10 days after the boost, and antibodies were purified with an
EZ Sep kit (Amersham Pharmacia Biotech). Monospecific antibodies
against the recombinant UbpA protein were obtained from the crude
antibody preparation by blot affinity purification essentially as
described by Talian et al. (41); elution of the antibody
bound to the antigen/polyvinylidene difluoride strip was done by use of
low pH shock. Affinity purified antibody was immediately neutralized, diluted 4-fold with 50 mM Tris, pH 7.9, 150 mM
NaCl, 0.05% Tween 20, 0.05% NaN3 (buffer A), then
concentrated and stored at 4 °C at a 1:10 dilution relative to the
starting antiserum volume.
Western Blots--
Dictyostelium cells harvested at
various developmental time points were directly solubilized with 2%
SDS sample buffer and heated at 100 °C for 5 min. For all Western
blots, the protein from 3 × 105 cells per lane was
resolved on 15 or 17.5% polyacrylamide-SDS gels and was transferred to
polyvinylidene difluoride membrane following Towbin et al.
(42). For the analysis of crude fractionated subcellular samples,
109 vegetative Ax4 cells were first collected by
centrifugation and resuspended to 3 × 108 cells/ml in
ice-cold MESES buffer (20 mM MES, pH 6.5, 1 mM
EDTA, 0.25 M sucrose). Cells were then homogenized with a
tight-fitting Dounce and centrifuged at 3,000 × g for
5 min to pellet nuclei. Post-nuclear supernatants (5 ml) were
centrifuged at 10,000 × g for 10 min, and the
resulting supernatant was spun again at 200,000 × g
for 30 min. Blots were blocked with 5% low-fat powdered milk in PBS (8 mM Na2HPO4, 1 mM
KH2PO4, pH 7.4, 0.14 M NaCl, 3 mM KCl) for 30 min, washed 3 min with buffer A, and then
incubated for 1.5 h with affinity purified UbpA antibody diluted
1:50 in buffer A at 25 °C. After a brief wash with buffer A, blots
were incubated with 0.230 µCi/ml 125I-protein A (Amersham
Pharmacia Biotech) in 20 mM Tris, 150 mM NaCl,
0.05% Tween 20, 1% bovine serum albumin, 0.05% NaN3
(buffer B) for 2.5 h at 25 °C and then washed with buffer B
five times (10 min each). Autoradiography was done using a Cronex
lightning plus amplifying screen (NEN Life Science Products) and Kodak
XAR-5 film.
To detect ubiquitin, Western blots were blocked for 1 h in PBS,
0.1% Tween 20 and then incubated with a 1:2000 dilution of anti-ubiquitin antibodies in PBS, 1% Tween 20, 1% Nonidet P-40, 0.1%
SDS for 1 h. To enhance resolution of low molecular mass proteins,
a Tricine gel system (43) was used. Proteins were transferred to
Immobilon-P membranes (Millipore); the filters were then boiled in
water and incubated with anti-ubiquitin antibodies. All blots used
anti-ubiquitin antibodies that had been affinity purified on a
ubiquitin affinity resin and were provided by C. Pickart or S. Swaminathan. Bound antibody was detected with the ECL Western blotting
kit (Amersham Pharmacia Biotech) following Pampori et al.
(44) using horseradish peroxidase-conjugated donkey anti-rabbit
antibodies (Amersham Pharmacia Biotech). Molecular size standards were
from the Life Technologies, Inc., 10-kDa protein ladder or ubiquitin
chain standards.
Determination of Isopeptidase Activity in Yeast
Extracts--
Overnight cultures of ubp14 cells
transformed with pUBP14 or pYEU1 plasmids were centrifuged and
resuspended in 3 volumes of disruption buffer (20 mM
Tris-HCl, pH 7.9, 10 mM MgCl2, 1 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 0.3 M
ammonium sulfate, 200 µg/ml aprotinin, 100 µg/ml pepstatin). Cells
were mixed with 4 volumes of chilled glass beads and vortexed five
times for 45 s each, leaving cells on ice for 1 min between
vortexings (35). The extracts were centrifuged, and supernatants were
stored at 
20 °C as 50% glycerol solutions. Assays of isopeptidase
activity were conducted at 37 °C in buffer containing 50 mM Tris-HCl, pH 7.3, 2 mM dithiothreitol in a
total volume of 20 µl. Reaction mixtures contained 2 µl of yeast
extract (160 ng of protein) and 1 µg of Ub-Ub. 3-µl aliquots were
removed at different times and analyzed by anti-ubiquitin
immunoblotting.
Immunofluorescence Staining--
All operations were at room
temperature unless specified. Vegetative DH1 and ubpA cells
were resuspended in PBM (20 mM
KH2PO4, pH 6.1, 1 mM
MgCl2, 10 µM CaCl2) and allowed
to settle and attach to 18 × 18 mm glass coverslips for 15 min.
After a brief wash with PBM, cells were fixed with 95% ethanol for 10 min on ice, air-dried, and placed in PBST (PBS, 0.1% Tween 20) for 15 min. Prior to use, antibodies were diluted 1:200 in PBST and
preadsorbed as follows. Six coverslips containing confluent, fixed
ubpA cells were consecutively incubated with 60 µl of
antibody for 15 min each. Experimental coverslips were then incubated
for 1 h with the preadsorbed antibody, washed twice (5 and 15 min)
with PBST, and incubated with 1:200 fluorescein
isothiocyanate-conjugated goat anti-rabbit (Cappell/Organon Teknika,
Durham NC) for 1 h. Slides were then washed as above and mounted
as described previously (45). Cells were examined and photographed with
a Microphot FX (Nikon, Melvillie, NY) and Kodak Tmax p3200 film (Kodak,
New Haven, CT) using a × 60 oil immersion objective.
Assays for cAMP Relay Response, Chemotaxis, and EDTA-resistant
Cell-Cell Adhesion--
The cAMP relay response was measured following
Van Haastert (46). Briefly, 5 × 106 cells in 0.1 ml
were stimulated with 10 µM 2'-deoxy-cAMP (a functional cAMP analog) in the presence of 10 mM dithiothreitol. At 0 and 3 min after stimulation, the cells were lysed, and total cAMP was
measured using the Amersham Pharmacia Biotech cAMP [3H]
assay system. For cAMP binding assays, cells starved on filters or in
shaking cultures were harvested, washed in phosphate buffer, resuspended to 1 × 108 cells/ml in ice-cold phosphate
buffer, and assayed for [3H]cAMP binding in 90% ammonium
sulfate (47). Chemotaxis was determined by the single spot small
population assay using a modification of Soll and Mitchell (48). Cells
starved for 4 h were washed twice in MES-PDF (8.2 mM
MES, pH 6.5, 20 mM KCl, 0.6 mM
MgSO4), and the final pellet was vortexed into a dense
suspension. Small droplets of cells were placed on 2% agarose
containing 0 or 1 µM cAMP. Diameters of the droplets were
measured using a Nikon microplot Fx with a × 4 objective and a
video system (allowing the drop diameter to be measured on the monitor)
just after plating (zero time) and at 3 h. EDTA-resistant
cell-cell adhesion was monitored as described by Soll and Mitchell
(48).
| |
RESULTS |
UbpA Has Similarity to a Deubiquitinating Enzyme--
A mutant
generated by an insertion into the ubpA gene was found in a
screen of REMI (restriction enzyme-mediated integration) transformants
as cells that never aggregated when grown on bacteria and then allowed
to starve on agar plates. When grown in axenic culture and then starved
on filters, wild-type cells begin to visibly aggregate (ripple stage)
6 h after starvation. Under identical conditions, ubpA
cells required at least 24 h to form ripples. After 3 days,
ubpA cells formed a small number of aberrant structures but
never formed fruiting bodies.
Genomic ubpA DNA was cloned from the mutant strain
(designated M7) in two fragments. Plasmid pM7E contained an
EcoRI fragment containing the insertion site and plasmid
pM7N contained an NsiI fragment from one side of the
insertion (Fig. 1A). The pM7E
plasmid was linearized with EcoRI and used to transform a
fresh wild-type host strain. The resulting gene disruption by
homologous recombination successfully recapitulated the delayed
aggregation phenotype.
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Fig. 1.
The ubpA gene.
A, genomic map of the ubpA region. The
thick bar represents the transcribed region, which is
divided into two exons; the black portion of the bar
designates the open reading frame. Restriction sites are as follows:
B, BspDI; E, EcoRI;
N, NsiI; P, PvuII. The
location of the DIV2 insertion is identified; DIV2 (not drawn to scale)
is the Dictyostelium pyr5-6 gene (cross-hatched
portion of bar) carried by the pGEM3 vector (spotted portion
of bar). The striped bar represents the PCR-generated
cDNA fragment used to construct the UbpA expression clone for
antibody production. B, the deduced amino acid sequence of
UbpA. The regions with identity to the Cys (amino acids 321-335),
"QQD" (amino acids 426-444), and His (amino acids 780-798) boxes
of the UBP family of deubiquitinating enzymes are
underlined. Two copies of the UBA domain are indicated by
double underlines. The DNA sequence of ubpA is
available in GenBankTM as accession number U48271.
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A probe prepared from the region flanking the insertion site was used
to screen a cDNA library, and several cDNA clones were isolated. These clones were sequenced as were the genomic fragments from pM7E and pM7N. Sequence analysis revealed a single long open reading frame interrupted by one intron (Fig. 1A). The
deduced amino acid sequence predicts a protein of 837 residues with a molecular mass of 95 kDa (Fig. 1B). The sequence has 44%
identity (63% similarity) to human isopeptidase T (ISOT1), and 29%
identity (50% similarity) to the yeast deubiquitinating enzyme Ubp14
(1, 7, 29, 49). The regions of similarity include the Cys and His
boxes, which are found in all members of the UBP family. These data
therefore suggest that UbpA is a UBP enzyme.
UbpA Is a Deubiquitinating Enzyme--
To test the potential
deubiquitinating activity of UbpA, the protein was expressed in
bacteria as a glutathione S-transferase (GST) fusion
protein. Enzymatic activity was assayed in bacterial cell extracts
using either a Lys-48-isopeptide bond-linked diubiquitin substrate
(Ub-Ub) or a derivative of this substrate that lacks the two C-terminal
glycine residues of the unanchored ubiquitin moiety (Ub-UbGG).
Extracts prepared from GST-UbpA-expressing E. coli cells
efficiently cleaved the diubiquitin substrate to ubiquitin monomers
(Fig. 2). The activity was comparable to
that of extracts from bacterial cells expressing a GST-Ubp14 fusion. Recently, yeast Ubp14 and its human homolog isopeptidase T were shown
to have very similar substrate specificities (7). Most strikingly,
neither enzyme can cleave the truncated diubiquitin, Ub-UbGG. We
conclude that the Dictyostelium UbpA enzyme, yeast Ubp14,
and human isopeptidase have closely related or identical substrate
specificities, with activity apparently restricted to ubiquitin chains
with a normal, unanchored C terminus.
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Fig. 2.
The Dictyostelium UbpA protein is
a deubiquitinating enzyme. Enzymatic activity toward Ub-Ub
(A) and Ub-UbGG (B) was measured in cell-free
extracts prepared from E. coli cells carrying pGEX,
pGEX-UBP14, or pGEX-ubpA plasmids. Reaction products were separated by
SDS-PAGE and analyzed by anti-ubiquitin immunoblotting. Positions of
ubiquitin dimers and monomeric ubiquitin are indicated. The small
amount of cleaved ubiquitin in B is due to a slight
contamination of the Ub-UbGG substrate with Ub-Ub (B. Krantz,
personal communication).
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UbpA Is a Functional Homolog of the Yeast Ubp14 Enzyme--
The
finding that UbpA is a deubiquitinating enzyme prompted us to compare
intracellular levels of ubiquitin and ubiquitin-protein conjugates in
wild-type and ubpA cells by anti-ubiquitin immunoblot analysis. At all stages of development, ubpA cells were
found to contain small ubiquitinated species that were absent in
wild-type cells, the most prominent of which comigrated with unanchored Lys-48-linked ubiquitin oligomers (Fig. 3
and data not shown). In addition, ubpA cells contained
increased levels of >80-kDa ubiquitin-containing species. Yeast
ubp14 cells show a similar accumulation of ubiquitin
oligomers (7). However, the yeast mutant does not display an obvious
accumulation of high molecular mass ubiquitinated species, and the
unanchored ubiquitin tetramer accumulates to a higher relative level
than any other species.
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Fig. 3.
Ubiquitin conjugates in parental and
ubpA cells. Total protein prepared from the parental
strain, DH1 (P), and the mutant ubpA strain ()
during axenic growth (0) and at 3 h of development
(3) was resolved by SDS-PAGE and analyzed by anti-ubiquitin
immunoblotting. Size standards were free ubiquitin and ubiquitin
oligomers (Ub).
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The high degree of similarity in sequence and in substrate specificity
between UbpA and Ubp14 and the comparable accumulation of unanchored
ubiquitin chains in Dictyostelium ubpA and yeast ubp14 cells led us to investigate whether the
Dictyostelium enzyme could functionally replace Ubp14 in
yeast cells. The ubpA gene was subcloned into the yeast
expression vector pYES2, and the resulting plasmid was transformed into
yeast ubp14 cells. ubp14 cells have a number of
characteristic phenotypic abnormalities, including hypersensitivity to
amino acid analogs and defects in the degradation of a variety of
ubiquitin system substrates (7). Anti-ubiquitin immunoblot analysis of
ubp14 cells expressing ubpA revealed that the
levels of unanchored ubiquitin chains were reduced to wild-type levels
(Fig. 4A). In addition, these
cells grew indistinguishably from wild-type cells on plates containing
the arginine analog canavanine (Fig. 4B). We previously
found that cell-free extracts prepared from ubp14 mutants
display a greatly reduced activity against the Lys-48-linked
diubiquitin test substrate (but not against Ub-UbGG) relative to
wild-type cells, indicating that Ubp14 is the major yeast DUB capable
of cleaving unanchored ubiquitin chains in yeast cells (7).
Transformation of ubp14 cells with the UbpA expression
plasmid restored activity against diubiquitin in cell-free extracts to
wild-type levels (data not shown).
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Fig. 4.
Expression of Dictyostelium UbpA
in yeast ubp14 cells restores the wild-type
phenotype. A, anti-ubiquitin immunoblot analysis of
wild-type and ubp14 mutant cells containing various
expression plasmids. Accumulation of unanchored ubiquitin chains is
suppressed to wild-type level in ubp14 mutants carrying the
UbpA-expressing plasmid pYEU1. Positions of free ubiquitin and
unanchored ubiquitin chains are indicated. B, growth on the
media containing 1.0 mg/ml canavanine sulfate. Wild-type and
ubp14 mutant cells carrying the indicated plasmids were
streaked onto canavanine plates and incubated for 3 days at 30 °C.
As in A, wild-type yeast MHY501 cells carried the vector
YEplac195 and the ubp14 strain MHY840 carried YEplac195,
YEplac195UBP14, or pYEU1. Cells were grown in media containing 3%
galactose.
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We also investigated whether the yeast ubp14 could restore
wild-type development in ubpA cells. Expression of the
ubp14 cDNA downstream from the Dictyostelium
act15 promoter complemented the aggregation defective phenotype of
ubpA cells; fruiting bodies formed but they were fewer in
number and somewhat aberrant when compared with wild-type (data not
shown). However, the phenotype of these cells was similar to
ubpA cells that contained the ubpA cDNA
expressed downstream from the act15 promoter. These data and
those of the previous section led us to conclude that the D. discoideum UbpA and S. cerevisiae Ubp14 enzymes are
functional homologs.
UbpA Is Present in Vegetative and Developing Cells--
The
temporal transcription pattern of ubpA was examined by
Northern analysis. A 2.9-kb ubpA mRNA was present in
vegetatively growing cells (0 h), increased in level during early
development to a maximum at 7.5 h, and continued to be present
through the remainder of development (Fig.
5A). ubpA RNA was
not present in ubpA cells (data not shown). Immunoblot
analyses indicated that a 95-kDa UbpA protein was present in wild-type
cells (Fig. 5B). The pattern of UbpA protein expression in
wild-type cells was similar to the mRNA pattern. A relatively high
level of the protein accumulated in growing cells; after the onset of
starvation, UbpA levels initially dropped, then increased to maximum
levels at 10-15 h, and continued to be present throughout the
remainder of development (Fig. 5B). The protein was not
detected in the ubpA mutant or when preimmune serum was used
in immunoblot analyses of either wild-type or mutant cells (data not
shown). Expression of ubpA RNA and protein in the parental
DH1 strain was similar to that seen in the wild-type cells (Ax4) (data
not shown).
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Fig. 5.
Expression of ubpA. Total
RNA and protein were prepared from wild-type cells (Ax4) during axenic
growth (0) and at the indicated stages of development (in
hours). A, RNA (5 µg) was resolved by electrophoresis
through 1.2% agarose/formaldehyde gels, blotted to nylon membranes,
and hybridized with a probe specific for ubpA. Size
standards are indicated on the left in kb. B,
protein was resolved on 17.5% polyacrylamide-SDS gels,
electrotransferred to polyvinylidene difluoride membrane, and the blots
stained with an antibody against a UbpA fusion protein. Size standards
are indicated on the left in kDa.
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To determine the subcellular location of UbpA, vegetative cells were
lysed and extracts were fractionated by differential centrifugation,
and the fractions were examined by anti-UbpA immunoblotting. UbpA was
present in the supernatant after a low speed spin to remove nuclei, and
a medium speed spin to pellet large organelles. The UbpA protein was
still present in the supernatant when the second supernatant was
centrifuged for 6 × 106 g/min (data not
shown). The subcellular and cell type distribution of UbpA was further
characterized by immunofluorescence analysis of fixed cells. Vegetative
and developing DH1 cells showed a diffuse staining throughout the
cytoplasm and nucleus, and all cells were stained (data not shown).
Only a weak, apparently nonspecific staining was observed for
vegetative ubpA mutant cells. No staining of either
wild-type or mutant cells was seen with preimmune sera. The
fractionation and immunofluorescence data thus suggest that UbpA is a
soluble protein present in all cells throughout growth and
development.
Growth and Initial Responses to Starvation Appear to be Normal in
ubpA Cells--
We next examined the general phenotype of growing and
starving ubpA cells. When grown axenically, DH1 parental and
ubpA cells doubled at essentially the same rate. When grown
on bacterial lawns, isolated ubpA cells formed plaques that
increased in diameter at approximately the same rate as DH1 cells.
Vegetative and starved ubpA cells were indistinguishable
from wild-type cells by phase-contrast light microscopy, and trypan
blue staining showed that lack of UbpA does not affect the viability of
cells. Using time lapse videomicroscopy, we determined that both
growing and starved ubpA cells have normal motility. As in
wild-type cells, cell division ceases in mutant cells upon starvation
(data not shown).
We found that ubpA cells secrete normal amounts of
biologically active CMF (data not shown). In addition, ubpA
cells were able to express both the CP2 prestalk and the SP70 prespore
markers within 18 h when starved at low cell density in the
presence of CMF and then cAMP and that there was a normal percentage of
the two cell types. This indicated that starved ubpA cells
are able to differentiate into the proper initial ratio of cell types, sense CMF, sense the cAMP added at 6 h, and to express mRNAs
and proteins many hours after starvation.
Expression of cAMP Pulse-induced Genes Is Altered in Cells That
Lack UbpA--
Since all the initial responses to starvation that we
examined are apparently normal in ubpA cells, we examined
whether there was a successful transition to the next developmental
stage. This stage is marked by chemotaxis to cAMP, cAMP relay, and
acquisition of EDTA-resistant cell-cell adhesion. To assay for
chemotaxis, we used the single spot small population method. The
ability of ubpA cells to chemotax is somewhat impaired
(Table I). We also examined the ability
of cells to relay a pulse of cAMP. In the parental DH1 cells, cAMP
accumulation in response to a pulse of a cAMP analog was similar to
that previously seen for wild-type cells (50), whereas ubpA
cells exhibited a 3.4-fold less cAMP production. Also during early
aggregation, EDTA-resistant cell-cell binding sites begin to appear,
enabling the cells to form tight multicellular aggregates (51). The
formation of these binding sites is mediated by gp80, a cell-surface
glycoprotein. EDTA-resistant cell-cell adhesion in wild-type cells
begins after 3 h and peaks at roughly 12 h (see Ref. 54; Fig.
6). For ubpA cells,
EDTA-resistant cell-cell adhesion only first appears at 24 h and
reaches a peak at 36 h (when the cells are aggregating) (Fig. 6).
The data thus suggest that during the time when wild-type cells are
aggregating, ubpA cells show an abnormally low level of both
chemotaxis to cAMP and cAMP relay and undetectable EDTA-resistant
cell-cell adhesion.
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Table I
Chemotaxis in response to a gradient of cAMP
Aliquots of parental DH1 or ubpA mutant cells were placed on
thin agar plates, with or without cAMP added to the agar, and the
percentage increase in droplet diameter after 3 h was determined.
The means ± S.D. for three separate experiments are shown. The
fold increase (+cAMP increase/cAMP increase) was calculated
separately for each experiment, and the means ± S.D. for the
three experiments are shown.
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Fig. 6.
Acquisition of EDTA-resistant cell-cell
adhesion is delayed in ubpA cells. Axenically grown
parental DH1 and ubpA mutant cells were developed on
filters, and EDTA-resistant cell-cell adhesion was determined for the
various time points.
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Northern blot analysis was then used to determine whether the above
defects correlated with lowered expression of cAR1, PDE, and gp80. In
wild-type cells, cAR1 and PDE mRNAs are present during growth and
early development, whereas a basal level of gp80 transcription is
initiated soon after the onset of starvation. The levels of all three
mRNAs greatly increase during the cAMP pulse-mediated aggregation
and then decline as development proceeds (see Refs. 52-54; Fig.
7A). In ubpA cells,
all three mRNAs were also expressed at a low level during early
development. However, the levels remain very low until 36 h, when
the ubpA cells began forming small aggregates. These data
suggest that the defective aggregation of ubpA cells is
associated with a failure to increase expression of some
aggregation-specific mRNAs that normally occurs in response to
pulses of cAMP.
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Fig. 7.
ubpA cells have delayed expression of
early developmental genes. A, total RNA was prepared
from the parental strain (DH1) and the mutant transformant
(ubpA) during axenic growth (0), at 3 h of
development, and at 7, 11, and 14 h when development of DH1 had
reached the ripple, tight aggregate, and early culminate stages,
respectively. In addition, RNA was prepared from ubpA cells
at 24, 36, and 42 h when the mutant cells were at the ripple,
loose aggregate, and tipped aggregate stages, respectively. RNA (5 µg) was resolved by electrophoresis through 1.2%
agarose/formaldehyde gels, blotted to nylon membranes, and hybridized
with a probe specific for cAR1 cAMP receptor (cAR1), cAMP
phosphodiesterase (PDE), or transcripts encoding the
cell-cell adhesion molecule gp80. B, total RNA was prepared
from parental DH1 and mutant ubpA cells that were starved
for 6 h in shaking culture in the presence (P) or
absence (S) of exogenous pulses of cAMP. RNA (5 µg) was
resolved by electrophoresis through 1.2% agarose/formaldehyde gels,
blotted to nylon membranes, and hybridized with a probe specific for
cAR1, PDE, or gp80.
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UbpA Is Required for Generating, but Not Sensing, Pulses of
cAMP--
Two possible explanations for why the ubpA cells
do not up-regulate cAMP pulse-induced genes are either the cells are
insensitive to cAMP pulses or they are not producing the pulses. To
determine if ubpA cells can respond to pulses of cAMP, cells
were starved in shaking culture in the presence or absence of exogenous
pulses of cAMP. After 6 h, the cells were harvested and plated on
non-nutrient agar. As shown in Fig. 8,
ubpA cells starved in the absence of pulses do not
aggregate, whereas the same cells starved in the presence of exogenous
cAMP pulses formed aggregates that developed to (but not beyond) the
early culminate stage. DH1 parental cells starved in the presence or
absence of cAMP pulses formed fruiting bodies (Fig. 8 and data not
shown). The ability to aggregate in response to exogenous cAMP pulses
was paralleled by an enhanced accumulation of aggregation-specific
mRNAs under the same conditions (Fig. 7B). Binding of
cAMP to both DH1 and ubpA cells was increased similarly as
well (Fig. 9A); therefore, the
extra cAR1 mRNA induced in pulsed ubpA cells
was translated, resulting in an up-regulation of the cAR1 receptor.
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Fig. 8.
Pulsing ubpA cells with cAMP
rescues the aggregation defective phenotype. Parental DH1
(WT) and mutant ubpA cells were starved in
shaking culture in the presence (WT, ubpA pulsed) or absence
(ubpA) of exogenous pulses of cAMP. After 6 h, the
cells were harvested and plated on non-nutrient agar.
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Fig. 9.
Pulsing ubpA cells with cAMP
partially rescues their inability to relay a pulse of cAMP. DH1
parental and ubpA mutant cells were starved on filters or in
shaking cultures in the presence or absence of exogenous pulses of
cAMP. A, after 1 and 6 h of starvation, cells were
harvested and assayed for cAMP binding. The means ± S.E. for five
separate experiments are shown. B, after 6 h, the cells
were harvested, washed, and resuspended to 6.25 × 107
cells/ml. Total cAMP was determined in duplicate 3 min after addition
of 2'-deoxy-cAMP (a cAMP analog which stimulates cAR1 but which is not
detected by the cAMP assay). The means ± S.E. for four separate
experiments are shown.
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Finally, we directly examined whether the defective aggregation of
ubpA cells was due to a defect in the ability of these cells
to generate pulses of cAMP (Fig. 9B). The parental DH1 cells starved in the absence of exogenous cAMP pulses will generate a relay
pulse of cAMP in response to a cAMP stimulus; this response increases
for cells exposed to cAMP pulses. ubpA cells have a significantly attenuated capacity for constitutive and stimulated cAMP
production, but when exposed to exogenous pulses of cAMP, the cAMP
product increases strongly but is still significantly reduced from
wild-type levels (Fig. 9B). This suggests that
ubpA cells have a partial defect in cAMP-induced cAMP
production, which can account for their failure to aggregate
normally.
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DISCUSSION |
In this report, we have characterized the Dictyostelium
UbpA protein, a new member of the UBP family of deubiquitinating or DUB
enzymes, and we have shown that deletion of UbpA from cells, while of
little consequence during growth, prevents cell aggregation and
fruiting body development. UbpA bears strong sequence similarity to the
yeast Ubp14 and mammalian isopeptidase T enzymes and shares with these
well characterized enzymes a specificity for unanchored polyubiquitin
chains. Moreover, we demonstrate by cross-species complementation that
UbpA and Ubp14 are in fact functional homologs. This is the first time
a DUB enzyme with a well characterized substrate specificity has been
directly implicated in multicellular development. A central question
raised by these data is how a deubiquitinating enzyme whose activity is
apparently restricted to free ubiquitin chains can be crucial to a
specific stage (or stages) in Dictyostelium
development.
The UbpA Enzyme Is Responsible for Polyubiquitin Chain Disassembly
in Vivo--
UbpA belongs to the Ubp14/isopeptidase T subfamily of
DUBs. There are three known isozymes of isopeptidase T in humans, and UbpA is over 40% identical to the two that have been studied in vitro, IsoT-1 and IsoT-2 (1, 29). Although the conservation with
yeast Ubp14 is weaker, sequence identity (~29% overall) extends over
the entire lengths of the two proteins (7). Whereas all UBPs share
conserved sequences in the Cys and His boxes and a few short stretches
between these two motifs, there is little further similarity among them
as a group, particularly in the generally large regions N-terminal to
the Cys box (3).
The conservation of primary sequence among members of the
Ubp14/isopeptidase T subfamily is paralleled by their comparable substrate specificities. A dramatic reduction in the rate of
disassembly of a Lys-48-linked diubiquitin molecule by both
isopeptidase T (3) and Ubp14 (7) is observed when the last two glycines are deleted from the proximal ubiquitin monomer (Ub-UbGG). The same
block to disassembly is also observed with UbpA (Fig. 2) but not
observed with other DUB enzymes (e.g. see Ref. 7). Most
telling, cross-species complementation experiments verify that UbpA and
Ubp14 are functional homologs (Fig. 4); analogous data had been
obtained when human isopeptidase T was expressed in yeast
ubp14 cells (7). The functional interchangeability of
Dictyostelium, human, and yeast enzymes indicates that the more detailed mechanistic analysis done in yeast cells can be used to
infer how other members of the Ubp14 subfamily participate in the
ubiquitin pathway in their respective cell types.
Consistent with this last point, we observe a comparable profile of
ubiquitin-containing species in yeast ubp14 (7) and in
Dictyostelium ubpA mutants (Fig. 4). The bulk of these
species comigrate with Lys-48-linked ubiquitin chain standards and, in the case of ubp14 cells, were shown to share the same
isoelectric point as free ubiquitin monomer, indicating that the
majority of ubiquitin conjugates that accumulate abnormally in these
mutants are unanchored ubiquitin chains. Interestingly, ubpA
cells, unlike yeast ubp14 cells, accumulate ubiquitinated
material of very high molecular mass in addition to the easily
identified ubiquitin oligomers (Fig. 3). These larger species may be
either very large free polyubiquitin chains or extensively
ubiquitinated proteins (or both). If the former were true, it might
reflect a more active or more processive polyubiquitin chain
synthesizing machinery in Dictyostelium than in yeast,
resulting in greater average chain lengths. This view is consistent
with the observation that yeast ubp14 cells accumulate
particularly high levels of ubiquitin tetramer and only small amounts
of larger species (7), whereas this bias is not seen in the
Dictyostelium mutant.
On the other hand, it is very likely (see below) that, like yeast
ubp14 cells, the ubpA mutant is impaired in
proteasome-mediated protein degradation, which can lead to the
accumulation of high molecular mass polyubiquitinated proteins. A more
severe proteolytic defect in the Dictyostelium mutant than
in the corresponding yeast mutant might account for the difference in
conjugated accumulation between taxa. However, this would predict a
considerable growth impairment in ubpA cells given that
proteasomes are essential for viability, and this was not observed.
Alternatively, yeast might have a more potent protein deubiquitination
potential than Dictyostelium, resulting in more rapid
deubiquitination of the polyubiquitinated substrates that fail to get
targeted to the proteasome. These various explanations are not mutually
exclusive, and in any event, the similarities in unanchored
polyubiquitin chain accumulation between the yeast and
Dictyostelium mutants remain striking.
A Specific Developmental Defect Is Associated with Loss of
UbpA--
The absence of UbpA affects neither the growth rate of
Dictyostelium cells nor the gross appearance or motility of
vegetative cells. UbpA does not appear to be required for sensing
starvation since ubpA cells exhibit certain normal responses
to starvation such as CMF secretion, a decrease in motility, the
ability to sense CMF, initiation of a basal level of gp80
transcription, and differentiation into CP2-positive prestalk,
SP70-positive prespore, and null cells. Even though the absence of UbpA
delays aggregation for several days, UbpA is not required for several aspects of development that occur 10-15 h after onset of starvation, such as sensing high continuous levels of cAMP, or prestalk, and prespore protein expression.
In growing wild-type cells, certain cAMP pulse-induced genes, such as
cAR1, are expressed at low levels (53). This presumably allows the
cells to have a small amount of the cAMP pulse signal transduction
machinery in place when they encounter starvation conditions, so that
when a high level of CMF informs the cells that there is a local high
density of starved cells (11, 12), the cells can begin to relay pulses
of cAMP. A bootstrap process then permits the pulses of cAMP to
up-regulate the cAMP signal transduction system to a level that allows
the cells to aggregate. The bootstrap routine in
Dictyostelium cells is such that a small random pulse of
extracellular cAMP can trigger cells to relay the pulse and to produce
more of the proteins required to sense and relay the cAMP pulses. Each
time a cell sees a cAMP pulse during the bootstrap phase of
development, it will respond by generating a slightly greater cAMP
pulse, which provides an enhanced signal that causes nearby cells to
increase production of the cAMP relay machinery, and so forth.
Like wild-type cells, ubpA cells have an initial low level
of cAR1 transcription, and their partial ability to chemotax toward cAMP and relay a pulse of cAMP indicates that they do have a functional cAMP signal transduction mechanism. However, the cAMP pulse-induced increase in the accumulation of mRNAs encoding proteins such as cAR1, PDE, and gp80 does not occur. The ability of exogenously added
pulses of cAMP to rescue this bootstrap process in the ubpA cells so that they are able to increase the accumulation of
pulse-induced mRNAs, increase cell-surface cAR1, increase the cAMP
pulse relay, and aggregate suggests that the lack of UbpA does not
cause a block in the signal transduction machinery that senses the
pulses of cAMP and increases the expression of the pulse-induced genes. The rescue of the various responses in ubpA cells by pulsing
them with cAMP, and the observation that ubpA cells have a
poor cAMP relay efficiency, thus indicates that these cells do not
generate sufficiently large pulses of extracellular cAMP to elicit a
normal bootstrap response. Because there is some relay of cAMP pulses in the ubpA cells, one would then predict, and we indeed
observe, that the bootstrap process will be present but slower,
resulting in the cells eventually aggregating. This suggests that one
function of UbpA is to permit starved cells to generate a large pulse
of cAMP in response to sensed cAMP early in the aggregation phase of
development.
Providing exogenous pulses of cAMP to starving ubpA cells
makes the cells competent to aggregate when plated onto non-nutrient agar; however, these cells do not develop beyond the aggregate stage.
This result and the fact that UbpA protein accumulates to a high level
just prior to and during aggregation suggest that ubpA cells
are also deficient in at least one other developmental transition.
Alternatively, UbpA may be required only for aggregation, but ubiquitin
conjugates that accumulate in the ubpA mutant may interfere
with development beyond the aggregate stage. It is possible that UbpA
functions throughout development; but, in its absence, ubiquitin
conjugates accumulate and block development only at specific
stages.
Polyubiquitin Chain Metabolism, Proteolysis, and
Development--
The primary, if not exclusive, physiological role of
Ubp14 in yeast is the disassembly of unanchored ubiquitin chains (7). In principle, such ubiquitin chains can be generated by DUB enzymes during 26 S proteasome action on ubiquitin-protein conjugates and/or
by release from protein substrates independent of the proteasome. In
addition, ubiquitin chains can be synthesized de novo from free ubiquitin monomer (see Ref. 7 and references therein). Free
ubiquitin chains can inhibit ubiquitin-dependent
proteolysis in vitro, presumably by competing for binding to
specific polyubiquitin-binding sites on the 26 S proteasome (28, 55).
Our recent work in yeast strongly supports the idea that such
inhibition can occur in vivo as well. Degradation of
multiple ubiquitin system proteolytic substrates is inhibited in
ubp14 mutants, suggesting that Ubp14 functions to keep
polyubiquitin levels low enough to avoid inhibition of the proteasome
(7).
We have shown here that yeast Ubp14 and Dictyostelium UbpA
are functional homologs with strong similarities in sequence and in
substrate specificity. Hence, UbpA is also very likely to be important
for maximal rates of ubiquitin/proteasome-dependent proteolysis of a broad range of substrates. Knowledge of the enzymatic specificity of the UbpA/Ubp14/isopeptidase T family of enzymes argues
against the alternative hypothesis that UbpA regulates the turnover of
specific protein substrates by selective protein deubiquitination. In
the one other example of a UBP enzyme clearly implicated in
development, the Drosophila Faf protein, the genetic evidence implied that Faf acts to antagonize proteasomal degradation of
specific proteins by deubiquitinating them (8). Unlike UbpA and Ubp14,
little is known about the substrate specificity of Faf.
Despite the striking accumulation of unanchored ubiquitin chains in
yeast ubp14 mutants, the proteolytic defect in these cells in relatively mild. For the ubiquitin system substrates tested, inhibition was only ~2-5-fold (7). It is reasonable to assume that
similarly modest but significant proteolytic defects will be obtained
in the Dictyostelium ubpA mutant inasmuch as the yeast and
Dictyostelium mutants have similar ubiquitin chain
accumulation profiles and neither has a growth defect. This would imply
that development in D. discoideum is extremely sensitive to
even minor inhibition of the ubiquitin system. The yeast sporulation
pathway, which is also induced by starvation, can be regarded as an
analog of the Dictyostelium developmental pathway leading to
fruiting body formation. Homozygous ubp14 yeast diploid
cells are unable to sporulate normally at 30 °C (but sporulate at
near wild-type frequency at 25 °C), indicating that this process is
also sensitive to small perturbations of
ubiquitin-dependent proteolysis (7). Progression through
the bootstrap phase and probably additional stages of
Dictyostelium development may require overcoming inhibition by specific negative regulators or "checkpoint" factors. In the simplest model, regulated ubiquitin-dependent degradation
of such factors would allow the proper coordination of the molecular
and cellular events required for development.
In support of the above ideas, it was found recently that a
Dictyostelium strain carrying a mutant allele of the
prtC gene, which encodes a C2-like subunit of the 20 S
proteasome, also suffers from specific developmental defects (see
GenBankTM accession number U60168), although not at the
aggregation stage. Drosophila melanogaster is known to have
at least three variants of another -type 20 S proteasome subunit,
and two of these are specifically expressed in the testes during
spermatogenesis (56). One can anticipate additional examples of
ubiquitin system involvement, particularly the participation of
UbpA-like enzymes, in specific developmental processes. In this regard,
it is interesting that at least three isoforms of isopeptidase T exist
in humans, two of which derive from tissue-specific alternative
splicing and one encoded by a separate gene (see (7).
§,
Top
Abstract
Introduction
Associate investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed. Tel.: 713-527-4872; Fax:
713-285-5154; E-mail: richard{at}bioc.rice.edu.
