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• W2009596467 abstract "Although cell differentiation usually involves synthesis of new proteins, little is known about the role of protein degradation. In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. This process is regulated by deubiquitinating enzymes, which can disassemble ubiquitin polymers or ubiquitin-substrate conjugates. We find that a deubiquitinating enzyme, UbpA, is required for Dictyostelium development.ubpA cells have normal protein profiles on gels, grow normally, and show normal responses to starvation such as differentiation and secretion of conditioned medium factor. However,ubpA cells have defective aggregation, chemotaxis, cAMP relay, and cell adhesion. These defects result from low expression of cAMP pulse-induced genes such as those encoding the cAR1 cAMP receptor, phosphodiesterase, and the gp80 adhesion protein. Treatment of ubpA cells with pulses of exogenous cAMP allows them to aggregate and express these genes like wild-type cells, but they still fail to develop fruiting bodies. Unlike wild type, ubpAcells accumulate ubiquitin-containing species that comigrate with ubiquitin polymers, suggesting a defect in polyubiquitin metabolism. UbpA has sequence similarity with yeast Ubp14, which disassembles free ubiquitin chains. Yeast ubp14 cells have a defect in proteolysis, due to excess ubiquitin chains competing for substrate binding to proteasomes. Cross-species complementation and enzyme specificity assays indicate that UbpA and Ubp14 are functional homologs. We suggest that specific developmental transitions in Dictyostelium require the degradation of specific proteins and that this process in turn requires the disassembly of polyubiquitin chains by UbpA. Although cell differentiation usually involves synthesis of new proteins, little is known about the role of protein degradation. In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. This process is regulated by deubiquitinating enzymes, which can disassemble ubiquitin polymers or ubiquitin-substrate conjugates. We find that a deubiquitinating enzyme, UbpA, is required for Dictyostelium development.ubpA cells have normal protein profiles on gels, grow normally, and show normal responses to starvation such as differentiation and secretion of conditioned medium factor. However,ubpA cells have defective aggregation, chemotaxis, cAMP relay, and cell adhesion. These defects result from low expression of cAMP pulse-induced genes such as those encoding the cAR1 cAMP receptor, phosphodiesterase, and the gp80 adhesion protein. Treatment of ubpA cells with pulses of exogenous cAMP allows them to aggregate and express these genes like wild-type cells, but they still fail to develop fruiting bodies. Unlike wild type, ubpAcells accumulate ubiquitin-containing species that comigrate with ubiquitin polymers, suggesting a defect in polyubiquitin metabolism. UbpA has sequence similarity with yeast Ubp14, which disassembles free ubiquitin chains. Yeast ubp14 cells have a defect in proteolysis, due to excess ubiquitin chains competing for substrate binding to proteasomes. Cross-species complementation and enzyme specificity assays indicate that UbpA and Ubp14 are functional homologs. We suggest that specific developmental transitions in Dictyostelium require the degradation of specific proteins and that this process in turn requires the disassembly of polyubiquitin chains by UbpA. deubiquitinating enzyme(s) conditioned medium factor base pair phosphodiesterase polymerase chain reaction kilobase pair(s) rapid amplification of cDNA ends polyacrylamide gel electrophoresis ubiquitin 4-morpholineethanesulfonic acid phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine ubiquitin-specific processing protease. 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. 1Wilkinson K.D. Tashayev V.L. O'Connor L.B. Larsen C.N. Kasperek E. Pickart C.M. Biochemistry. 1995; 34: 14535-14546Crossref PubMed Scopus (259) Google Scholar and 2Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1443) Google Scholar). 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 ε-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 (3Wilkinson K.D. Hochstrasser M. Peters J.M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell Cycle. Plenum Publishing Corp., New York1998: 99-125Google Scholar). 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 (4Baker R.T. Tobias J.W. Varshavsky A. J. Biol. Chem. 1992; 267: 23364-23375Abstract Full Text PDF PubMed Google Scholar, 5Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (339) Google Scholar). Several additional short sequences are also moderately well conserved (3Wilkinson K.D. Hochstrasser M. Peters J.M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell Cycle. Plenum Publishing Corp., New York1998: 99-125Google Scholar). 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 (2Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1443) Google Scholar). 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 (6Lam Y.A. Xu W. DeMartino G.N. Cohen R.E. Nature. 1997; 3852: 737-740Crossref Scopus (365) Google Scholar). 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 (5Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (339) Google Scholar). On the other hand, Ubp14 has recently been found to be the major yeast DUB that disassembles free ubiquitin chains (7Amerik A.Y. Swaminathan S. Krantz B.A. Wilkinson K.D. Hochstrasser M. EMBO J. 1997; 16: 4826-4838Crossref PubMed Scopus (196) Google Scholar). 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 (5Papa F.R. Hochstrasser M. Nature. 1993; 366: 313-319Crossref PubMed Scopus (339) Google Scholar). 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 (8Huang Y. Baker R. Fischer-Vize J.A. Science. 1995; 270: 1828-1831Crossref PubMed Scopus (240) Google Scholar). 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 (9Gomer R.H. Yuen I.S. Firtel R.A. Development. 1991; 112: 269-278PubMed Google Scholar, 10Jain R. Gomer R.H. Murtagh Jr., J.J. BioTechniques. 1992; 12: 58-59PubMed Google Scholar, 11Yuen I.S. Gomer R.H. J. Theor. Biol. 1994; 167: 273-282Crossref PubMed Scopus (35) Google Scholar, 12Jain R. Gomer R.H. J. Biol. Chem. 1994; 269: 9128-9136Abstract Full Text PDF PubMed Google Scholar, 13Yuen I.S. Jain R. Bishop J.D. Lindsey D.F. Deery W.J. Van Haastert P.J.M. Gomer R.H. J. Cell Biol. 1995; 129: 1251-1262Crossref PubMed Scopus (64) Google Scholar). 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. 14Firtel R.A. van Haastert P.J.M. Kimmel A.R. Devreotes P.N. Cell. 1989; 58: 235-239Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 15Peters D.J.M. Cammans M. Smit S. Spek W. Campagne M.M.V. Schaap P. Dev Genet. 1991; 12: 25-34Crossref PubMed Scopus (21) Google Scholar, 16Dottin R.P. Bodduluri S.R. Doody J.F. Haribabu B. Dev. Genet. 1991; 12: 2-5Crossref PubMed Scopus (17) Google Scholar, 17Van Haastert P.J.M. Janssens P.M.W. Erneux C. Eur. J. Biochem. 1991; 195: 289-303Crossref PubMed Scopus (39) Google Scholar for review). The pulses of cAMP also function as a chemoattractant that causes the cells to aggregate (reviewed in Ref. 18Robertson A.D.J. Grutsch J.F. Cell. 1981; 24: 603-611Abstract Full Text PDF PubMed Scopus (36) Google Scholar). 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. 19Schaap P. Dworkin M. Intercellular Interactions during Dictyostelium Development. American Society for Microbiology, Washington, D. C.1991: 147-178Google Scholar, 20Loomis W.F. Dictyostelium discoideum: A Developmental System. Academic Press, New York1975Google Scholar, 21Loomis W.F. Curr. Top. Dev. Biol. 1993; 28: 1-46Crossref PubMed Scopus (64) Google Scholar, 22Devreotes P. Science. 1989; 245: 1054-1058Crossref PubMed Scopus (227) Google Scholar, 23Firtel R.A. Gene Dev. 1995; 9: 1427-1444Crossref PubMed Scopus (146) Google Scholar, 24Gross J. Microbiol. Rev. 1994; 58: 330-351Crossref PubMed Google Scholar for review). The entire developmental process takes only 24 h. InDictyostelium, ubiquitin genes have been identified, and ubiquitin mRNA species were shown to be developmentally regulated, suggesting a role for ubiquitin in development (25Muller-Taubenberger A. Westphal M. Jaeger E. Noegel A. Gerisch G. FEBS Lett. 1988; 229: 273-278Crossref PubMed Scopus (32) Google Scholar, 26Ohmachi T. Giorda R. Shaw D.R. Ennis H.L. Biochemistry. 1989; 28: 5226-5230Crossref PubMed Scopus (29) Google Scholar). In addition, a ubiquitin-conjugating enzyme, UBC1, has been implicated in Dictyostelium development (27Clark A. Nomura A. Mohanty S. Firtel R. Mol. Biol. Cell. 1997; 8: 1989-2002Crossref PubMed Scopus (21) Google Scholar). 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 (7Amerik A.Y. Swaminathan S. Krantz B.A. Wilkinson K.D. Hochstrasser M. EMBO J. 1997; 16: 4826-4838Crossref PubMed Scopus (196) Google Scholar) and mammalian isopeptidase T (1Wilkinson K.D. Tashayev V.L. O'Connor L.B. Larsen C.N. Kasperek E. Pickart C.M. Biochemistry. 1995; 34: 14535-14546Crossref PubMed Scopus (259) Google Scholar, 28Hadari T. Warms J.V.B. Rose I.A. Hershko A. J. Biol. Chem. 1992; 267: 719-727Abstract Full Text PDF PubMed Google Scholar, 29Falquet L. Paquet N. Frutiger S. Hughes G.J. Hoang-Van K. Jaton J.-C. FEBS Lett. 1995; 359: 73-77Crossref PubMed Scopus (43) Google Scholar), and that the UbpA enzyme is crucial for development. We used a random mutagenesis protocol (30Kuspa A. Loomis W.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8803-8807Crossref PubMed Scopus (397) Google Scholar) 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. D. discoideum Ax4 cells were grown as described previously (31Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (100) Google Scholar). The uracil-lacking strain DH1 (a gift of Peter Devreotes, Johns Hopkins University. Baltimore), apyr5-6 derivative of Ax4, was grown in HL5 medium (32Sussman M. Spudich J.A. Cultivation and Synchronous Morphogenesis of Dictyostelium Under Controlled Experimental Conditions. Academic Press, Orlando, FL1987: 9-29Google Scholar) supplemented with 20 μg/ml uracil. Cells were developed on filters (31Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (100) Google Scholar) or starved in shaking cultures as described (13Yuen I.S. Jain R. Bishop J.D. Lindsey D.F. Deery W.J. Van Haastert P.J.M. Gomer R.H. J. Cell Biol. 1995; 129: 1251-1262Crossref PubMed Scopus (64) Google Scholar). 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 Clayet al. (33Clay J.L. Ammann R.A. Gomer R.H. Dev. Biol. 1995; 172: 665-674Crossref PubMed Scopus (27) Google Scholar). Time lapse videomicroscopy, motility assays, and trypan blue exclusion were done as described in Yuen et al. (13Yuen I.S. Jain R. Bishop J.D. Lindsey D.F. Deery W.J. Van Haastert P.J.M. Gomer R.H. J. Cell Biol. 1995; 129: 1251-1262Crossref PubMed Scopus (64) Google Scholar). The yeast strains used in this work were MHY501 (MATα his-Δ200 leu2-3, 112 ura3-52 lys2-801 trp1-1) and MHY840 (MATα his-Δ200 leu2-3, 112 ura3-52 lys2-801 trp1-1 ubp14-Δ1::HIS3i). Yeast-rich and minimal media were prepared as described, and standard yeast genetic methods were used (34Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). E. coli strains used were JM101 and SURE (Stratagene, La Jolla, CA), and standard procedures for recombinant DNA work were used (35Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology.in: Chandra V.B. John Wiley & Sons, Inc., New York1989Google Scholar). Restriction enzyme-mediated integration (REMI) was carried out following Kuspa and Loomis (30Kuspa A. Loomis W.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8803-8807Crossref PubMed Scopus (397) Google Scholar) by electroporating EcoRI-linearized DIV2 (an insertion vector carrying the Dictyostelium pyr5-6 gene, see Fig. 1 A) 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-bpPvuII-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 λgt11 library of Dictyostelium cDNA (CLONTECH, Palo Alto, CA) following Jain et al. (31Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (100) Google Scholar). 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 λgt11 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 (10Jain R. Gomer R.H. Murtagh Jr., J.J. BioTechniques. 1992; 12: 58-59PubMed Google Scholar,36Frohman M.A. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. RACE: Rapid Amplification of cDNA Ends. Academic Press, San Diego1990: 28-38Google Scholar). 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. 1 A), 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. For ubpA cloning, the ubpA coding sequence was amplified by PCR from a λgt11 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 (37Barnes W.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2216-2220Crossref PubMed Scopus (972) Google Scholar) 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. TheubpA insert of pYEU1 was removed by digestion withBamHI, blunt-ended using Klenow, and digested withXbaI, and then subcloned into similarly cut pDXA-3H, aDictyostelium expression vector (38Manstein D.. Schuster H. Morandini P. Hunt D. Gene (Amst.). 1995; 162: 129-134Crossref PubMed Scopus (192) Google Scholar). For expression in E. coli, the complete ubpA coding sequence, contained on a λgt11 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 withEcoRI, blunt-ended using Klenow, and digested withBamHI to generate pGEX-ubpA. To express the yeastubp14 in Dictyostelium, the ubp14cDNA obtained by digesting pGEX-UBP14 (7Amerik A.Y. Swaminathan S. Krantz B.A. Wilkinson K.D. Hochstrasser M. EMBO J. 1997; 16: 4826-4838Crossref PubMed Scopus (196) Google Scholar) with XhoI, blunt-ended using Klenow, followed by digesting with BamHI, was cloned into similarly cut pDXA-3H. 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 A 600 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 mmdithiothreitol in a total volume of 20 μl following Amerik (7Amerik A.Y. Swaminathan S. Krantz B.A. Wilkinson K.D. Hochstrasser M. EMBO J. 1997; 16: 4826-4838Crossref PubMed Scopus (196) Google Scholar). Reaction mixtures contained 2 μl of bacterial extracts and 0.5 μg of Ub-Ub or Ub-UbΔGG. After 1 h at 37 °C, aliquots were removed and analyzed by anti-ubiquitin immunoblotting. RNA isolation, electrophoresis, and transfer to Duralon UV membrane (Stratagene) were performed as described (31Jain R. Yuen I.S. Taphouse C.R. Gomer R.H. Genes Dev. 1992; 6: 390-400Crossref PubMed Scopus (100) Google Scholar). 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 (39Wu L. Franke J. Gene (Amst.). 1990; 91: 51-56Crossref PubMed Scopus (26) Google Scholar). Chi-Hung Siu (University of Toronto, Toronto, Canada) provided the gp80 clone which carried a 1.0-kb EcoRI cDNA insert (40Wong L.M. Siu C. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4248-4252Crossref PubMed Scopus (32) Google Scholar). 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 mNa2HPO4, pH 7.2, 0.25 m NaCl, 5% SDS, 1 mm EDTA, 10% PEG (M r 8,000). Blots were washed with 50 mmNa2HPO4, 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. 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 addedBamHI 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 (33Clay J.L. Ammann R.A. Gomer R.H. Dev. Biol. 1995; 172: 665-674Crossref PubMed Scopus (27) Google Scholar). 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. (41Talian J.C. Olsted J.B. Goldman R.D. J. Cell Biol. 1983; 97: 1277-1282Crossref PubMed Scopus (128) Google Scholar); 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 mmNaCl, 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. 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.(42Towbin H. Staehelin R. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44635) Google Scholar). 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 mmEDTA, 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 × gfor 30 min. Blots were blocked with 5% low-fat powdered milk in PBS (8 mm Na2HPO4, 1 mmKH2PO4, 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 (43Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10405) Google Scholar) 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.(44Pampori N.A. Pampori M.K. Shapiro B.H. BioTechniques. 1995; 18: 588-589PubMed Google Scholar) 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. Overnight cultures of ubp14Δ cells transformed with pUBP14 or pYEU1 plasmids were centrifuged and resuspended in 3 volumes of disruption buffer (20 mmTris-HCl, pH 7.9, 10 mm MgCl2, 1 mmEDTA, 5% glycerol, 1 mm dithiothreitol, 0.3 mammonium 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 (35Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman" @default.
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• W2009596467 title "A Deubiquitinating Enzyme That Disassembles Free Polyubiquitin Chains Is Required for Development but Not Growth in Dictyostelium" @default.
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