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, ubpA cells 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.
Highlights
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
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
Mammalian 26 S proteasomes contain a DUB enzyme that removes ubiquitin molecules from the distal ends of polyubiquitin chains attached to protein substrates [6]
Summary
Growth and Manipulation of Dictyostelium, Yeast, and Escherichia coli Cells—D. discoideum Ax4 cells were grown as described previously [31]. The cDNA sequence matched the sequence obtained from the genomic clones except for the 5Ј-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. For expression in E. coli, the complete ubpA coding sequence, contained on a gt 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 5Ј-CGGGATCCTAATGGAATTATTCCCAGAATTAAAAAATATTAAAGTACC-3Ј and 5Ј-CGGGGTACCTAAATTAAAATTTAATTTAGTTGTC-3Ј. To express the yeast ubp in Dictyostelium, the ubp cDNA obtained by digesting pGEX-UBP14 [7] with XhoI, blunt-ended using Klenow, followed by digesting with BamHI, was cloned into cut pDXA-3H
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