Dominant-negative mutation in the β2 and β6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage

Abstract

In Drosophila, dominant-negative mutations in the ␤2 and ␤6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, ␤2 and ␤6 DTS mutations confer lethality at the pupal stage. I investigate here the role of proteasome activity in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperatureshift experiments in ␤2 and ␤6 DTS mutant pupae occasionally resulted in external sense organs with two sockets and no shaft. This double-socket phenotype was strongly enhanced in conditions in which Notch signaling was up-regulated. Furthermore, conditional overexpression of the ␤6 dominantnegative mutant subunit led to shaft-to-socket and to neuronto-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the ␤6 dominant-negative mutant subunit led to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila.In eukaryotic cells, degradation of many proteins involves their covalent modification by conjugation with ubiquitin. Ubiquitinated proteins can be rapidly degraded by a large multisubunit complex called the 26S proteasome (1). This complex is present in the nucleus and in the cytosol of all cells. The 26S proteasome consists of a 20S core particle capped by two 19S regulatory complexes. The 20S proteasome is a barrel-shaped cylinder composed of four stacked rings of seven subunits each (2). The two external rings are composed of seven ␣ subunits (␣1-7), and the two inner rings comprise seven ␤ subunits (␤1-7) that catalyze the hydrolysis of polypeptide substrates.In Drosophila, the DTS7 and DTS5 dominant temperaturesensitive (DTS) mutations affect the ␤2 and ␤6 proteasome subunit genes, respectively (3, 4). DTS5 and DTS7 heterozygous flies develop perfectly at the permissive temperature (25°C), but die as undifferentiated pupae with failures in head eversion at the restrictive temperature (29°C). The DTS5 and DTS7 mutations behave genetically as antimorphic mutations, and they correspond to substitutions in residues that are conserved from flies to vertebrates (3, 4). The structure proposed for the yeast 20S proteasome indicates that ␤2 directly interacts with ␤6 in the adjacent ring (2), and it predicts that the amino acids mutated in the DTS5 and DTS7

The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila

“…), RNAi or pharmacological inhibition have been used to study proteasome function. 8,22,27,51,57,64 In other approaches, whole embryos mutant for proteasome subunits were characterized for defects in dendrite pruning in sensory neurons in Drosophila. [65][66][67] However, a specific analysis characterizing recessive alleles for defects in proteasome activity has not been reported.…”

Proteasomal Regulation of the Proliferation vs. Meiotic Entry Decision in the Caenorhabditis elegans Germ Line

“…Proteasome-mediated protein degradation has previously been implicated in other systems as a regulatory mechanism controlling the level of Notch signaling. For example, dominant-negative temperature-sensitive mutations in two Drosophila proteasome subunits result in shaft-to-socket cell fate transformations and a double-socket phenotype; both of these phenotypes are associated with an increase in Notch signaling (Schweisguth 1999). The increase in Notch signaling observed in these dominant-negative proteasome subunit mutations in Drosophila is likely due, at least in part, to a decrease in degradation of the cleaved intracellular form of the Notch receptor; an activated version of the intracellular domain is stabilized in one of the dominant-negative proteasome subunit mutants (Schweisguth 1999).…”

“…For example, dominant-negative temperature-sensitive mutations in two Drosophila proteasome subunits result in shaft-to-socket cell fate transformations and a double-socket phenotype; both of these phenotypes are associated with an increase in Notch signaling (Schweisguth 1999). The increase in Notch signaling observed in these dominant-negative proteasome subunit mutations in Drosophila is likely due, at least in part, to a decrease in degradation of the cleaved intracellular form of the Notch receptor; an activated version of the intracellular domain is stabilized in one of the dominant-negative proteasome subunit mutants (Schweisguth 1999). Proteasome-mediated degradation of the intracellular domain of the Notch receptor has also been shown or suggested to occur in other systems, including C. elegans (Mango et al 1991;Hubbard et al 1997;Gupta-Rossi et al 2001;Oberg et al 2001;Wu et al 2001;McGill and McGlade 2003;Tsunematsu et al 2004).…”

The ubiquitin–proteasome system postsynaptically regulates glutamatergic synaptic function

“…Mutant subunits act in a dominant negative manner to interfere with proteasome function; in multiple previous studies, they have been shown to inhibit UPS-mediated degradation of known proteasome substrates (Schweisguth, 1999;Speese et al, 2003;Neuburger et al, 2006). Transgenic co-expression of both proteasome subunit mutants in the Drosophila eye using the UAS-GAL4 system has a synergistic effect (Belote JM and Fortier, 2002).…”