Developmental-stage-specific regulation of the polyubiquitin receptors inDrosophila melanogaster

Abstract

Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

“…Polyclonal rat aWOC (IF: 1/500) and rabbit aROW (WB: 1/10,000, ChIP/IP: 1 ml) antibodies were raised against recombinant proteins aa230-626 and aa588-956, respectively. Other antibodies used are described in the indicated references: rat aHP1c (WB: 1/10,000, IF: 1/500, ChIP/IP: 1 ml), aHP1b (WB: 1/ 10,000, ChIP/IP: 1 ml) and aROW (WB: 1/10,000, IF: 1/400, ChIP/IP: 1 ml) 18 , rabbit aWOC (WB: 1/10,000, ChIP/IP: 1 ml) 62 , rabbit and rat aDDP1 (ChIP/IP: 1 ml) 63 , rabbit adDsk2 (WB: 1/12,000, IF: 1/1,000, ChIP/IP: 1 ml) 16 , mouse ap54 (WB: 1/50) 64 , rabbit aNELF-E (WB: 1/500, ChIP/IP: 3 ml) 65 and rabbit aRPB3 (WB: 1/1,500, ChIP: 15 ml) 26 . Commercially available antibodies used were as follows: rabbit aH3K4me3 (Abcam, ab8580, ChIP: 1 ml), rabbit aIIoser5 (Abcam, ab5131, ChIP: 3 ml), rabbit aBre1 (NOVUS, 40280002, WB: 1/1,000, ChIP: 2 ml) and aRad6 (SANTA CRUZ, sc-30078, WB: 1/500, ChIP: 2 ml), mouse aGST (glutathione S-transferase; Novagen, 71097-3, WB: 1/10,000), mouse aGFP (Roche, 1814460, IF: 1/50), mouse aH2Bub1 (Millipore, 05-1312, WB: 1/4,000, ChIP/IP: 1 ml), mouse aTubulin (Millipore, MAB3408, WB: 1/2,000), rabbit aActin (Sigma, A2066, WB: 1/10,000), mouse aV5 (Invitrogen, 460705, WB: 1/5,000), mouse aUbiquitin (SANTA CRUZ, sc-8017, WB: 1/500), rabbit aPanAc (Abcam, Ab193-100, ChIP: 1 ml) and rabbit aH4Ac (Millipore, no.…”

“…dDsk2 belongs to the UBA/UbL family of extraproteasomal receptors (reviewed in refs 10,11), where the ubiquitin-associated (UBA) domain directly binds the ubiquitin moiety of ubiquitylated substrates 12,13 and the ubiquitin-like (UbL) domain mediates interaction with the proteasome 14 . dDsk2 is highly evolutionarily conserved from yeast to humans 10,15,16 . We show that, in addition to regulating protein stability, Drosophila dDsk2 interacts with the complex formed by the euchromatic dHP1c isoform and the transcription factors WOC and ROW [17][18][19] , localizes at promoters and is required for transcription via nonproteolytic mechanisms.…”

Effects of aging and reproduction on protein quality control in soma and gametes of Drosophila melanogaster

“…The extracts were prepared by homogenization on ice using a Teflon pestle either in buffer 1 containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl 2 , 5 mM ATP, 1 mM DTT, and 10% glycerol (Vernace et al, 2007) or in a similar buffer 2 containing 25 mM Tris-HCl, 100 mM NaCl pH 7.4, 5 mM MgCl 2 , 1 mM ATP, 1 mM DTT, and 5% glycerol (Lipinszki et al, 2009). Both buffers were supplemented with the protease inhibitors, such as 0.7 lg mL )1 pepstatin (Sigma-Aldrich, Stockholm, Sweden), 0.5 lg mL )1 leupeptin, 0.5 lg mL )1 aprotinin, and 1 mM PEFAblock (Roche, Bromma, Sweden), which preserve the 26S proteasome assembly and native protein conformations and gave equal results for all analyses performed.…”

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a

“…14, 15 A substantial proportion of S5a is extraproteasomal. 16, 17, 18, 19 It remains to be established whether this participates in bringing ubiquitinated substrates to the proteasome. There are also other ubiquitin receptors that associate transiently with the proteasome in substoichiometric amounts.…”