Biochem. J. (2013) 454, 571–583 (Printed in Great Britain) doi:10.1042/BJ20130040 571 A novel interplay between the ubiquitin–proteasome system and serine proteases during Drosophila development Zolt´ an LIPINSZKI* 1,2 , Eva KLEMENT*, Eva HUNYADI-GULYAS*, KatalinF. MEDZIHRADSZKY*, R´ obert M ´ ARKUS†, Margit P ´ AL*, P´ eter DE ´ AK* and Andor UDVARDY* 2 *Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary, and †Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary The concentrations of the Drosophila proteasomal and extrapro- teasomal polyubiquitin receptors fluctuate in a developmentally regulated fashion. This fluctuation is generated by a previously unidentified proteolytic activity. In the present paper, we describe the purification, identification and characterization of this protease (endoproteinase I). Its expression increases sharply at the L1–L2 larval stages, remains high until the second half of the L3 stage, then declines dramatically. This sharp decrease coincides precisely with the increase of polyubiquitin receptor concentrations in late L3 larvae, which suggests a tight developmental co-regulation. RNAi-induced down-regulation of endoproteinase I results in pupal lethality. Interestingly, we found a cross-talk between the 26S proteasome and this larval protease: transgenic overexpression of the in vivo target of endoproteinase I, the C-terminal half of the proteasomal polyubiquitin receptor subunit p54/Rpn10 results in transcriptional down-regulation of endoproteinase I and consequently a lower level of proteolytic elimination of the polyubiquitin receptors. Another larval protease, Jonah65A-IV, which degrades only unfolded proteins and exhibits similar cross-talk with the proteasome has also been purified and characterized. It may prevent the accumulation of polyubiquitylated proteins in larvae contrary to the low polyubiquitin receptor concentration. Key words: Drosophila, gastric caeca, larval serine protease, poly- ubiquitin receptor, 26S proteasome, transcriptional cross-talk, ubiquitin–proteasome system. INTRODUCTION The UPS (ubiquitin–proteasome system) is the major proteolytic route responsible for the controlled intracellular degradation of short-lived and misfolded proteins. The substrate selection, which is the first critical step in this highly regulated process, is ensured by the members of the ubiquitylating enzyme cascade [1]. Ubiquitin ligases, alone or in combination with ubiquitin- conjugating enzymes, recognize degrons within short-lived proteins and mediate the assembly and covalent attachment of the polyubiquitin chain to the protein designated for degradation. The 26S proteasome, a large proteolytic complex which is responsible for the elimination of these proteins, is assembled in an ATP-dependent reaction from two subcomplexes [2], the CP (catalytic particle), which executes the enzymatic cleavage of substrate proteins, and the multifunctional RP (regulatory particle). The RP selectively binds polyubiquitylated proteins, unfolds them via its anti-chaperone activity [3,4], reprocesses the ubiquitin residues of the substrate proteins [5,6], regulates the opening of the gated channel of the CP [7] and feeds the substrate into the catalytic chamber. Polyubiquitin receptors also co-operate in the processing of polyubiquitylated proteins before their proteasomal degradation by selective recognition and binding of polyubiquitylated substrates and targeting them to the RP. Two different classes of proteins have been identified that meet all of the criteria required of a polyubiquitin receptor. The proteasomal ubiquitin receptors Rpn10/S5a/p54 [8– 11] and Rpn13/ADRM1/p42E [12–15] (yeast/human/Drosophila orthologues) are genuine subunits of the RP, whereas the extraproteasomal polyubiquitin receptors are monomeric proteins which carry two essential domains involved in their receptor function: the UBA (ubiquitin-associated domain) ensures the selective recognition and binding of the polyubiquitin chain [16], whereas the UBL (ubiquitin-like domain) interacts with the 26S proteasome [17,18]. Rad23, Dsk2 and Ddi1 are well-characterized members of the UBA–UBL-type polyubiquitin receptors [19]. We have reported previously that the proteasomal and extraproteasomal polyubiquitin receptors are under strict developmental regulation in Drosophila melanogaster [20]. The concentration of p54/Rpn10 suddenly falls relative to that of other RP subunits at the beginning of larval development, remains low throughout the larval stages, starts to increase again in the wandering third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the two main extraproteasomal polyubiquitin receptors (Dsk2 and Rad23) functioning in Drosophila. Depletion of the polyubiquitin receptors is not accompanied by an increase in the concentration of polyubiquitylated proteins, suggesting that a hitherto unidentified proteolytic system may partially substitute for or complement the function of the UPS in larvae. Semi-quantitative RT (reverse transcription)–PCR analysis revealed that the elimination of subunit p54 in instar stages is not a consequence of the selective transcriptional down-regulation of Pros54, which encodes the p54 protein, but is rather due to the developmentally regulated emergence of a proteolytic activity Abbreviations used: CBD, chitin-binding domain; CDS, coding sequence; CHO, Chinese-hamster ovary; CP, catalytic particle; CTH, C-terminal half; endo-I, endoproteinase I; ER, endoplasmic reticulum; ERAD, endoplasmic-reticulum-associated degradation; ERD-10, early responsive to dehydration 10; NA, numerical aperture; NACP, non-amyloid-β component of Alzheimer’s disease precursor; RP, regulatory particle; RT, reverse transcription; UBA, ubiquitin-associated domain; UBL, ubiquitin-like domain; UPS, ubiquitin–proteasome system. 1 Present address: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, U.K. 2 Correspondence may be addressed to either of these authors (email zl295@cam.ac.uk or udvardy.andor@brc.mta.hu). c  The Authors Journal compilation c  2013 Biochemical Society Biochemical Journal www.biochemj.org