Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8842-8846, October 1993 Genetics Identification of an essential gene, 1(3)73Ai, with a dominant temperature-sensitive lethal allele, encoding a Drosophila proteasome subunit (multicatalytic prte/antmorph/dnnt negtie muttion) KENNETH J. SAVILLE* AND JOHN M. BELOTEt Department of Biology, Biological Research Laboratories, Syracuse University, Syracuse, NY 13244 Communicated by Dan L. Lindsley, June 3, 1993 ABSTRACT Proteasomes are multicatalytic protelnase alteration in proteasome subunit composition (8). These complexes that function as a major nonlysosomal proteolytic observations suggest that proteasome function is under de- system in all eukaryotes. These partces are made up of 13-15 velopmental control and that proteasomes might be involved nonidentical subunits, and they exhibit multiple endopeptidase in controlling specific processes related to development, such activities that promote the intracellular turnover of abnormal as cell division or morphogenesis. polypeptides and short-lived regulatory proteins. Although the To better understand the biological function(s) of protea- biochemical characterization of proteasomes has been quite some-regulated protein degradation, genetic studies would be extenive, and although a number of the genes encoding of considerable value. Although such studies have been proteasome subunits have been doned from various organisms, initiated in yeast (5, 9-12), no proteasome mutants have yet there is sil much to be learned about their function in Wivo and been described for any other organism. In this paper we what role(s) they might play during development. Here, we report the identification and initial characterization ofmutant report the Identffication of the 1(3)73Ai' allele of Drosophila alleles ofa gene, 1(3)73Ai, encoding aDrosophila proteasome melanogaster as a dominant temperature-ensitive lethal mu- subunit. One allele, 1(3)73Ail, is a previously discovered tation in a gene encoding a component ofthe proteasome, thus dominant temperature-sensitive lethal mutation (13) that be- opening the way for future genetic and developmental studies haves genetically as an antimorphic mutation and that ex- on this Impornt proteolytic system in a higher eukaryote. hibits an intriguing lethal phenotype at the restrictive tem- perature. Two other alleles, 1(3)73Ai2 and 1(3)73Ai3, are Proteasomes are highly conserved proteinase complexes that nonconditional recessive lethals (14). The identification of have been found in archaebacteria and in all eukaryotes 1(3)73Ai as a gene encoding a proteasome subunit, and the examined (for reviews, see refs. 1 and 2). In eukaryotes, availability of a dominant conditional mutant allele, have these complexes can be found in the cytosol and the nucleus allowed us to initiate a genetic study ofthe biological function as large (20S, =700 kDa) particles consisting of a family of of proteasomes in the Drosophila model system.t subunits arranged in a barrel-shaped configuration. Biochem- ical studies have shown that proteasomes -exhibit multiple MATERIALS AND METHODS endopeptidase activities and that they can exist in active and latent forms. Further in vitro analyses indicate that the 20S Standard Techniques. All standard molecular techniques proteasome particles can assemble with two other factors to were done as described (15). Flies were reared on cornmeal/ form 26S particles that are responsible for degrading ubiqui- dextrose/sucrose/yeast/agar medium containing propionic tin-tagged proteins (3, 4). The role of proteasomes in the acid as a mold inhibitor. Genetic variants of Drosophila ubiquitin proteolytic pathway is supported by genetic obser- melanogaster are described in ref. 16. vations in yeast, where it has been found that a temperature- P-Element-Mediated Germ-line Transformation. Microin- sensitive mutation in a proteasome subunit gene causes jection of Drosophila embryos was done using standard ubiquitin-protein conjugates to accumulate at the nonper- methods (17). Restriction fragments to be introduced into the missive temperature (5). Thus, the proteasome is an integral genome were subcloned into the P-element transformation component ofan important nonlysosomal proteolytic system vector pUChsneo (18) or into pW8 (19). responsible for removing unnecessary and abnormal proteins Cloning of the l(3)73Ail Mutant Alkle. Genomic DNA was from eukaryotic cells. isolated from 1(3)73Ai'/Dff3L)st-j7 flies that had been reared During the last few years, there has been a great deal of at 180C. Size-selected EcoRI fragments (3-4 kb) were ligated information gathered on the biochemical and physical prop- into Agtll and, after packaging, the resultant bacteriophage erties ofproteasomes, and, recently, molecular studies have were plated and screened with a 3.5-kbEcoRI fragmentprobe been initiated (see ref. 1). Despite the impressive body of containing the 1(3)73Ai gene. The 3.5-kb EcoRI insert of a experimental data, however, the in vivo function of protea- representative phage clone was subcloned into the plasmid somes remains obscure. In Drosophila, different develop- pIBI76 (International Biotechnologies) for sequencing. This mental stages exhibit different patterns of proteasome sub- insert was also cloned into pW8 to yield P[3.5R,DTSJ, which unit proteins, as seen in two-dimensional gels (6), and im- was used for germ-line transformation experiments. munostaining experiments reveal region-specific changes in TheP[Actin-1.2BR,DTSJ construct, containingthe 1(3)73Ail proteasome abundance during early embryogenesis (7). Sim- gene driven by the actin 5C promoter, was made by replacing ilarly, in chicken embryonic muscle cells, the proteolytic the 2.4-kb BamHI/EcoRI fragment of P[3.5R,DTSJ with the activities of proteasomes change during development, and this is correlated with a developmental- and tissue-specific *Present address: Center for Agricultunl Biotechnology, Maryland Biotechnology Institute, 2111 Agriculture/Life Sciences Surge Building, College Park, MD 20742. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U00790). 8842 Downloaded by guest on September 30, 2021 Genetics: Saville and Belote Proc. Natl. Acad. Sci. USA 90 (1993) 8843 actin SC promoter region from the plasmid pUC18ARI- In l(3)73Ail/+ flies raised at the restrictive temperature of actin5CSRS (20). 29°C, embryonic and larval development appears to proceed ProteasoI e Purification and Immunoblot Analysis. Protea- normally, but metamorphosis is abnormal and death occurs somes were isolated from adult flies (Oregon R strain) using at the late pupal stage (Table 1, A and B). The most the sucrose density gradient centrifugation method as de- conspicuous defect is a failure of the adult abdominal seg- scribed (6, 21). ments (i.e., the tergites and sternites) to develop. At mar- For the generation of anti-1(3)73Ai protein antibodies, a ginally restrictive temperatures, the anterior structures ofthe trpEl(3)73Ai fusion gene was produced by ligating a cDNA adult fly appear to develop normally, while the abdomen restriction fragment containing the entire 1(3)73Ai coding region fails to differentiate and histolyzes, resulting in a dead region into the expression vector pATH21 (22). The fusion pharate adult that lacks abdominal structures. At slightly protein was gel purified as described (23) and used to immu- higher temperatures, the anterior structures also show gross nize rabbits (Pocono Rabbit Farm, Canadensis, PA). Proteins defects, such as reduced size and a frequent failure of head from the sucrose gradient fractions were separated by SDS/ eversion. In addition to its dominant conditional phenotype, PAGE and electrophoretically transferred to poly(vinylidene 1(3)73Ail also acts as a recessive lethal-that is, 1(3)73Ail difluoride) membrane according to the method ofthe supplier homozygotes or hemizygotes [e.g., 1(3)73Ai'/Dfl3L)st-j7], (Bio-Rad). Filters were incubated for 2 hr with purified raised at 25°C, die during the first larval instar stage (Table 1, IgG in incubation solution (phosphate- C). The lethal phenotype of hemizygotes can be suppressed anti-trpE-(3)73Ai by rearing the flies at 18°C, although surviving adults often buffered saline/5% Carnation dry milk/0.3% Tween 20) and exhibit abdominal tergite defects and are sterile (Table 1, D). then treated with secondary antibody [alkaline phosphatase- The 1(3)73Ail allele can be classified as a "gain-of-function" conjugated goat anti-rabbit IgG (Bio-Rad)]. Antibody binding mutant, since a deletion of the chromosomal region contain- was detected using the Bio-Rad AP conjugate staining kit. ing the 1(3)73Ai locus has no dominant phenotypic effect Immunoblot controls were done with IgG purified from (Table 1, E). An additional observation concerning 1(3)73Ail preimmune serum. is that its dominant conditional lethal phenotype can be rescued by an additional wild-type copy of the locus (Table RESULTS 1,F). Two other mutant alleles of this locus, 1(3)73Ai2 and Genetic Properties of 1(3)73Ai. As part of a comprehensive 1(3)73Ai3, behave as nonconditional recessive lethal alleles, study on temperature-sensitive mutations in D. melano- having lethal periods during the first larval instar (14). The gaster, Holden