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Volume 270, Number 4, Issue of January 27, 1995 pp. 1850-1858
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmental Changes of the 26 S Proteasome in Abdominal Intersegmental Muscles of Manduca sexta during Programmed Cell Death (*)

(Received for publication, September 2, 1994)

Simon P. Dawson,  Jane E. Arnold,  Nicholas J. Mayer,  Stuart E. Reynolds (1),  Michael A. Billett ,  Colin Gordon (4),  Laurence Colleaux (4),  Peter M. Kloetzel (2),  Keiji Tanaka (3),  R. John Mayer (§)

From the  (1)Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom, the Department of Biochemistry and Biological Sciences, University of Bath, Claverton Down, Bath, BA2 7AY, United Kingdom, the (2)Institut fur Biochemie, Medizinische Fakultat Charite-der Humboldt-Universitat zu Berlin, Hessische Str. 3/4, 10115 Berlin, Germany, the (3)Institute for Enzyme Research, University of Tokushima, Tokushima 770, Japan, and the (4)Medical Research Council, Human Genetics Unit, Western General Hospital, Creve Road, Edinburgh EH4 2XU, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

cDNA clone MS73 codes for an ATPase that is a regulatory subunit of the 26 S proteasome. Reverse transcriptase polymerase chain reaction analysis demonstrates that the expression of the gene dramatically increases in the pre-eclosion period. Western analyses show increases in other related ATPases including MS73, MSS1, and mts2 but not TBP1. A similar increase in the 30-kDa subunit of the 20 S proteasome occurs. There are accompanying large changes in the peptidase activities of the 26 S proteasome. Relative to the 30-kDa subunit, there is no change in MSS1 and MS73, a 3-fold increase in mts2, and a 5-fold decline in TBP1. A large increase in the concentration of 26 S proteasomes together with extensive regulatory reprogramming may facilitate rapid muscular proteolysis.

INTRODUCTION

Programmed neuromuscular death is a characteristic of some abdominal motor neurones and muscles at eclosion in the tobacco horn moth Manduca sexta(1, 2, 3) . The cells are destroyed and debris is removed within 24-30 h(4) . The programmed elimination of motor neurones and muscles is hormonally orchestrated by a decrease in 20-hydroxyecdysone(4) . Previous studies have shown that eclosion is preceded by a massive increase in the expression of a polyubiquitin gene and accumulation of ubiquitinated proteins in the muscles and some nerves(5, 6, 7) .

Protein ubiquitination is emerging as a widespread pleotypic process with roles in non-lysosomal (8) and lysosomal (9, 10, 11, 12, 13) protein degradation, cell cycle control(14, 15) , regulation of transcription (16, 17) , chromatin structure(18) , receptor function(19, 20) , viral replication(21) , and synaptogenesis(22) . The covalent addition (23, 24, 25) and removal of ubiquitin (26, 27) from proteins appear to be widespread regulatory post-translational modifications of proteins like phosphorylation and dephosphorylation.

A series of elegant studies has characterized the enzymes involved in the activation and conjugation of ubiquitin to proteins(8) . Ubiquitinated proteins are then degraded by an enormous 26 S protease (28, 29, 30) . The residues catalyzing proteolysis in the complex are unknown and are different to those in any other known protease. The proteasome has multicatalytic endoproteolytic activities, presumably to degrade a large variety of proteins to small peptides or amino acids. Ubiquitin is released in the process to fuel further rounds of proteolysis(31) .

The 26 S protease (proteasome) consists of a 20 S protease core plus regulatory complexes containing several proteins including ATPases(32, 33) , which belong to a new superfamily with diverse functions(34, 35, 36) . The 20 S protease is also involved in antigen fragmentation for class I major histocompatibility complex presentation(37) . Interferon- treatment results in the replacement of housekeeping subunits of the 20 S proteasome with major histocompatibility complex-encoded LMPs (38, 39, 40) .

The studies reported here on regulatory ATPases of the 26 S proteasome during the programmed death of intersegmental abdominal muscles in Manduca show that the destruction of the muscles is not only dependent on increases in the concentration of the 26 S proteasomes but also on extensive changes in the complement of controlling ATPases in new proteasomes to facilitate rapid ubiquitin-dependent proteolysis.

EXPERIMENTAL PROCEDURES

Insect Culture and Staging

Tobacco hornworms, M. sexta (Lepidoptera, Sphingidae), were reared at 25 °C under a 17-h light (7-h dark photo period) on a wheatgerm-based artificial diet using standard procedures(41) . Humidity in the rearing room was not controlled.

Different stages of pre-ecdysial development were recognized by a staging scheme adapted from that of Schwartz and Truman (4) and described fully by Samuels and Reynolds(42) . Insects defined as stage 0 (greater than 100 h before eclosion), stage 1 (about 90 h before eclosion), stage 2 (about 80 h prior to eclosion), stage 4 (about 68 h prior to eclosion), and stage 5 (about 45 h prior to eclosion) had black, soft wings and firm abdomens; stage 6 insects (24 h before eclosion) had black, soft wings and soft abdomens; in stage 7 insects (6 h before eclosion), the old pupal cuticle had a ``crinkly'' feel, and moulting fluid resorption could be recognized by visibility of markings on the new cuticle through the old; stage 8 corresponded to the time of eclosion.

Abdominal intersegmental muscle (ISM) (^1)was collected from staged insects by dissection under a simple insect physiological saline solution (43) and immediately frozen in liquid nitrogen. They were stored at -80 °C until needed.

cDNA Libraries, Screening, and Plasmid Constructs

cDNA was synthesized from poly(A) RNA of stage 7 ISM using Superscript reverse transcriptase (Life Technologies, Inc.), ligated to EcoRI linkers, and cloned into ZAP II (Stratagene). The primary library titre was approximately 3.5 times 10^5 plaque-forming units with an average insert size of 750 base pairs. The library was amplified once prior to use.

High specific activity P-labeled cDNA from poly(A) RNA was generated as previously described(44) . The library was screened with radioactive cDNAs from precommitted stage 0 ISM and stage 7 ISM (± screening). Recombinants that displayed differential labeling with the two probes were rescreened for verification. Inserts were isolated by in vivo excision with the helper phage M13K07.

Polymerase chain reaction (PCR) amplification of the N-terminal region of MS73 cDNA from the stage 7 cDNA library was achieved using one primer flanking the EcoRI site in ZAP II and one primer corresponding to the 5`-region of the C-terminal fragment originally isolated from the library. Sequences are as follows: forward primer (vector flanking region), 5`-GTAAAACGACGGGCCAGTGAA-3`; reverse primer, 5`-TCTGTGTGTCCATGCCACCA-3`. The 600-base pair amplified fragment was cloned into the SmaI site of pBluescript KS (Stratagene).

Nucleotide sequences were determined by the method of (45) in both the forward and reverse directions. Sequence analysis was performed using the University of Wisconsin Genetics Computer Group and Clustal software packages(46, 47) .

Fusion protein constructs for antibody generation were made as follows. pRSETA and pRSETB (Invitrogen) were digested with EcoRI, and the ends were filled in with Klenow DNA polymerase. Blunt-ended plasmids were then digested with KpnI. The SmaI/KpnI fragments of pMS73c and pMS73n were cloned into pRSETA and pRSETB, respectively, to yield pSMS73c and pSMS73n.

The RT-PCR control plasmid (pMS73cDeltaMA) was constructed from pMS73c in the following way. An 87-base pair deletion was introduced by digestion with AatII/MscI, blunt-ending with Klenow DNA polymerase, and religation.

RNA Isolation and RT-PCR

Total RNA was isolated from Manduca intersegmental muscles using the method described (48) .

In brief, the RT-PCR analysis was performed as follows. The RNA used as an internal control was prepared from pMS73cDeltaMA by linearization with KpnI and in vitro transcription using T3 RNA polymerase (Life Technologies, Inc.). DNA template was removed by treatment with RNase-free DNase I (Pharmacia Biotech Inc.). RNA was purified by phenol/chloroform extraction and precipitation. Primers used for PCR amplification had the following sequence: forward primer (bases 561-580), 5`-GCTGTAGAGCTGCCTCTCAC-3`; reverse primer (complementary to bases 1202-1219), 5`-AAGTCTTTAGGCAAGACG-3` (see Fig. 1).

Figure 1: cDNA and putative amino acid sequence of MS73. Amino acid residues forming part of the potential leucine zipper region (labeled I), the general ATPase A and B boxes (labeled II and III, respectively), and the putative RNA helicase motifs (labeled IV) are highlighted.


Reverse transcription reactions were set up as follows. Approximately 1 µg of total RNA from each developmental stage was reverse transcribed together with 0.15 pg of control RNA transcribed from pMS73DeltaMA in addition to 100 pmol of random hexamers, 50 mM TrisbulletHCl, pH 8.3, 75 mM KCl, 3 mM MgCl(2), 10 mM dithiothreitol, 10 units of placental ribonuclease inhibitor (Pharmacia), and 200 units of Superscript reverse transcriptase. Reaction mixes were made up to 20 µl with RNase-free H(2)O and incubated at 37 °C for 2 h.

PCR reactions were typically set up as follows. 1 µl of the reverse transcription mixture was amplified in a mixture containing the following: 20 pmol of each primer, 50 mM each dNTP, 10 mM TrisbulletHCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl(2), 0.1% Triton X-100, 0.185 mBq [alpha-P]dCTP, and 1 unit of Taq DNA polymerase (Life Technologies, Inc.). Reaction mixtures were made up to 50 µl with H(2)O and 75 µl of light mineral oil applied to prevent evaporation.

PCR was performed for 32 complete cycles with annealing at 55 °C. P-labeled PCR reaction products (658-base pair DNA from ISM RNA and 571-base pair DNA from control RNA) were separated using denaturing polyacrylamide gel electrophoresis(45) , excised from the gel, and quantitated by Cerenkov counting. The concentration of MS73 mRNA in each reaction was calculated by comparison of amounts of PCR product obtained with that amplified from known amounts of control RNA template in the same reaction(49) . Small variations in amounts of RNA template present in each reaction were corrected by independently measuring the poly(A)) contents of each sample by dot blot hybridization(50) , and results were expressed as copies/ng total cellular RNA, assuming 0.002 ng of poly(A)/ng of total cellular RNA(50) .

Antibody Production

Polyclonal antisera were generated against the fusion proteins produced from both pSMS73n and pSMS73c. Fusion proteins were typically prepared as follows. Constructs were transformed into Escherichia coli strain BL21(DE3) (Invitrogen), and cells were grown to mid-log phase (A = 0.5). Fusion proteins were induced by the addition of isopropyl beta-D-thiogalactopyranoside to a final concentration of 0.1 mM and a further 3-h incubation at 37 °C. Cells were harvested by centrifugation, resuspended in lysis buffer (6 M guanidine hydrochloride, 20 mM sodium phosphate, 500 mM NaCl, pH 7.8, 0.2 volume), and sonicated to lyse cells. After removal of cell debris by centrifugation, the lysate was bound, with agitation at room temperature, to ProBond nickel resin (Invitrogen) in batches contained in a column. The column was then sequentially washed with 2 times 0.1 volume of denaturing binding buffer (8 M urea, 20 mM sodium phosphate, 500 mM NaCl, pH 7.8), denaturing wash buffer 6.0 (8 M urea, 20 mM sodium phosphate, 500 mM NaCl, pH 6.0), denaturing wash buffer 5.3 (8 M urea, 20 mM sodium phosphate, 500 mM NaCl, pH 5.3), and then eluted in 0.1 volume of denaturing elution buffer (8 M urea, 20 mM sodium phosphate, 500 mM NaCl, pH 4.0). Eluted protein was dialyzed against 10 mM TrisbulletHCl, pH 8.0, 0.1% Triton X-100 overnight at 4 °C to remove excess urea. Dialyzed protein was freeze-dried, checked for purity on SDS-polyacrylamide gel electrophoresis, and then used for immunization of New Zealand White rabbits.

Two young New Zealand White rabbits were each injected subcutaneously with 100 µg of purified hybrid protein. 3 weeks after initial injection, boost injections commenced at intervals of 14 days using 100 µg of protein emulsified in Freund's incomplete adjuvant. Antisera were initially tested for their ability to react with the expressed fusion protein by Western analysis of whole cell extract of E. coli BL21(DE3) transformed with pSMS73c or pSMS73n.

Preparation of M. sexta Muscle Homogenates

Frozen ISM (0.3 g) taken from different developmental stages of M. sexta were homogenized in 4 volumes of homogenizing buffer (20 mM TrisbulletHCl, 2 mM ATP, 5 mM MgCl(2), 1 mM EDTA, pH 7.5) using a polytron homogenizer. The homogenate was centrifuged at 9000 times g for 20 min at 4 °C, and the supernatant was collected (soluble muscle extract).

Separation of 26 and 20 S Proteasomes on Glycerol Gradients

Soluble muscle extract (3.5 mg of protein) prepared from stage 0 and stage 7 ISM was loaded onto a 14-ml 10-40% (v/v) glycerol gradient according to the method of Tanaka and others(51) . Samples were centrifuged at 24000 rpm in a SW 6 times 16.5 rotor for 22 h (R 70000 times g). Fractions of 0.5 ml were collected by displacement with Maxidens (Nycomed).

Assay of Peptidase Activities

The chymotrypsin, trypsin, and peptidylglutamyl peptide-hydrolyzing activities of the 20 and 26 S proteasomes were measured using the fluorogenic substrates Suc-LLVY-MCA, Boc-LRR-MCA, and Cbz-LLE-betaNA (Sigma), respectively. Chymotrypsin and trypsin activities were assayed by incubating glycerol gradient fractions with 0.1 mM substrate in 100 mM TrisbulletHCl (pH 8.0) for 15 min at 37 °C in a final volume of 120 µl. The reaction was stopped by the addition of 80 mM acetate (pH 4.3), and the fluorescence was measured on a Shimadzu RF5000 fluorimeter at an excitation of 360 nm and an emission of 460 nm. Peptidylglutamyl peptide-hydrolyzing activity was assayed by incubating glycerol gradient fractions with 0.3 mM substrate in 100 mM TrisbulletHCl (pH 8.0) for 30 min at 37 °C. The reaction was terminated by the addition of 0.3 ml of ethanol followed by water to a volume of 2.5 ml(52) . Fluorescence was measured at an excitation of 335 nm and an emission of 450 nm. Protein was determined according to the method of Bradford(53) .

Western Blot Analysis

Samples were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane (Hybond C, Amersham Corp.). The membrane was blocked by incubation in 5% (w/v) Marvel milk powder in Tris-buffered saline for 1 h, followed by a 2-h incubation at room temperature in primary antibody at the indicated dilutions. Incubation with the secondary antibody (peroxidase-conjugated swine anti-rabbit, 1:1000 dilution, Dako) was carried out for 2 h at room temperature and developed by enhanced chemiluminescence.

The resulting Western blots were quantitated by densitometry using a Molecular Dynamics laser densitometer, and the pixel values obtained were used to calculate changes in the levels of the ATPases. Due to the low levels of some of the ATPases at stage 0 and the inherent inaccuracies of quantitating such values, stage 2 muscle was routinely used to calculate the fold changes.

A proportional relationship between the amount of antigen present and the signal detected on the x-ray film was found under the conditions used.

Antibodies

Anti-human MSS1 antibody (equivalent to the S7 subunit of the 26 S proteasome(33) ) and anti-human TBP1 antibody were raised against their recombinant proteins (in K. T.'s laboratory). Anti-Drosophila 20 S proteasome antibody was raised against the purified 20 S proteasome (in P. M. K.'s laboratory), and an anti-yeast mts2 antibody, equivalent to the S4 subunit of the 26 S proteasome(54) , was donated by C. Gordon (MRC Human Genetics, Edinburgh). Anti-MS73 antibodies were raised in this laboratory against recombinant expressed N-terminal and C-terminal regions of MS73.

Anti-MS73 C-terminal antibody cross-reacted with the recombinant expressed C-terminal region of MS73 but not with the other recombinant ATPases. In the soluble extracts of Manduca ISM, this antibody detected a protein with a molecular mass of 50 kDa. Similarly, antisera raised to the human ATPases MSS1 and TBP1 reacted with a protein of approximately 50 kDa, while an antibody to the yeast ATPase, mts2, detected a Manduca protein of 58 kDa. Anti-MSS1 did not react with the recombinant C-terminal region of MS73. Anti-TBP1 gives a developmental profile distinct from that with the antibody to the C terminus of MS73 in ISM. These two antibodies (anti-MSS1 and anti-TBP1) therefore detect distinct antigens to anti-MS73 in ISM extracts. Antisera to the Drosophila 20 S proteasome cross-reacted with two proteins of the Manduca 20 S proteasome with molecular masses of 30 and 25 kDa. It is not known if the Drosophila antibody recognizes only two of the 20 S subunit proteins or if Manduca contains a simplified 20 S subunit similar to that of the archaebacteria Thermoplasma acidophilum(55) .

RESULTS

Sequence Analysis of MS73

One clone corresponding to the 3`-end of the mRNA (pMS73c), identified during differential screening of a Manduca stage 7 ISM cDNA library for developmentally regulated sequences, was chosen for further study, and an overlapping further clone (pMS73n) corresponding to the 5`-end of the mRNA was isolated.

The two overlapping cDNA clones, pMS73c and pMS73n, yielded a sequence of 1341 base pairs (Fig. 1). The longest open reading frame is 1248 base pairs and gives a predicted amino acid sequence of 415 amino acids with a molecular mass of 47 kDa. The methionine residue at the start of the longest open reading frame is derived from an ATG codon whose sequence context conforms to the rules suggested by Kozak (56) . The amino acid sequence obtained for MS73 showed good homology with two similar sequences (Fig. 2), demonstrating 82% similarity and 68% identity with a sequence, YTA2, from Saccharomyces cerevisiae(57) and 91% similarity and 85% identity with the human TBP7 (S6) sequence(58) . YTA2 and TBP7 (S6) belong to a rapidly expanding superfamily of putative ATPases (59) that all contain one or two copies of a conserved ATPase domain spanning approximately 200 amino acids (Fig. 1, bases 624-1029). Two regions (Fig. 2, boxesII and III) of MS73 correspond to the A and B boxes of a general ATPase motif(60) . A region of MS73 comprising a heptad repeat (Fig. 2, boxesI) is reminiscent of that seen in the leucine zipper motif involved in protein-protein interactions of transcription factors and other proteins(61) . The predicted MS73 protein sequence also contains within the conserved ATPase domain a similar motif to that present in a group of putative RNA helicases (Fig. 2, MAT, LDPALXRPGRXDRK, boxesIV) (62) .

Figure 2: Multiple alignment of MS73, YTA2, and S6 sequences. Alignment was performed using the Clustal algorithm (47) . Default gap penalties were used (k-tuple = 1, pairwise gap penalty = 3, fixed gap penalty = 10, floating gap penalty = 10). Specific regions of amino acid sequence are highlighted as in Fig. 1. Conservative substitutions are depicted by a dot beneath the residue, while identical residues shared by all three sequences are depicted by the presence of an asterisk.


Analysis of MS73 Expression during the Onset of Programmed Cell Death

Recent evidence presented by Schwartz and co-workers (5) has shown a significant increase in the expression of a polyubiquitin gene at the onset of muscle atrophy (day 15) with a much larger increase during degeneration (day 18) in the abdominal ISM of Manduca. In addition, it has been shown by immunohistochemical methods that there is a concomitant increase in ubiquitin-protein conjugates in both the ISM and the innervating neurones (7) during the onset of programmed cell death in these tissues. Quantitative RT-PCR was used to assess accurately the abundance of MS73 mRNA during the progression of the ISM into programmed cell death. The method of staging developing pupae used in the current study, although based on similar criteria, is somewhat different to that previously described(6) . Thus, days 15, 17, and 18 (4, 6) correspond to stages 3-4, 7, and 8, respectively, in this study.

mRNA corresponding to MS73 was detectable at all stages examined (stages 0-2, 4-8). The lowest concentration detected was at stage 0, corresponding to late day 13/early day 14. At this time point, 254 ± 51 copies mRNA/ng total cellular RNA (n = 4) were detected (Fig. 3). During progression of the ISM through development to eclosion at stage 8, there is a general increase in the concentration of MS73 mRNA. The highest concentration (906 ± 70 copies mRNA/ng total cellular RNA, n = 4) was detected at stage 7 (day 17), immediately prior to eclosion. This represents a 3.6 relative -fold increase over stage 0. At stage 8 (day 18), the concentration of MS73 mRNA had dropped to 756 ± 63 copies mRNA/ng total cellular RNA (n = 4). From stage 2 (382 ± 57 copies, n = 4) to stage 4 (798 ± 65 copies, n = 4), a sharp increase in mRNA concentration occurs, followed by a slight decrease at stage 5 (704 ± 58 copies, n = 4), before increasing again through stage 6 (736 ± 53 copies, n = 4) to stage 7. Stage 4 (day 15) is the stage of ISM atrophy when a transient increase in polyubiquitin expression was observed(5) .

Figure 3: Expression of MS73 mRNA in Manduca ISM during the onset of programmed cell death. RT-PCR reactions were performed, in quadruplicate, as described under ``Experimental Procedures.'' One quarter of each reaction was ethanol precipitated, vacuum dried, and resuspended in H(2)O prior to electrophoresis on denaturing polyacrylamide gel electrophoresis gels. The position of PCR products was determined by autoradiography, and bands were excised from the gel and Cerenkov counted. Amounts of mRNA are expressed as copies/ng cellular RNA, assuming 0.002 ng of poly(A)/ng of cellular RNA.


These results show that there is a developmental regulation of MS73 mRNA coincident with the onset of ISM atrophy and degeneration.

Developmental Changes of the ATPase Subunits in Manduca

Antisera raised to the C-terminal region of MS73 protein detected, by Western blot analysis, a 50-kDa protein in soluble extracts of ISM. The size of the protein is consistent with the predicted amino acid sequence of the longest open reading frame of MS73 (Fig. 1). No ATPase could be detected in the 9000 times g pellet. The amount of MS73 was measured by Western blot analysis in Manduca muscle at various stages of development and was shown to greatly increase prior to eclosion (Fig. 4). Quantitation of the Western blots by laser densitometry showed a 5.6-fold increase of this ATPase at stage 7 compared with stage 2 (Fig. 5, Table 1).

Figure 4: Developmental changes in the 26 S proteasome ATPase subunits in the ISM of M. sexta. Soluble muscle extracts (50 µg of protein) from different stages of development were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane as described under ``Experimental Procedures.'' After blocking for 1 h, blots were incubated for 2 h with anti-MSS1 antibody (1:500 dilution), anti-TBP1 antibody (1:500 dilution), anti-MS73 antibody (1:1000 dilution), anti-mts2 antibody (1:250 dilution), or anti-20 S antibody (1:250 dilution). This was followed by incubation with peroxidase-conjugated swine anti-rabbit (1:1000) dilution and developed by enhanced chemiluminescence.


Figure 5: Changes in the relative amounts of ATPases during ISM development. Pixel values obtained from the quantitation of Western blots (Fig. 3) were used to calculate the -fold changes in Manduca muscle extracts for each ATPase and the 20 S proteasome relative to the corresponding values for stage 2.


Western analyses of other ATPases known to be components of the regulatory complexes of the 26 S proteasome were carried out. An increase in the amount of MSS1 and mts2 was detected while the amount of TBP1 remained unchanged (Fig. 4). Antisera to Drosophila 20 S proteasome similarly showed a progressive increase in the amounts of two Manduca 20 S proteins (Fig. 4).

Quantitation of the amounts of ATPases showed a 5-fold increase in MSS1 at stage 7 compared with stage 2, which was mirrored by similar changes in MS73 and in the 30-kDa subunit of the 20 S proteasome ( Fig. 5and Table 1). However, mts2 increased by 15-fold at stage 7 relative to stage 2 and declined somewhat by stage 8. In contrast, TBP1 showed little change. If the 30-kDa subunit of the 20 S proteasome is taken to be representative of the Manduca 20 S complex, then a comparison of the -fold changes of the ATPases to that of the 20 S proteasome can be made (Table 1). Both MSS1 and MS73 increase in a similar manner to the 20 S proteasome, suggesting an increase in the concentration of the 26 S proteasome prior to eclosion. However, mts2 shows a 3-fold increase while TBP1 undergoes a 5-fold decline in amount relative to the 20 S proteasome. This suggests that the proportions of the ATPases present in the 26 S proteasome population alter during programmed development.

Comparison of the Properties of 26 S Proteasomes at Stages 0 and 7 of Muscle Development

Glycerol gradient analysis was performed on soluble extracts from stage 0 and stage 7 to demonstrate that the changes in ATPases measured in the soluble muscle extracts were associated with the 26 S proteasome. Measurement of chymotrypsin, trypsin, and peptidylglutamyl peptide-hydrolyzing activities (Fig. 6) at stage 0 detected two distinct peaks of enzyme activity corresponding to the 20 S (fractions 8-11) and 26 S proteasomes (fractions 11-16), consistent with the results of other workers(63) . At stage 7, the activities of the 20 S species remain unchanged while the activities of all of the peptidases in the 26 S species dramatically increased (Fig. 6, Table 2).

Figure 6: Glycerol gradient analysis of proteasomes at stage 0 and stage 7 of development. Soluble muscle extract (3.5 mg of protein) was fractionated on 10-40% glycerol gradients as described under ``Experimental Procedures.'' Aliquots from each fraction were assayed for protein (panel A), chymotrypsin activity (panel B), trypsin activity (panel C), and peptidylglutamyl peptide-hydrolyzing activity (panel D) as described under ``Experimental Procedures.''


Western blot analyses of proteasomes in stage 7 muscle show that the 26 S species (fractions 11-16) contains the ATPases (TBP1, MSS1, and MS73) and the 20 S components (Fig. 7). The mts2 subunit (S4) (54) was also present (results not shown). The low levels of ATPases detected in slower sedimenting fractions (e.g. fraction 10) in both early and late stages of muscle development may reflect an incomplete separation of the 26 S species from the 20 S proteasome, or, more likely, the ATPases may be present in the 19 S regulatory complex prior to assembly with the 20 S core particle to form the 26 S proteasome(64, 65) . The three peptidase activities of the 26 S proteasome showed a 5-8-fold increase between stage 0 and 7 ( Fig. 6and Table 2). This is reflected by an increase in the amounts of MSS1 and MS73 in the 26 S particle over the same period (Fig. 7, Table 1) and corroborates the findings in muscle extracts (Fig. 4). Analyses of Coomassie-stained gels of gradient fractions show the presence of proteins with molecular masses between 40 and 100 kDa associated with the 26 S proteasome at stage 7, which could not be detected at stage 0 (results not shown). These data suggest that other regulatory components of the 26 S proteasome increase in amount during development.

Figure 7: Western analysis of proteins on glycerol gradients. 1 µg of protein from the glycerol gradient fractions (Fig. 5) was applied to a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membrane as described under ``Experimental Procedures.'' Blots were blocked, treated with primary and secondary antibody, and developed as in Fig. 4.


The results show that the ATPases detected in Manduca muscle are components of the 26 S proteasome and also that the amount of this protease increases during the programmed elimination of ISM. The differential changes of the ATPase components (Table 1) during muscle development may change the substrate specificity of the 26 S proteasome.

DISCUSSION

We have cloned, sequenced, and analyzed the expression of a Manduca ATPase (MS73), which is a member of a rapidly expanding new superfamily of ATPases (59) involved in such diverse cellular processes as the control of the cell cycle(54, 66, 67, 68, 69) , vesicle-mediated protein transport(70, 71, 72) , peroxisome biogenesis (73) , intramitochondrial protein sorting(74) , modulation of the human immunodeficiency virus tat gene expression(75, 76) , and transcriptional regulation(77) .

Nucleotide sequence analysis of MS73 identified a single large open reading frame with the potential to code for a protein of 47 kDa (Fig. 1). The detection of a polypeptide of approximately 50 kDa by two different polyclonal antibodies raised to different parts of the expressed MS73 in soluble extracts of Manduca ISM is consistent with the assigned open reading frame.

There is remarkable sequence similarity shared between MS73 and other members of this ATPase family. MS73 shares 49.2% identity (71.2% similarity) and 41.8% identity (64% similarity) with S4 (mts2) and MSS1(32, 33) , known subunits of the regulatory complex of the 26 S proteasome. A lesser, although still significant similarity, is apparent between MS73 and members of the ATPase family containing two ATPase domains(70, 71, 73) . With the exception of TBP7 (S6) and YTA2, homology between MS73 and other members of the ATPase family is confined almost exclusively to the conserved 200 amino acids of the ATPase domain. The sequence of MS73 is much closer to that of the S6 (TBP7, 85% identity, 91.5% similarity) subunit of the human 26 S proteasome (58) than any other member of the ATPase superfamily, suggesting that MS73 corresponds to the Manduca homologue of TBP7 (S6).

Outside the conserved ATPase domain is a heptad repeat region (Fig. 2, boxesI) similar to that of a leucine zipper, a feature present in only some members of the ATPase family (57) . The leucine zipper region is known to be important in the dimerization of transcription factors, defining their DNA binding capabilities and controlling specificity of binding(61) . In the ATPase TBP1(78) , the heptad repeat region is required for homo- and heterodimerization (with TBP7). The regulatory complex of the 26 S proteasome is known to contain a number of ATPases(32, 33, 58) , and therefore dimerization may be involved in the assembly of some regulatory ATPases of the complex. It may also be possible, as has been suggested(79) , that such regions may be involved in binding to unfolded ubiquitinated target proteins, ``shuffling'' them between the different catalytic sites of the proteasome. ATPases with leucine zippers are located predominantly in the nucleus(75, 77, 78) . Proteasomes are known to be involved in the degradation, at least in vitro, of transcription factors and oncoproteins (80, 81, 82) , e.g. c-Fos and c-Jun, which also have leucine zippers(83) . It is possible that heterodimerization of a proteosomal ATPase subunit with a ubiquitinated transcription factor may bind the substrate protein to the proteasome, ensuring rapid specific degradation.

Two further conserved elements (Fig. 2, boxesII and III) are examples of the general ATPase A and B boxes (60) . The presence of such elements suggests that members of this ATPase family are responsible in part for defining the ATP dependence of the 26 S proteasome(29, 84, 85, 86) . Such ATP dependence might be needed for the assembly of intact 26 S proteasomes (65) or be involved in the proteolysis of ubiquitinated proteins(79) . A third conserved element (Fig. 2, boxesIV) bears a striking resemblance to the basic amino acid region of the DEAD family of putative RNA helicases(62) . It seems unlikely that an RNA helicase function of MS73 could be involved in the proteolytic activities of the 26 S proteasome. However, some reports on the isolation of proteasomes describe their association with translationally repressed messenger ribonucleoprotein particles and also document the presence of small RNA molecules within the particles(87, 88, 89, 90, 91) .

Expression of MS73 mRNA was detectable at all stages investigated. A general increase in MS73 mRNA concentration was apparent during development of the ISM from stage 0 to a point immediately prior to eclosion (Fig. 3, stage 7). From stage 2 to stage 7, a 2.4-fold increase in the concentration of MS73 mRNA was apparent. This is approximately half that seen for the increase in MS73 protein over the same time period (see below). The increase in the amounts of MS73 mRNA and MS73 protein occurs before the increase in polyubiquitin expression (5, 6) . An increase in the production of ubiquitin and presumably protein ubiquitination following the synthesis of new proteasomes and/or the adaptation of pre-existing proteasomes may be one of the last synthetic events before the programmed elimination of abdominal ISM.

The anti-MS73 antibody detects a 50-kDa protein in Manduca ISM extracts, which is a component of the 26 S proteasome (Fig. 7). During the programmed elimination of Manduca ISM, the amount of this ATPase increases approximately 5.6-fold and is mirrored by similar increases in MSS1 and a 30-kDa subunit of the 20 S proteasome. This indicates that there is an overall increase in the amount of the 26 S proteasome complex during programmed muscular cell death. Previously, Schwartz and co-workers (5, 7) showed that there is not only an increase in polyubiquitin gene expression but also a 10-fold increase in ubiquitin-protein conjugates in ISM at eclosion. The increase in the 26 S proteasome described in this paper provides the vital and appropriate machinery to remove such ubiquitinated proteins.

The work reported here shows for the first time that TBP1 is a component of the 26 S proteasome (Fig. 7). Although TBP1 has been identified as an ATPase(78) , it has not been detected in the 26 S proteasomes of human erythrocytes(58) . This correlates with the observations of others(75, 78) , which suggests that TBP1 may be predominantly localized in the nucleus. The amount of this ATPase does not change during eclosion despite the overall increase in the amounts of the 26 S proteasome. In contrast, the mts2 ATPase increases 3-fold relative to the 30-kDa subunit of the 26 S proteasome, suggesting that developmental reprogramming of the ATPases in the regulatory complexes of the 26 S proteasome may occur during eclosion. Although alternative explanations are possible, the simplest notion is that new proteasomes containing different regulatory ATPases are synthesized during programmed muscular cell death. Such changes in the regulatory ATPases of the new 26 S complexes may facilitate the degradation of new ubiquitinated proteins, which are generated during programmed elimination of abdominal ISM. The differential increases of the peptidase activities (Table 2) are consistent with the observed reprogramming of the ATPases in newly synthesized 26 S proteasomes.

Changes in the subunits of the 20 S proteasome have been widely reported; interferon- treatment results in increased synthesis of the LMP2 and LMP7 subunits of the 20 S proteasome, leading to altered peptidase activities(38, 39, 40, 63) . Displacement of components of the 20 S proteasome seem essential for protein fragmentation for class I major histocompatibility complex antigen fragment presentation(40) . Similarly, there are changes in the protein subunits of the 20 S Drosophila proteasome during embryogenesis(92) . The results reported in this paper constitute the first demonstration of extensive regulatory reprogramming of the 26 S proteasome.

Protein ubiquitination is increasingly being shown to be involved in muscle protein degradation. The degradation of proteins in muscles undergoing atrophy during starvation, following denervation, metabolic acidosis, and after treatment with tumor necrosis factor alpha is a ubiquitin-dependent process(93, 94, 95, 96, 97) . Clearly, the complexity of regulation of the 26 S proteasome suggests that changes in regulatory components will be seen in a variety of physiological and pathological states in muscle as well as in other tissues. Activation of the 26 S proteasome (98) as well as inhibition and modulation of specificity (99) may occur in a number of homeostatic mechanisms. Interestingly, heat shock protein 90 (100) has recently been shown to inhibit the peptidase activity of the proteasome; interaction of proteasomes with members of the heat shock family of proteins may be a general phenomenon.

Programmed cell death is a property of most cells, and yet the intracellular mechanisms involved in cell death are still not fully characterized(101) . Cell death is probably controlled by central switch genes such as reaper in Drosophila(102) , ced-9/bcl-2 homologues(103) , c-myc(104) , MTS1(105) , or nur77(106) . The genes that code for the enzymes that destroy cellular macromolecules and organelles are not fully characterized but should include nucleases and proteases. DNA fragmentation has been described in several model systems(107, 108) . However, proteases should have a powerful central role in the generation of cellular corpses for engulfment by phagocytic cells. The extent of action of proteases may vary between cell types depending on, for example, the accessibility of cells to phagocytosis and the speed of the process.

Elegant studies on programmed cell death in the nematode Caenorhabditis elegans have indicated that the ced-3 gene codes for the nematode homologue of the human interleukin 1-beta converting enzyme, although the protein substrates for this enzyme are unknown(109) . The studies described here and those of others (5, 6) show that ubiquitin-dependent protein degradation destroys abdominal ISM in the tobacco horn moth, M. sexta, around eclosion. Developmentally controlled reciprocal changes in the regulatory ATPases of the 26 S proteasome may alter the spectrum of ubiquitinated proteins, which can be degraded to ensure the rapid demise of the muscles.

Premature activation of p34 serine-threonine kinase occurs by the action of a serine protease, fragmentin-2, which is produced by natural killer cells and enters target cells in the presence of perforin(110) . Activation of the kinase results in events resembling mitotic catastrophe with nuclear dissolution and DNA fragmentation. Following fragmentin-2 action, the p34 kinase is activated and also tyrosine dephosphorylated at the beginning of apoptosis. Degradation of a tyrosine kinase could trigger the process. The specific proteolytic action of a protease, fragmentin-2, is followed by DNA fragmentation(110) ; the destruction of DNA may be a late event in programmed cell death. Widespread destruction of protein also appears to precede DNA degradation when nerve growth factor is withdrawn from cultured rat sympathetic neurones(111) .

Two proteases and both the major non-lysosomal (26 S proteasome) and lysosomal proteolytic systems have now been shown to be involved in mediating programmed cell death in different model systems. Specific and general proteolytic action, combined with subsequent nucleic acid fragmentation, may provide a pattern of hydrolytic activities that either independently or concertedly eliminate cytoplasm from eukaryotic cells before the phagocytic ingestion of cell corpses by neighboring cells. Clearly, proteolytic activities now appear central to mechanisms of apoptosis.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 0602-709369; Fax: 0602-709969.

(^1)
The abbreviations used are: ISM, abdominal intersegmental muscle; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; LMP, low molecular weight protein.

ACKNOWLEDGEMENTS

We acknowledge the support of the SERC (to S. D.) and Wellcome Trust (to J. A.) in these studies.

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