Pathways for degradation of the catalytic subunit of cAMP-dependent protein kinase differ in wild-type and kinase-negative S49 mouse lymphoma cells.

The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [35S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of ∼9.2 h in unstimulated cells and ∼4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.

The catalytic subunit of cAMP-dependent protein kinase radiolabeled with [ 35 S]methionine in wild-type S49 mouse lymphoma cells was degraded with half-lives of ϳ9.2 h in unstimulated cells and ϳ4.5 h in cells stimulated with a membrane-permeable cAMP analog. Turnover in kinase-negative mutant cells was about three times faster than in stimulated wild-type cells and appeared to involve a unique 47-kDa intermediate. Levels of catalytic subunit protein revealed by Western immunoblotting were consistent with the measured differences in turnover, but whereas the protein was mostly soluble in wild-type cell extracts, it was almost entirely insoluble in the mutant cell extracts. A substantial fraction of the catalytic subunit labeled in a 5-min pulse was soluble in kinase-negative cell extracts, but most of this material was rendered insoluble by incubating the cells for an additional 30 min before extraction. Degradation of the catalytic subunit in kinase-negative, but not in wild-type, cells was inhibited strongly by two specific peptide aldehyde inhibitors of the proteasomal chymotrypsin-like activity. An inhibitor of the proteasomal protease that prefers branched-chain amino acids had less of an effect on catalytic subunit degradation in the mutant cells.
Kinase-negative (kin Ϫ ) 1 mutants of S49 mouse lymphoma cells are defective in post-translational maturation and/or accumulation of the catalytic (C) subunit of cAMP-dependent protein kinase with the result that the cells have no detectable C subunit activity and little or no C subunit protein (1)(2)(3)(4). Because the kin Ϫ phenotype is fully dominant in somatic cell hybrids between wild-type and kin Ϫ cells and can be reverted to give cells that are temperature-dependent for C subunit expression, it would appear that the underlying mutation is in a protein-encoding regulatory gene that is in some way critical for expression of a functional C subunit (1,2). Expression of mRNAs for the two major isoforms of the C subunit (C␣ and C␤) is normal in kin Ϫ cells, as is synthesis of the C subunit proteins (3,4). The coding region of the C␣ subunit gene amplified from kin Ϫ cell cDNA can direct expression of a functional C subunit when introduced into a suitable host cell via a mammalian expression plasmid (3), and the coding sequences of C␣ subunit cDNAs from wild-type and kin Ϫ cells are identical. 2 As noted by Orellana and McKnight (3), the normal rate of C subunit production in kin Ϫ cells implies that the C subunit deficiency results from enhanced C subunit degradation. Such accelerated turnover could be either the immediate cause of the mutant phenotype or a consequence of the production of defective C subunit protein. At least two post-translational maturation steps appear to precede the appearance of a functional C subunit in wild-type S49 cells: the first results in solubilization of the newly synthesized protein, and the second is phosphorylation at Thr-197, which is required for efficient catalysis (4,5). In kin Ϫ cells, the majority of newly synthesized C subunit remains insoluble (4). From this observation, we have hypothesized that the primary defect in kin Ϫ cells involves a failure to fold properly the newly synthesized C subunit protein (4). In this view, enhanced C subunit turnover might reflect the activity of a pathway specific for clearance of aberrant cell proteins.
For further analysis of the kin Ϫ phenotype, we decided to measure rates of C subunit turnover in wild-type and kin Ϫ S49 cells. Although several studies have reported enhanced degradation of the C subunit in cells treated chronically with cAMP (6 -9), there are no published data on the rates of C subunit turnover in mammalian cells. We report here that cAMP-dependent kinase activation enhances turnover in wild-type cells by ϳ2-fold. In kin Ϫ cells, not only is C subunit turnover faster than in wild-type cells, but also the pathway for C subunit turnover appears to involve a unique intermediate.

Materials
Chemicals and Radiochemicals-Deoxyribonuclease I, Nonidet P-40, sodium deoxycholate, Triton X-100, Tween 20, spermidine, and spermine were from Sigma; ribonuclease A was from Promega (Madison, WI); and 8-(2-chlorophenylthio)-cAMP (CPT-cAMP) was from Boehringer Mannheim. Alkaline phosphatase-conjugated anti-goat immunoglobulin G (whole molecule) was from the Cappel Products Division of Organon Teknika Corp. (Durham, NC), and Pansorbin was from Calbiochem. [ 3 H]Leucine and [ 35 S]methionine were from DuPont NEN. Benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO), benzyloxycarbonyl-Gly-Pro-Phe-leucinal (Z-GPFL-CHO), and benzyloxycarbonyl-* This work was supported by Grant BE-178 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Submitted this work in partial fulfillment of the degree of Doctor of Philosophy.

Methods
Culture and Radiolabeling of S49 Cells-Wild-type (subline 24.3.2) and kin Ϫ (subline 24.6.1) S49 mouse lymphoma cells were grown in suspension culture in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum, preincubated, and labeled with [ 35 S]methionine in low methionine medium at 37°C as described previously (4,10). The details of the labeling protocols are provided in the figure legends. For pulse-labeled samples, incorporation was stopped by adding 20 l of the cell culture to 700 l of phosphatebuffered saline containing 2 mM L-methionine, and cells were collected by centrifugation. Cell pellets were frozen on dry ice and either extracted immediately or stored at Ϫ70°C for later extraction. For pulsechase experiments, labeled kin Ϫ cells were diluted 40-fold with chase medium, and incubation was continued at 37°C. After various times of chase, cells from 800-l cultures were harvested by centrifugation through 500-l cushions of phosphate-buffered saline containing 10% glycerol. Because the residual isotope was toxic over the longer periods required for turnover experiments with wild-type cells (see Fig. 2 and Table I), these cells were washed by centrifugation through 2 or more volumes of heat-inactivated horse serum before suspending with chase medium (with or without 100 M CPT-cAMP). For the experiments of Fig. 6 and Table I, chase medium contained dimethyl sulfoxide-solubilized inhibitors at the concentrations indicated; the final concentration of dimethyl sulfoxide in all cultures was adjusted to 0.2%.
Expression and Labeling of Recombinant C Subunit-Recombinant murine C␣ subunit was expressed in Escherichia coli BL21(DE3) and purified as described previously (5). A carboxyl-terminally truncated, 33-kDa fragment of the C␣ subunit was expressed and labeled with [ 3 H]leucine as described (11) for use as an internal standard in immunoadsorptions.
Immunoadsorption-Pellets of radiolabeled cells were thawed on ice, extracted by suspension in 20 l of RIPA buffer containing ϳ10,000 acid-precipitable cpm of the [ 3 H]leucine-labeled 33-kDa C subunit fragment, and clarified by centrifugation for 12 min at 178,000 ϫ g in a Beckman Airfuge. The supernatant fractions were then immunoadsorbed with an affinity-purified goat anti-C subunit antibody using a two-cycle procedure described elsewhere (11). In brief, radiolabeled extracts were precleared with Pansorbin and then immunoadsorbed using 3.2 g of anti-C subunit antibody and Pansorbin. After washing the Pansorbin-bound complexes several times with RIPA buffer, they were dissociated by incubation for 10 min at 0°C in a 1% (w/v) solution of SDS containing 1 M 2-mercaptoethanol. The Pansorbin was removed by centrifugation, and the supernatant fractions were diluted and readsorbed with fresh antibody and Pansorbin. For antibody blocking (see Fig. 1, lane d), the antibody was preincubated with 20 g of recombinant C subunit for 15 min on ice before the second immunoadsorption cycle. Immunocomplexes were solubilized with SDS gel sample buffer (12), Pansorbin was removed by centrifugation, and supernatant fractions were saved for scintillation counting and electrophoresis. Incorporated [ 35 S]methionine in extracts and cell fractions was measured by scintillation counting after acid precipitation and filtration (10). For label-chase and/or cell fractionation experiments, equal amounts of tritium radioactivity from each sample were loaded onto gel lanes.
For fractionation experiments (see Fig. 5), radiolabeled cells harvested as described above were extracted directly with 200 l of FB buffer (without freezing). Pellet fractions from low and high speed centrifugations (details in the figure legend) were dissolved with RIPA buffer, and the final supernatant fractions were diluted 2-fold with twice concentrated RIPA buffer. ϳ10,000 cpm of the [ 3 H]leucine-labeled 33-kDa C subunit fragment were added to each fraction, and C subunits were immunoadsorbed as described above.
Western Immunoblots-For immunoblots, cells were harvested by centrifugation, washed once with phosphate-buffered saline, and extracted directly into ice-cold FB buffer at a density of 5 ϫ 10 8 cells/ml. Deoxyribonuclease I and ribonuclease A were added to concentrations of 250 g/ml, and samples were incubated for 2 h on ice with occasional gentle shaking. Portions of the extracts were diluted directly with an equal volume of twice concentrated SDS gel sample buffer for analysis, and the remainder was centrifuged for 10 min at 10,000 ϫ g. Supernatant fractions were diluted 2-fold with twice concentrated SDS gel sample buffer, and pellet fractions were dissolved with SDS gel sample buffer. Protein concentrations were determined by the method of Lowry et al. (13) using bovine serum albumin as a standard. After fractionation by SDS-polyacrylamide gel electrophoresis (PAGE), proteins were transferred electrophoretically to Immobilon-P membranes (Millipore Corp., Bedford, MA), and C subunits were visualized using affinitypurified goat anti-C subunit antibody as the primary antibody, alkaline phosphatase-coupled anti-goat immunoglobulin G as the secondary antibody, and Rad-free Lumi-Phos 530 chemiluminescent substrate sheets (Schleicher & Schuell) as described (11).
Gel Electrophoresis, Fluorography, and Quantitation of Radioactivity or Chemiluminescence in Gel Patterns-SDS-PAGE was carried out as described by Laemmli (14) using 10% polyacrylamide gels. For fluorography, gels were impregnated with 2,5-diphenyloxazole in dimethyl sulfoxide and dried as described by Bonner and Laskey (15). X-ray films from fluorograms or Western immunoblots were scanned with a Molecular Dynamics Model 300A computing densitometer, and integrated absorbances corresponding to the various C subunit species were determined as described (11). Absorbances for the 39-kDa C subunit species and for the putative 47-kDa C subunit conjugate were normalized to that in the 33-kDa marker protein. Fig. 1 shows SDS-PAGE patterns of [ 35 S]methionine-labeled proteins immunoadsorbed from wild-type and kin Ϫ S49 cells with an anti-C subunit antibody. The patterns from both cell lines had a cluster of bands of ϳ39 kDa (C) that correspond to phosphorylated and nonphosphorylated forms of C␣ and C␤ subunits (4,16). An additional species of ϳ47 kDa (C J ) was seen only in the patterns from kin Ϫ cells. The 33-kDa band (M) seen in Fig. 1 (lanes a and b) is a tritium-labeled marker Extracts were immunoadsorbed with anti-C subunit antibody, and the immunoadsorbed species were visualized by SDS-PAGE and fluorography as described under "Experimental Procedures." For the sample in lane d, the antibody was blocked by preincubation with purified recombinant C␣ subunit before the second immunoadsorption cycle (see "Experimental Procedures"). C and M indicate the positions of the ϳ39-kDa C subunit bands and the ϳ33-kDa truncated C subunit marker, respectively; C J indicates the position of a putative ϳ47-kDa C subunit conjugate that was unique to the kin Ϫ cell samples.

RESULTS
protein added to cell extracts to monitor recoveries. Immunoadsorption of both the 39-kDa C subunit forms and the 47-kDa species seen in kin Ϫ samples was blocked by preincubation of the antibody with purified recombinant C subunit (Fig. 1, lane  d). The minor species that ran slightly slower than the 47-kDa species in some wild-type patterns (e.g. Fig. 1, lane a) was not blocked by the C subunit (data not shown), but several minor higher molecular mass species in the kin Ϫ samples were blocked (Fig. 1, lanes c and d; and data not shown).
Figs. 2 and 3 show results from label-chase experiments designed to monitor the intracellular degradation of the C subunit in wild-type and kin Ϫ cells. In wild-type cells, the C subunit label decreased exponentially after ϳ1 h of chase with half-lives of ϳ9.2 h in untreated cells and 4.5 h in cells treated with sufficient CPT-cAMP to activate fully the endogenous kinase holoenzyme (Fig. 2). The C subunit label in kin Ϫ cells disappeared with a half-life of ϳ1.5 h, and the 47-kDa species was degraded at a similar rate (Fig. 3). There were no apparent differences in turnover of total radiolabeled protein among the mutant, drug-free wild-type, and CPT-cAMP-treated wild-type cells (data not shown). Fig. 4 shows Western immunoblot patterns that compare levels of C subunit protein in extracts of wild-type and kin Ϫ cells. Consistent with the radiolabeling studies, a cluster of bands corresponding to the various forms of the C subunit was detected in extracts from both cell lines, and an extra 47-kDa species was detected only in the mutant preparation. C subunit protein was mostly soluble in wild-type extracts, but almost entirely particulate in kin Ϫ extracts. In additional experiments, various amounts of extracts from the two cell lines were analyzed, and the proportions of protein in the C subunit were estimated by densitometry using a standard curve of purified recombinant C subunit for reference (11). The wild-type cell extracts had approximately five times as much C subunit/unit protein as did the kin Ϫ cell samples (data not shown). Fig. 5 shows the results of experiments comparing the distribution of the C subunit and total protein label in extracts from wild-type and mutant cells. Centrifugation conditions were chosen to fractionate the extracts roughly into nuclei and large particulates (fraction P1), polysomes (fraction P2), protein complexes larger than ϳ900 kDa but smaller than polysomes (fraction P3), and soluble proteins (fraction S3). In ex-tracts of pulse-labeled wild-type cells, ϳ15-30% of the total protein label was found in each fraction, but the majority of the C subunit label was in the soluble fraction (Fig. 5A). After a 30-min chase, there was little change in the distribution of either the C subunit or total protein label except for a decrease of the total label in the polysome fraction with corresponding increases in fractions P1 and S3 (Fig. 5B). The behavior of the total protein label in kin Ϫ extracts was similar to that in the wild-type extracts, but that of the C subunit label was distinct. Although ϳ50 -60% of the C subunit label was soluble in extracts of kin Ϫ cells pulse-labeled for 5 min (Fig. 5C), Ͻ30% remained soluble by 30 min of chase (Fig. 5D). The 47-kDa species (here treated as an alternative form of the C subunit) was seen only after the chase and accounted for ϳ40 -50% of the total C subunit radioactivity in each fraction (Fig. 5D).
Since the 47-kDa immunoreactive protein was not seen in samples from kin Ϫ cells labeled in a short pulse (Fig. 5), we undertook experiments to monitor the appearance of this spe- cies. Fig. 6 shows that the 47-kDa species accounted for ϳ20% of the immunoadsorbed label from cells pulsed for 10 min with [ 35 S]methionine. Over the next 20 min, the label in the 47-kDa species increased at the expense of the label in the ϳ39-kDa forms of the C subunit to an apparent steady-state proportion of 0.55 that was maintained for at least 2.5 h. The absence of labeling of the 47-kDa species in a 5-min pulse (Fig. 5) and the reciprocal changes in labeling of the 47-and 39-kDa species at early times of chase are consistent with identification of the 47-kDa species as an intermediate in C subunit degradation.
The multicatalytic proteinase complex or 20 S proteasome is a major extralysosomal proteolytic system of eukaryotic cells that serves as the catalytic core of the 26 S proteasome, which degrades ubiquitin-conjugated proteins (17)(18)(19)(20). For the experiment of Fig. 7, we investigated the effects of selective peptide aldehyde inhibitors of either the chymotrypsin-like or the branched-chain amino acid-preferring activities of the proteasome on turnover of the C subunit in kin Ϫ cells. MG-132 and Z-LLF-CHO, potent inhibitors of the chymotrypsin-like activity (21,22), markedly stabilized the 47-kDa species, which was otherwise mostly degraded after a 4.5-h chase (Fig. 7, compare  lanes c and d with lane b). The 39-kDa C subunit species, however, were only slightly stabilized by these inhibitors. Z-GPFL-CHO, an inhibitor selective for the branched-chain amino acid-preferring activity (23), had a weaker stabilizing effect (Fig. 7, lane e). Z-PP-CHO, a prolylendopeptidase inhibitor used to protect Z-GPFL-CHO from intracellular degradation (23), had no apparent effect by itself (Fig. 7, lane f). Densitometry of the gel patterns of Fig. 7 and those from replicate experiments revealed that, while only 25-35% of the label in all C subunit species (C ϩ C J ) from pulse-labeled samples was recovered in control or Z-PP-CHO chase samples, ϳ85-95% was recovered in samples chased in the presence of MG-132 or Z-LLF-CHO; ϳ40 -60% was recovered in samples chased in the presence of Z-GPFL-CHO and Z-PP-CHO. None of these inhib-itors affected the recovery of acid-precipitable radioactivity in total extracts of the chase samples (data not shown).
In light of a report that inhibitors of the proteasomal proteases could stabilize long-lived proteins that might be degraded by ubiquitin-independent pathways (22), we attempted to assess the effects of the inhibitors used above on turnover of the C subunit in wild-type cells. Analysis of inhibitor effects on C subunit turnover in unstimulated wild-type cells was precluded by the toxicity of the drugs. Judging by the release of acid-precipitable radioactivity from [ 35 S]methionine-labeled cells, both MG-132 and Z-LLF-CHO caused apparent cell lysis after treatments in excess of 7 h. 2 Table I shows data from an experiment testing the effects of proteasomal protease inhibitors on the enhanced C subunit turnover observed in cells treated with CPT-cAMP. None of the inhibitors appeared to stabilize the C subunit significantly in this or three related experiments. (The apparent destabilizing effect of Z-GPFL-CHO in the experiment of Table I  Cells were then extracted without freezing; the extracts were fractionated by centrifugation; and the radioactivity in acid-precipitable protein (open bars) and immunoadsorbable C subunit species (closed and crosshatched bars) was analyzed as described under "Experimental Procedures." The 47-kDa species (cross-hatched bars) was detected only in extracts of kin Ϫ cells that underwent a chase (D). Data shown are means Ϯ S.D. from three or four independent experiments. Fraction P1 is the pellet after centrifugation for 1 min at 1000 ϫ g in a microcentrifuge; fraction P2 is the pellet obtained by centrifuging the supernatant from fraction P1 for 4 min at 178,000 ϫ g in an Airfuge; and fractions P3 and S3 are pellet and supernatant fractions, respectively, obtained after centrifuging the supernatant fraction from fraction P2 for an additional 30 min at 178,000 ϫ g.
FIG. 6. The 47-kDa species behaves kinetically as an intermediate in the degradation of C subunit. kin Ϫ cells were preincubated and labeled with [ 35 S]methionine as described for Fig. 3, but labeling was for only 10 min. Cells were diluted into chase medium, incubated for the indicated times, harvested, and extracted. Radioactivity in the 39-kDa (A) and 47-kDa (B) forms of the C subunit was determined by immunoadsorption, SDS-PAGE, fluorography, and densitometry as described for Figs. 2 and 3. For A and B, data from three independent experiments (circles, squares, and triangles) were normalized to give the same initial values; the scale is arbitrary. C shows the accumulation of the radiolabeled 47-kDa species (C J ) as a fraction of the total immunoreactive C subunit label (C ϩ C J ).

DISCUSSION
This study reports, for the first time, quantitative data on the intracellular turnover of the C subunit of cAMP-dependent protein kinase. The protein was degraded with a half-life of ϳ9.2 h in unstimulated cells and about twice as quickly in cells stimulated with CPT-cAMP. This difference in turnover rates accounts quite well for the 2-3-fold reductions in C subunit activity and/or protein reported in previous studies on a number of different cell systems treated chronically with either agents that elevate intracellular cAMP or cAMP analogs (6 -9). We suspect that the enhanced turnover of free C subunit results from greater accessibility to degradative enzymes and represents a general mechanism for down-regulation of the enzyme under persistent activation conditions. C subunit turnover in kin Ϫ cells was three times faster than that in wild-type cells activated with CPT-cAMP and about six times faster than that in unstimulated wild-type cells. The 5-fold difference in C subunit protein levels found in extracts of wild-type and kin Ϫ cells by Western immunoblotting was roughly consistent with the observed differences in turnover rates. On the other hand, the differences in C subunit turnover were not sufficient to account for the 50 -100-fold reductions in C subunit activity found in kin Ϫ cells (1). Consistent with our previous observation that newly synthesized [ 35 S]methioninelabeled C subunit was found mostly in an insoluble fraction of kin Ϫ cell extracts (4), the unlabeled C subunit found in kin Ϫ cell extracts was essentially all insoluble (Fig. 4).
Unexpectedly, immunoadsorption or Western immunoblot analysis of C subunits from kin Ϫ cells revealed a novel 47-kDa species that was not seen in extracts from wild-type cells. This component was missed in previous studies because of high backgrounds in single cycle immunoadsorptions and low sensitivity in Western immunoblots (2,4). Based on the following evidence, we believe that this species is a covalent (non-disulfide-bonded) conjugate of the C subunit with another molecule of ϳ8 kDa. The 47-kDa species was stable to boiling in SDS gel sample buffer (Figs. 1 and 4); it was immunoreactive with an affinity-purified anti-C subunit antibody (Figs. 1 and 4); and the immunoreactivity of this species in both immunoadsorption and Western immunoblotting experiments was blocked with purified recombinant C subunit ( Fig. 1 and data not shown). Based on immunoadsorption alone (Fig. 1), it remained possible that the 47-kDa species was not itself immunoreactive, but rather associated in a complex with the C subunit. This possi-bility was effectively ruled out by the Western immunoblotting experiment of Fig. 4, in which samples boiled in SDS gel sample buffer were subjected to SDS-PAGE, electroblotted to membranes, and only then reacted with antibody. The label-chase experiments of Figs. 5 and 6 provide further evidence against the possibility that the 47-kDa protein is either noncovalently associated with the C subunit or an unrelated protein sharing antigenic determinants with C subunit. In either of these cases, the protein should have been seen in pulse-labeled samples, but there was no detectable labeling of the 47-kDa protein in immunoprecipitates from kin Ϫ cells pulse-labeled for 5 min with [ 35 S]methionine (Fig. 5). Furthermore, there was a reciprocal decrease of label in the 39-kDa C subunit species as the labeled 47-kDa protein accumulated during a chase (Fig. 6). The kinetics of the appearance and disappearance of the 47-kDa species imply that it is an intermediate whose formation is rate-limiting for C subunit degradation in the kin Ϫ mutant strain (24).
The molecular mass of the 47-kDa species and its putative role as an intermediate in C subunit degradation suggest that it is a monoubiquitinated conjugate of the C subunit. The ubiquitin would target the protein for degradation by the 26 S proteasome either as the monoubiquitinated form or after further ubiquitination to form a polyubiquitinated protein (19,25,26). We used an affinity-purified rabbit anti-ubiquitin antibody (27) in several attempts to detect the presence of ubiquitin in the 47-kDa protein, but we were unable to visualize either the labeled protein by sequential immunoadsorption with anti-C subunit and anti-ubiquitin antibodies or the unlabeled protein by immunoblotting of anti-C subunit-adsorbed material with anti-ubiquitin antibodies. 2 Because both expression of the 47-kDa protein and the sensitivity of conjugate detection with the anti-ubiquitin antibody were very low, these experiments neither confirmed nor ruled out the identity of the species as a ubiquitin conjugate.
Stabilization of the 47-kDa species by MG-132 and Z-LLF-CHO implicates the chymotrypsin-like proteasomal protease in turnover of the conjugate (21,22). That higher molecular mass conjugates were not observed in cells treated with the proteasomal protease inhibitors argues against polyubiquitination of the C subunit in the kin Ϫ cells, but it is also possible that polyubiquitinated forms did not accumulate because of the activity of isopeptidases selective for such species (28). Turnover of the C subunit in wild-type cells neither involved a protein conjugate nor was affected appreciably by inhibitors of the proteasomal proteases. It appears, therefore, that C subunit turnover pathways in wild-type and mutant cell lines are distinct. The overall turnover of [ 35 S]methionine-labeled proteins was equivalent in wild-type and kin Ϫ cells, and the proteasomal protease inhibitors had no apparent effect on turnover of acid-precipitable label from either cell type. 2 It thus seemsunlikelythatkin Ϫ cellshaveanunusuallyactiveubiquitindependent proteolysis system.
Although a number of normal proteins are degraded by the pathway involving ubiquitination and the 26 S proteasome, the pathway was identified initially by its involvement in the rapid turnover of denatured proteins or proteins containing amino acid analogs (29 -31). Selective use of this pathway for C subunit degradation in kin Ϫ cells reinforces the notion that the folded structures of C subunits in this cell line are in some way abnormal despite their having wild-type amino acid sequences (3). 2 The rapid apparent aggregation of newly synthesized C subunits into insoluble complexes suggests that hydrophobic regions of the protein might be unusually exposed after synthesis in the mutant cell line. Both 39-and 47-kDa C subunit forms were found in soluble and insoluble fractions of the kin Ϫ cells, so the aggregation and conjugation processes are apparently independent of one another. We had thought previously that phosphorylation of the C subunit at Thr-197, which is required for full activity, was limited to wild-type cells (4). Using conditions that prevent dephosphorylation of the C subunit (11), we found recently that at least some of the C subunit synthesized in kin Ϫ cells is phosphorylated at Thr-197. 2 This observation argues that lack of phosphorylation is not the critical factor preventing accumulation of soluble C subunit in the mutant cells.
The hypothesis that the kin Ϫ mutation prevents proper folding of newly synthesized C subunit is problematical in view of both the dominance of the phenotype and its apparent specificity for the C subunit. The dominance of the kin Ϫ phenotype in somatic cell hybrids between wild-type and kin Ϫ cells would imply either that the mutation alters the function of an unknown cellular component so that it actively prevents proper C subunit folding or that the mutation alters the structure of a normal component of the protein folding machinery in such a way that it can inhibit proper folding of the C subunit even when it is present with an excess of wild-type copies of the same component. The latter situation seems plausible for the chaperonin proteins, which function in higher order oligomeric ring structures (32)(33)(34), but it is difficult to imagine that C subunit folding requires a specific chaperonin species that is not also essential for proper folding of many other cellular proteins. Specificity cofactors have been demonstrated for folding of tubulin by the chaperonin complex containing t complex polypeptide 1 (35), but mutations in such accessory factors would be expected to produce recessive enzyme deficiency phenotypes. Assuming that different proteins require different levels of chaperonin function for efficient folding, an alternative model could explain the apparent specificity of the kin Ϫ mutation for the C subunit. For proteins like the C subunit that apparently cannot fold properly without chaperonins, 2 thresholds of chaperonin activity might exist below which folding essentially fails. If the level of chaperonin activity (or, perhaps, activity of some particular chaperonin complex with selectivity for the C subunit) were just sufficient in S49 cells for C subunit folding to keep pace with expression, a moderate reduction in activity might have a disproportionate effect on C subunit folding. The defect would appear to be specific for the C subunit if other proteins using the same chaperonin complex(es) had lower critical thresholds.