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Mol Biol Cell. Nov 1997; 8(11): 2281–2290.
Degradation of Hepatic Stearyl CoA Δ9-Desaturase
Juris Ozols
Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030
Guido Guidotti, Monitoring Editor
Received March 18, 1997; Accepted August 5, 1997.
Δ9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt–washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.
The formation of monounsaturated fatty acids is catalyzed by Δ9 desaturase (EC1.14.99.5) in a reaction requiring acyl-CoA, NADH, NADH-reductase, cytochrome b5, phospholipid, and oxygen (Strittmatter et al., 1974 blue right-pointing triangle). The desaturase, a 40-kDa intrinsic membrane protein, can be induced more than 50-fold in the endoplasmic reticulum by the administration of fat-free diet, insulin, or certain carbohydrate metabolites to animals (Oshino and Sato, 1972 blue right-pointing triangle). When the dietary regimen is stopped, the desaturase activity decreases to undetectable levels with a half-life of a few hours (Oshino and Sato, 1972 blue right-pointing triangle). The proteolytic system responsible for the rapid and selective degradation of desaturase is unknown. Eukaryotic cells contain multiple proteolytic systems including the lysosomal proteases, ATP-ubiquitin–dependent and ubiquitin-independent ATP-proteosome pathway (Hershko and Ciechanover, 1992 blue right-pointing triangle; Inoue and Simoni, 1992 blue right-pointing triangle). Some of these proteolytic events presumably occur on the cytoplasmic side (Gardner et al., 1993 blue right-pointing triangle), whereas others occur within the lumen of endoplasmic reticulum (Wikstrom and Lodish, 1992 blue right-pointing triangle). Several substrate-related peptidyl aldehydes such as N-acetyl-leucyl-leucyl norleucinal and N-acetyl-leucyl-leucyl-methional (ALLM) have been identified to inhibit the calpain, and the ubiquitin-proteosome–dependent proteolytic pathways (Jensen et al., 1995 blue right-pointing triangle). The Streptomyces metabolite, lactacystin, is a specific inhibitor of the proteosome (Fenteany et al., 1995 blue right-pointing triangle). It covalently modifies the highly conserved N-terminal threonine of the mammalian proteosome subunit X, a close homologue of the LMP7 proteosome subunit encoded by the major histocompatibility complex (Fenteany et al., 1995 blue right-pointing triangle). Notwithstanding, membrane proteins are selectively degraded. A number of short-lived membrane proteins, incorrectly synthesized proteins, and partially oligomerized complexes are rapidly degraded in the endoplasmic reticulum, but it is not understood how this degradation is achieved and regulated (Klausner and Sitia, 1990 blue right-pointing triangle; Bonifacino and Klausner, 1994 blue right-pointing triangle).
We previously derived the cDNA sequence of the desaturase (Thiede et al., 1986 blue right-pointing triangle) and constructed an expression vector for the production of active desaturase in Escherichia coli (Strittmatter et al., 1988 blue right-pointing triangle). DNA constructs expressing desaturase in mice (Kaestner et al., 1989 blue right-pointing triangle), Saccharomyces cerevisiae (Stukey et al., 1990 blue right-pointing triangle), and a soluble form of desaturase in plants (Thompson et al., 1991 blue right-pointing triangle; Shanklin and Somerville, 1991 blue right-pointing triangle) have also been reported. A form of desaturase up-regulated in response to cold has been described in fish (Tiku et al., 1996 blue right-pointing triangle).
To study the degradation of this membrane protein, desaturase was induced in rat liver membranes and isolated, and its degradation in subcellular fractions was investigated. In the present study, I report that selective degradation of desaturase can be readily monitored in microsomes and that complete degradation of this enzyme may be partially altered by a high-salt wash of the microsomes. Moreover, the inhibitors of known proteolytic systems such as lysosomes, cathepsins, or the nonlysosomal 26S proteosome complex failed to inhibit the specific degradation of desaturase. These findings should permit the design of experiments to identify the specific desaturase-degrading activity among the many previously characterized cellular proteolytic systems.
Detergents, enzyme substrates, cofactors, and chromatographic media were obtained from Sigma (St. Louis, MO). Cytochrome b5 and cytochrome b5 reductase were prepared from rabbit liver microsomes as described previously (Ozols, 1974 blue right-pointing triangle, 1989 blue right-pointing triangle; Strittmatter et al., 1993 blue right-pointing triangle). ALLM, pepstatin, leupeptin, and phenylmethylsulfonyl fluoride (PMSF) were from Sigma. Porcine erythrocyte calpain was obtained from Calbiochem (San Diego, CA). Lactacystin was obtained from Professor E.J. Corey, Department of Chemistry, Harvard University (Boston, MA).
Preparation of Desaturase Containing Subcellular Fractions
Male Sprague Dawley rats weighing 200–250 g were fasted for 48 h, fed regular diet for 48 h, fasted for second 48-h period, and refed for 20 h with Nutritional Biochemical (Cleveland, OH) “Fat Free” test diet on a schedule that permitted the animals to be killed at the beginning of a day. Control animals were refed regular diet. Subcellular fractionation of livers from induced and control animals was performed according to published procedure (Ozols, 1990 blue right-pointing triangle). The perfused livers were homogenized in a buffer containing 0.25 M sucrose, 10 mM Tris-acetate, pH 8.1, 1 mM EDTA (6 ml/g of liver) in a glass homogenizer. Pellet P-1 was obtained by centrifugation of the homogenate at 800 × g for 10 min. The resulting supernatant was then spun at 10,000 × g for 35 min yielding pellet P-2. Centrifugation of the P-2 supernatant at 130,000 × g for 1.5 h gave pellet P-3 and the supernatant (cytosol fraction). Pelleted microsomes (P-3) were suspended in 20 volumes of 0.1 M sodium pyrophosphate, pH 7.4, and recentrifuged at 130,000 × g. High-salt washed microsomes were prepared by suspending the pellet in 20 volumes of buffer containing 0.1 M Tris-acetate, 0.5 M NaCl, 10 mM EDTA and sedimenting at 130,000 × g for 1 h to obtain high-salt washed microsomes and the high-salt supernatant. The nuclear pellet was refractionated by the method of Fleisher and Krevina (1974). Concentration of the high-salt and cytosol fractions was accomplished on a Centricon-30 concentrator (Amicon, Danvers, MA). Subcellular fractions were stored at −70°C until use. Protein concentration in the samples was determined using the Coomassie dye binding reagent (Pierce, Rockford, IL), using bovine serum albumin as a standard.
Triton X-114 Fractionation of Pellet P-2
Precondensed 4% Triton X-114 in Tris-buffered saline, pH 7.5 (Oxford Glycosystems, Abingdon Oxon, United Kingdom) was added to 120 μg of P-2 to a final concentration of 0.8%. The reaction mixture was spun at 100,000 × g for 15 min at 4°C. The supernatant was layered over a cushion of 0.25 M sucrose, and the centrifuge tube was incubated for 5 min at 37°C. Centrifugation of the reaction mixture at 12,000 × g for 5 min at 37°C yielded detergent-containing lower phase and detergent-depleted aqueous upper phase.
Isolation of Desaturase
Desaturase from the P-1 and P-2 fractions was purified in the presence of sodium deoxycholate and Triton X-100 as described previously for the purification of microsomal desaturase (Strittmatter et al., 1974 blue right-pointing triangle, 1988 blue right-pointing triangle). Desaturase in the P-1 or P-2 fractions for sequencing purposes was subjected to 12% SDS-PAGE and electroblotted onto the Immobilon-P membrane. Sequence analysis on the electroblotted material was performed on an Applied Biosystems (Foster City, CA) model 470A sequenator on line with a model 120A phenylhydantoin analyzer.
Reconstitution of the Desaturase System
The reconstitution and desaturase assay was performed as described previously (Strittmatter et al., 1988 blue right-pointing triangle). Typically, 20 μl of 600 μM cytochrome b5, 6 μl of 100 μM cytochrome b5 reductase, and 20–40 μl of 30 mM egg phosphatidylcholine were added to 115 μl of desaturase sample in 2% Triton X-100 and 0.4% sodium deoxycholate. After incubation for 1.5 h at 4°C, enzyme activity was measured at 25°C by the rate of NADH oxidation in the presence and absence of stearyl-CoA.
Preparation of Antibody against Rat Liver Microsomal Desaturase
To a solution of desaturase, 20 volumes of cold acetone containing 0.2% (y/y) HCl were added. After several hours at −20°C, the protein was collected by low-speed centrifugation. About 0.5 mg of desaturase protein was mixed with 0.5 ml of Freund’s complete adjuvant and injected into rabbits. Rabbit immunoglobulin G (IgG) was prepared from serum by precipitation with ammonium sulfate as described (Hardy, 1986 blue right-pointing triangle). IgG fractions were diluted with an equal volume of 0.1 M potassium phosphate, pH 7, and the IgG was partially purified by affinity chromatography using a column of Protein A Sepharose CL-4B equilibrated in 0.1 M potassium phosphate, pH 7, and stored in 50% glycerol at −70°C.
Complete degradation of desaturase in microsomes and nuclear fractions was determined by Western Blotting (Toubin et al., 1979 blue right-pointing triangle). After incubation at 37°C the samples were subjected to SDS-PAGE using 12- or 10% acrylamide gel under reducing conditions in duplicate. One gel was stained with Coomassie blue, and an identical gel was electrotransferred to an Immobilon-P transfer membrane. The membrane was reacted with rabbit antidesaturase antibody, which was then complexed with antirabbit IgG-alkaline phosphatase (Sigma product A-3687). Immunoreactive desaturase bands were visualized using phosphatase substrate system detection kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Immunoblots were quantified by densitometry using a Kodak DCS 200 digital camera with the Image software (Center for Biomedical Imaging Technology).
Assay of Desaturase Degradation
The reaction mixtures contained 10–15 μg of protein in 50 μl of 50 mM Tris-acetate, pH 7.9, and 50 mM KCl. ATP, ATP-Mg2+, and various protease inhibitors were added where indicated in the figure legend. Cytosol or high-salt fractions were concentrated on a Centricon-30 membrane (Amicon) and added to the reaction samples where indicated. The protease inhibitors were dissolved in dimethylsulfoxide and added at the concentration indicated in the figure legend. Final dimethylsulfoxide concentrations in the incubation samples were 1.5% or less. Model reactions for the protease inhibitors in microsomes were as follows: leupeptin, pepstatin, and PMSF at the concentration indicated in the figure legend was used to inhibit pepsin, papain, or endoproteases Lys-C and Asp-N. Porcine erythrocyte calpain was used as the substrate for the ALLM inhibitor. The samples were incubated 4–18 h at 37°C. Control samples were prepared at 4°C and stored at −20°C. Desaturase degradation in high-salt washed microsomes was restored by supplementation of the reaction mixture with 2 μl of 30 mM egg lecithin liposomes, 2 μl of 50 μM cytochrome b5 reductase, 2 μl of 300 μM cytochrome b5, and 5 μl of stearyl-CoA. Before gel electrophoresis of the digests, 75–150 μl of the loading buffer, containing 60 mM Tris-acetate, pH 6.8, 3% SDS, 5% β-mercaptoethanol, 10% glycerol, and 0.25% bromophenol blue were added. Ten-microliter aliquots of the reaction mixtures were subjected to electrophoresis in duplicate for Coomassie blue staining and immunoblot analysis.
Localization of Desaturase in Microsomes
As the first step in examining the desaturase degradation of desaturase, the enzyme was induced in liver by feeding fasted rats a fat-free, high-carbohydrate diet. As seen in SDS-PAGE Coomassie blue and immunoblot analysis (Figure (Figure1,1, lanes 2 and 5), a 37-kDa band reacting with desaturase antibody is evident in microsomes from rat livers that had undergone dietary manipulation. In contrast, this band was not detected in liver microsomes from control animals (Figure (Figure1,1, lane 3). Purified liver microsomal desaturase migrates on SDS-PAGE as a 37-kDa band, which is lower than the predicted molecular size of 41-kDa (Thiede et al., 1986 blue right-pointing triangle). The cytosolic fraction obtained from desaturase-induced livers is shown in Figure Figure1,1, lane 6, revealing the absence of desaturase in the cytosol fraction.
Figure 1
Figure 1
Identification of desaturase in liver microsomes from rats that have undergone dietary alteration. (A) SDS-PAGE, Coomassie blue-stained gel. Lanes 1 and 7 denote molecular weight standards with the mass marked on the left of the panel. Lane 2, pyrophosphate-washed (more ...)
Association of Desaturase with the Particulate Cellular Fractions
Differential centrifugation of the liver homogenate showed that desaturase is present in the subcellular fractions P-1 and P-2 as well as microsomes. Desaturase activity measurements in the P-1 and P-2 fractions indicated the presence of substantial amounts of the enzyme (Table (Table1).1). The total activity of desaturase in these fractions was estimated to be about 25% that of the microsomes. Figure Figure22 shows the immunoblot analysis of the desaturase in the P-1 fraction. When subcellular fractionation was repeated on the P-1 and P-2 fractions, the desaturase was again present in both the low- and the high-speed sedimenting pellet, but was absent in all of the high-speed supernatants. Partitioning of proteins in Triton X-114 phase has been used to resolve membrane proteins from the soluble proteins (Barrett, 1981 blue right-pointing triangle). Triton X-114 is a nonionic detergent that forms small micelles at low temperature and large micelles above the cloud point temperature. Phase separation of P-2 fraction proteins in Triton X-114 is shown in Figure Figure2,2, lanes 5 and 6. Most of the desaturase partitioned in the detergent phase, although some was present in the aqueous supernatant phase.
Table 1
Table 1
Desaturase activities of hepatic subcellular fractions from animals induced for the enzyme
Figure 2
Figure 2
Association of desaturase with the components of subcellular fractions. Lane 2, P-1 (800 × g); lane 3, P-2 (10,000 × g) fractions. Lanes 5 and 6, Triton X-114 extract of P-2 fraction: lane 5, detergent-rich phase; lane 6, detergent-depleted (more ...)
Structure of Desaturase in the P-1 and P-2 Material
To define the relationship between the microsomal and the desaturase present in the nuclear fraction, isolation, SDS-PAGE, and the N-terminal sequence analysis of the two desaturase preparations were performed. The SDS-PAGE of the two preparations were indistinguishable (Figure (Figure3).3). The N terminus of the enzyme from microsomes was blocked, but sequence analysis of the desaturase in the nuclear fractions P-1 and P-2 indicated the N terminus to be open and identical to residues 3–10 of the microsomal enzyme (Table (Table2).2).
Figure 3
Figure 3
SDS-PAGE of desaturase preparations isolated from microsomes, P-1, and P-2 fractions. (A) Coomassie Blue-stained gel: lane 2, desaturase (Ds) isolated from microsomes; lane 3, desaturase isolated from P-1 fraction; and lane 4, from P-2 fraction. (B) Immunoblot (more ...)
Table 2
Table 2
N-terminal sequence analysis of desaturase in microsomes and fractions P-1/P-2
Selective Degradation of Desaturase Occurs in Microsomes and in P-1, P-2 Fractions
Incubation of microsomes at 37°C led to the disappearance of the desaturase as evidenced by the Coomassie blue staining and by the immunoblot (Figure (Figure4,4, lanes 2 and 3). Incubation of high-salt–washed microsomes at 37°C led to an incomplete degradation of the desaturase band upon the SDS-PAGE, confirmed on Western blot (Figure (Figure4,4, lanes 5 and 6). A major pathway for the intracellular degradation of proteins is an ATP-dependent reaction (Hershko and Ciechanover, 1992 blue right-pointing triangle). Addition of ATP and Mg2+ (5 mM) to the incubation reaction did not enhance the desaturase degradation in high-salt–washed microsomes (Figure (Figure4,4, lane 7). The degradation of desaturase in the P-1 or P-2 fractions was complete without supplementation, but fractions solubilized with Triton X-114 failed to be degraded under the standard incubation conditions.
Figure 4
Figure 4
Selective degradation of desaturase in subcellular fractions. Aliquots of the various subcellular fractions (10–15 μg) were incubated overnight at 0 and 37°C in 50 μl of 50 mM Tris-acetate, pH 7.9 containing 50 mM KCl. (more ...)
To further explore the incomplete desaturase degradation in the high-salt–washed microsomes, an aliquot of P-1 material was added to the salt-washed microsomal preparation before the incubation. As seen in Figure Figure4,4, lanes 12 and 13, when the quantity of desaturase in Figure Figure4,4, lane 5, was coincubated with the amount of P-1 fraction represented by lane 9, a complete desaturase degradation was observed. By comparison, the cytosolic fraction was unable to enhance the degradation in high-salt–washed microsomes, even at levels of 10 fold concentration (Figure (Figure5,5, lanes 4 and 5). Addition of a 10-fold concentrated high-salt wash supernatant also failed to affect the desaturase degradation (Figure (Figure5,5, lanes 6 and 7). Complete degradation of desaturase in the high-salt–washed microsomes could be restored by the addition of liposomes, cytochrome b5, and its reductase, components essential for the desaturase reaction (Figure (Figure5,5, lanes 9, 10, and 11). The time course of the degradation of the desaturase under various conditions is presented in Figures Figures4C4C and and5C.5C. The time course of desaturase degradation in high-salt–washed microsomes supplemented with P-1 fraction is similar to that observed in intact microsomes (Figure (Figure4C,4C, curves 1 and 3). In contrast, in high-salt– washed microsomes only some 30–40% of the desaturase is degraded (Figure (Figure4C,4C, curve 2). Supplementation of the high-salt–washed microsomes with cytosol or the high-salt wash fraction does not increase the extent of the degradation, (Figure (Figure5C,5C, curve 1). Whereas addition of the components of the desaturase system to the high-salt–washed microsomes yields a degradation rate similar to that observed with the intact microsomes (Figure (Figure5C,5C, curve 2 and Figure Figure4C,4C, curve 1).
Figure 5
Figure 5
Proteolysis of desaturase in the high-salt–washed microsomes cannot be reconstituted by the addition of cytosol or high-salt wash supernatant but can be restored by supplementation with lipids and cytochrome b5 reductase. Lanes 2 and 3, high-salt–washed (more ...)
Selective Degradation of Desaturase Is Not Inhibited by Lysosomal or Calpain Inhibitors
To test whether lysosomal enzymes are involved in the desaturase degradation, several lysosomal protease inhibitors were examined. No inhibition of desaturase degradation was observed with chlorquine, leupeptin, pepstatin, ALLM, or PMSF. Leupeptin and pepstatin inhibits hepatic cathepsin B and cathepsin D, respectively (Barrett, 1971). ALLM is a synthetic peptide inhibiting the activity of cathepsins B and L and calpains (Rock et al., 1994 blue right-pointing triangle). Figure Figure66 shows the lack of inhibition of microsomal desaturase degradation by leupeptin and pepstatin. A similar lack of inhibition of desaturase degradation was also observed in our unpublished results when the high-salt–washed microsomes were incubated with the above inhibitors. To explore whether lysosomal proteases were responsible for desaturase degradation in the nuclear associated membranes, the above inhibitors were incubated with P-1 or P-2 fraction. As seen in Figure Figure7,7, pepstatin (180 μg/ml), leupeptin (200 μg/ml), ALLM (170 μg/ml), or PMSF (1 mM) also failed to block the degradation of desaturase in the P-2 fraction.
Figure 6
Figure 6
Microsomal degradation of desaturase is not inhibited by pepstatin or leupeptin. Experimental conditions were as in Figure Figure44 except that pepstatin (240 μM) was present in reaction mixtures represented by lanes 4 and 5 and leupeptin (more ...)
Figure 7
Figure 7
Degradation of desaturase in P-2 fraction is not blocked by inhibitors of lysosomal enzymes or serine proteases. Experimental conditions were as in Figure Figure4,4, except for supplementation with the protease inhibitors. Lanes 4 and 5 represent (more ...)
Lactacystin Does Not Block the Degradation of Desaturase in Microsomes or P-1, P-2 Fractions
Currently, the degradation of rapid turnover of cellular proteins is thought to involve the proteosome system, which is the major proteolytic activity in both the cytosol and the nucleus (Tanaka et al., 1986 blue right-pointing triangle). Very recently, lactacystin, a Streptomyces metabolite, was demonstrated to be a highly specific inhibitor of multiple proteosome activities (Fenteany et al, 1995 blue right-pointing triangle). In view of the profound effect of lactacystin on protein processing, the influence of this reagent on the desaturase degradation in microsomes and the nuclear fractions was examined. The results of such an experiment on the desaturase degradation in microsomes and P-2 fractions are shown in Figure Figure8,8, lanes 2, 4, 6, 7, and 8. Clearly, lactacystin caused no inhibition of the desaturase degradation.
Figure 8
Figure 8
Proteosome inhibitor lactacystin does not inhibit desaturase degradation. Experimental conditions were as in Figure Figure44 except for addition of lactacystin in the indicated sample lanes. The incubation temperature and the final concentration (more ...)
It is now recognized that the endoplasmic reticulum is an important site for intracellular protein breakdown (for review, see Klausner and Sitia, 1990 blue right-pointing triangle; Bonifacino and Klausner, 1994 blue right-pointing triangle). Short-lived proteins, unassembled components of oligomeric complexes such as the T-cell receptor subunits (Wileman et al., 1993 blue right-pointing triangle), and asialoglycoprotein receptors (Wikstrom and Lodish, 1993 blue right-pointing triangle) are degraded in the endoplasmic reticulum. Apolipoprotein B-like proteins (Furukawa et al., 1992 blue right-pointing triangle) and the protein product of the cystic fibrosis-associated gene (CFTR) are degraded in the endoplasmic reticulum (Jensen et al., 1995 blue right-pointing triangle; Ward et al., 1995 blue right-pointing triangle). Although the list of proteins degraded in this organelle tends to grow, the proteolytic machinery of endoplasmic reticulum is poorly understood, and the responsible enzymes have not been identified.
Previously, we describe the isolation, cDNA sequence, and bacterial expression of rat liver desaturase (Thiede et al., 1986 blue right-pointing triangle; Strittmatter et al., 1988 blue right-pointing triangle). The results described in the current report provide a glimpse of the proteolytic processing of this short-lived membrane protein. As seen in Figure Figure1,1, fasting and refeeding a fat-free, high-carbohydrate diet induced high levels of desaturase in the liver microsomal membranes. When the dietary regimen was stopped, desaturase levels rapidly decreased to levels not detectable by immunoblots (Figure (Figure1,1, lane 3). An in vivo half-life of about 2 h has been estimated for the desaturase (Oshino and Sato, 1972 blue right-pointing triangle).
Surprisingly, a considerable amount of desaturase was present in the subcellular fractions P-1 and P-2, in addition to being present in the microsomes (Table (Table1).1). The postmicrosomal supernatant or the high-salt wash of the microsomes did not contain desaturase (Figure (Figure1,1, lane 6). While the P-1 fraction consisted essentially of nuclear material, P-2 subcellular fraction was heterogeneous organelle preparation. The outer nuclear membrane of hepatocytes is continuous with the endoplasmic reticulum, implying that some of the desaturase-containing membranes may traffick to the nuclear membrane. The amount of desaturase associated with P-1 and P-2 subcellular material was significant (Table (Table1)1) and unanticipated.
To determine the relationship between the microsomal and putative nuclear enzyme present in the P-1, P-2 subcellular fractions, desaturase from the latter fractions was purified for sequence analysis. N-terminal sequence analysis of the P-1, P-2 preparations showed an absence of three residues present in the microsomal protein, whereas the microsomal enzyme has a blocked N terminus (Table (Table2).2). Thus, the desaturase present in the nuclear fraction does not represent microsomal enzyme contamination, but appears to represent a specifically processed form of the enzyme. The relationship between the N-terminal processing and the nuclear localization remains to be elucidated, as discussed below.
Incubation of the microsomal membranes at 37°C resulted in the complete degradation of the desaturase, whereas in microsomes washed with a high-salt buffer, the degradation was incomplete as seen in Figure Figure4.4. The complete desaturase degradation, however, could not be restored by the addition of concentrated cytosol or high-salt wash fractions to the microsomes. Complete degradation of desaturase in the high-salt–washed microsomes could be restored by the addition of lipids, cytochrome b5 and its reductase, which constitutes functional desaturase activity. In these experiments, omission of lipid or any of the protein components limited proteolysis. It appears that in high-salt–washed microsomes, only 30–40% of the desaturase is degraded (Figure (Figure4C).4C). Although the possible effects of the salt wash on the desaturase degradation could be the result of many factors, one explanation may involve the formation of high-salt wash–induced conformations in the desaturase population that are resistant to the protease action. The data in Figure Figure5C5C suggest that supplementation of salt-washed microsomes with the lipid, cytochrome b5 reductase, and cytochrome b5 renders the resistant form of the desaturase to further proteolysis. The formation of insoluble desaturase aggregates have been observed during centrifugation on glycerol gradient detergent-solubilized microsomal preparations in the presence of high salt. Some of these forms retain enzymatic activity whereas others do not. Notwithstanding, the degradation reconstitution experiments imply that the procedure used to reconstitute an enzymatically active desaturase system may also yield to protein conformations that are susceptible to the proteolysis of the enzyme.
Desaturase in P-1 or P-2 fractions was degraded completely. Premixing of the salt-washed microsomes with the P-1, P-2 fraction also resulted in a complete degradation of the desaturase. Desaturase antigen bands of lower molecular mass than the desaturase could not be detected in the degradation mixtures, although the desaturase antibody can recognize the bacterial synthesis product lacking some 30 residues from the N terminus, corresponding to a decrease of 3000–5000 Da (Strittmatter et al., 1988 blue right-pointing triangle).
Hepatic lysosomal or endosomal proteases or their precursors are ubiquitous enzymes, and their presence in microsomes and in P-1 and P-2 fractions would not be surprising, since proteases such as the procathepsin B and L may exist in the microsomal membranes as latent precursors. To determine whether lysosomal proteases are involved in the desaturase degradation, several types of protease inhibitors were examined. Leupeptin and pepstatin, inhibitors of lysosomal and endosomal proteases, had no effect on the microsomal or the P-1, P-2 desaturase degradation (Figures (Figures66 and and7).7). The cysteine protease inhibitor ALLM and the serine protease inhibitor PSMF were also ineffective in blocking the desaturase degradation (Figure (Figure7).7). ALLM has been shown to inhibit the regulated degradation of microsomal HMG-CoA reductase (Inoue et al., 1991 blue right-pointing triangle), and a serine protease has been implicated in the rapid degradation of unassembled Ig light chains in endoplasmic reticulum (Gardner et al., 1993 blue right-pointing triangle). One nonlysosomal pathway present in the cytoplasm and nuclear components that mediates rapid elimination of proteins is the proteosome pathway. Multiple types of evidence suggest that the proteosome plays a key role in the processing of antigens for the major histocompatibility complex class I presentation (Chiechanover, 1994 blue right-pointing triangle) and is involved in generating the active forms of molecules such as the production of the 50-kDa subunit of the transcription factor NF-κB from the 105-kDa precursor (Palombella et al., 1994 blue right-pointing triangle). The proteosome is also thought to be responsible for the degradation of the HMG-CoA reductase (McGee et al., 1996 blue right-pointing triangle) and of the cystic fibrosis gene product (CFTR) in the endoplasmic reticulum (Rock et al., 1994 blue right-pointing triangle; Ward et al., 1995 blue right-pointing triangle). The proteosome is a 26S (2000-kDa) complex, containing the 20S proteosome as a key proteolytic component (Rechsteiner et al., 1993 blue right-pointing triangle; Jentsch and Schlenker, 1995 blue right-pointing triangle; Lowe et al., 1995 blue right-pointing triangle). The 20S (700 kDa) complex consists of seven different α-subunits and seven unrelated β-subunits with masses ranging from 24 to 32 kDa comprising about 1% of the protein in mammalian cells (Jentsch and Schlenker, 1995 blue right-pointing triangle). None of the individual subunits of the proteosome have proteolytic activity or show relationship to any known proteases. Recently, a highly specific, irreversible inhibitor of the proteosome, a Streptomyces metabolite–lactacystin has been identified (Fenteany et al., 1995 blue right-pointing triangle). Lactacystin modifies covalently the highly conserved N-terminal threonine of the mammalian proteosome subunit X, a close homologue of the LMP7 proteosome subunit encoded by the major histocompatibility complex (Fenteany et al., 1995 blue right-pointing triangle). Lactacystin has not been found to inhibit any other known protease (Fenteany et al., 1995 blue right-pointing triangle). In view of such a remarkable housekeeping proteolytic function of the proteosome, it was of interest to determine whether the proteosome is involved in the degradation of desaturase. Lactacystin had no effect on the microsomal desaturase degradation (Figure (Figure8).8). The experiment of Figure Figure88 also shows that lactacystin (100–350 μM) also failed to inhibit the desaturase degradation in the P-1 and P-2 subcellular fractions.
The studies reported here show that degradation of desaturase occurs in several subcellular fractions isolated by differential centrifugation. The degradation of desaturase, however, was insensitive to the lysosomal and proteosome inhibitors. If the lysosomal proteases or proteosome do not play a significant role in the desaturase degradation, what alternatives do we have to explain the desaturase degradation? Proteolytic activities such as the ER-60 protease have been detected in detergent-solubilized microsomal preparations (Otsu et al., 1995 blue right-pointing triangle). The proteolytic activity of ER-60, however, is inhibited by leupeptin and ALLM (Otsu et al., 1995 blue right-pointing triangle).
The observation that desaturase is present and readily degraded in subcellular fractions other than the microsomes implies that degradation of native desaturase may also involve targeting of the enzyme to compartments containing specific proteolytic machinery, which constitute a sorting pathway or the reverse process of protein targeting to the membranes. Of interest is that the amino acid sequence of desaturase has two segments that contain a potential nuclear localization sequence (NLS). In residues 33–36, Lys-Met-Lys-Lys and Arg-Lys-Lys-Val-Ser-Lys, residues 335–340 constitute potential consensus sequences for the import of proteins to the nucleus. Import of proteins to the nuclear pore complex is specified by short stretches of amino acids known as the NLSs (see review in Melchior and Gerace, 1995 blue right-pointing triangle; Gorlich and Mattaj, 1996 blue right-pointing triangle). Site-directed mutagenesis of desaturase in the two putative NLS segments should clarify the significance of this finding. Are posttranslation modifications involved in this process? Structure analysis of desaturase in the P-1 and P-2 fractions showed that it lacked three residues at the N terminus (Figure (Figure22 and Table Table2).2). The cDNA sequence predicts a Met-Pro-Ala sequence at the N terminus of the microsomal desaturase (Strittmatter et al., 1988 blue right-pointing triangle). The N terminus of the enzyme present in microsomes is blocked, and the nature of the blocking group remains to be determined. The N-terminal–blocking groups of the two upstream essential components of the desaturase pathway, cytochrome b5 and its reductase, are an acetyl and myristoyl residue, respectively (Ozols et al., 1984 blue right-pointing triangle; Ozols, 1989 blue right-pointing triangle). The presence of a myristoylated residue at the N terminus of desaturase is unlikely because of the absence of a consensus Gly residue in the proximity of its N terminus. The removal of an N-blocked terminus and ProAla segment from the native desaturase is of interest because we are not aware of any reports on hepatic aminopeptidases capable of cleaving residues from N-acetylated proteins. The hepatic acylpeptide hydrolase (E.C. acts only on N-acetylated peptides that are shorter than 10 to 15 residues (Tsunasawa et al., 1983 blue right-pointing triangle). Cathepsins that function as aminopeptidases act only on proteins with a free N terminus.
The complete degradation of desaturase in microsomes can be inhibited by a high-salt wash of the microsomes. This inhibition cannot be restored by the addition of the proteins present in the high-salt wash fraction. The partial degradation of desaturase in high-salt–washed microsomes, however, could be restored by the addition of the components essential for the in vitro catalytic activity of the desaturase. This finding implies that desaturase degradation system may necessitate a specific membrane protein assembly, similar to that observed in reconstitution of the desaturase catalytic activity in vitro. In summary, degradation of the microsomal membrane desaturase was demonstrated in this study. This specific degradation may involve several degradation pathways including removal of the N-terminal residues and the targeting of the modified desaturase to cellular components such as the nuclear material. The possibility that a short-lived protein can be degraded according to different pathways, however, would be unprecedented. Whether the removal of the N-terminal residues from the desaturase results in the formation of a specific determinant that acts as a mediator for the observed trafficking remains to be investigated.
I am grateful to George Korza for his outstanding technical assistance. I thank Professor E.J. Corey, of Harvard University, for providing lactacystin. This work was supported by grant R01-Gm-26351 from The National Institutes of Health.
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