Intracellular protein degradation in cultures of dystrophic muscle cells and fibroblasts

We have analysed protein degradation in primary cultures of normal and dystrophic chick muscle, in fibroblasts derived from normal and dystrophic chicks, and in human skin fibroblasts from normal donors and from patients with Duchenne muscular dystrophy (DMD). Our results indicate that degradative rates of both short- and long-lived proteins are unaltered in dystrophic muscle cells and in dystrophic fibroblasts. Longer times in culture and co-culturing chick fibroblasts with the chick myotubes do not expose any dystrophy-related abnormalities in protein catabolism. Furthermore, normal and dystrophic muscle cells and fibroblasts are equally able to regulate proteolysis in response to serum and insulin. We conclude that cultures of chick myotubes, chick fibroblasts, and fibroblasts derived from humans afflicted with DMD are not appropriate models for studying the enhanced protein degradation observed in dystrophy.     

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte- mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.     

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.     

The ATP dependence of the degradation of short- and long-lived proteins in growing fibroblasts

To characterize the system(s) responsible for degradation of short-lived and long-lived proteins in mammalian cells, we compared the concentrations of ATP required for the degradation of these classes of proteins in growing hamster fibroblasts. By treating CHEF-18 cells with increasing concentrations of dinitrophenol and 2-deoxyglucose, it was possible to reduce their steady-state ATP content by different amounts (up to 98%). These treatments caused a rapid decrease in the degradation of both short- and long-lived proteins. Removal of the inhibitors led to a prompt restoration of ATP and proteolysis. As ATP content fell below normal levels (about 3.1 mM), rates of proteolysis decreased in a graded biphasic fashion. Reduction in ATP by up to 90% (as may occur in anoxia or injury) decreased proteolysis up to 50%; and with further loss of ATP, protein breakdown fell more sharply. Degradation of both classes of proteins was inhibited by 80% when ATP levels were reduced by 98%. The levels of ATP required for the breakdown of short- and long-lived proteins were indistinguishable. Protein synthesis was much more sensitive to a decrease in ATP content than protein breakdown and fell by 50% when ATP was reduced by only 15%. Chloroquine, an inhibitor of lysosome function, did not reduce the degradation of either class of proteins in growing cells, but it did inhibit the enhanced degradation of long-lived proteins upon removal of serum (in accord with previous studies). Thus, in growing fibroblasts, an ATP-dependent nonlysosomal process appears responsible for the hydrolysis of both short- and long-lived proteins.     

Comparison of the control and pathways for degradation of the acetylcholine receptor and average protein in cultured muscle cells

In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (¹²⁵I)-bungarotoxin. The apparent average half-life of cell protein was 38 ± 2 hours and that of the receptor-toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor-toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor-toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11-fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10-fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate-limiting steps in these processes have different activation energies. Incubation with 2-deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor-toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy.     

Investigation of the mechanism of protein turnover in HeLa S-3 cells by incubation at elevated temperatures

An apparent paradox relating to the degradation of endogenous proteins in HeLa S-3 cells occurs at 45 degrees C, at which their proteolysis is considerably enhanced in vitro but completely inhibited in vivo. No significant differences in rates of degradation of short-lived (nascent) and long-lived ('existing') proteins synthesised at 37 degrees C were found when chased at temperatures up to 43 degrees C, but at 45 degrees C degradation of both categories was reduced to zero in vivo. Synthesis of protein was suppressed at temperatures above 41 degrees C, being reduced by up to 60% at 43 degrees C. Proteolysis in vitro proceeded 1.6-1.7 times faster at 45 degrees C than at 37 degrees C and neutral pH. Evidence is presented for the involvement of the basal system; the findings both in vivo and in vitro do not seem to implicate the lysosomal system, no firm indication being obtained of its 'induction' at elevated temperatures. The results are discussed in terms of the arrest of intracellular circulation at elevated temperatures, thereby reducing the delivery rate of proteins as substrates of the intracellular basal proteolytic enzyme system to negligible levels (i.e., to the frequency of encounters due solely to the diffusion of protein molecules with the cytoplasm).     

Kinetics of protein degradation in diploid and trisomic human fibroblasts

The degradation rate of long-lived and short-lived proteins was determined in diploid fibroblasts and fibroblasts with trisomy 7 derived from human embryos. Two fractions of proteins were detected in the exponentially growing diploid fibroblasts with half-lives (T 1/2) 37 and 19 hours. The rate of protein degradation increases in diploid fibroblasts as they approach confluence and protein fractions with T 1/2 30, 18 and 12 hours appear. The rate of protein degradation in trisomic fibroblasts does not change for the long-lived and short-lived proteins and is the same in both exponential (T 1/2 31 and 14 hours) and stationary phase (T 1/2 33 and 17 hours). The relative amount of the short-lived proteins in trisomic fibroblasts in the stationary phase decreased as compared with the one in diploid fibroblasts. It is apparent that a mechanism of regulation of protein catabolism in trisomic fibroblasts is impaired.     

Intracellular protein catabolism. IX. Hydrophobicity of substrate proteins is a molecular basis of selectivity.

Double-labeled cytosol proteins from rat liver (3H in short-lived, 14C in long-lived proteins) were fractionated by using siliconized glass-beads, phenylsepharose and octylsepharose. Always the short-lived proteins are more tightly bound to the hydrophobic matrix. The same distribution was found with monkey liver substrate proteins. Therefore it is concluded that the different degrees of exposure of superficial hydrophobic areas on substrate protein molecules are a molecular basis of selectivity of the intracellular protein catabolism.     

Effects of aging in vitro on intracellular proteolysis in cultured rabbit lens epithelial cells in the presence and absence of serum

Summary Alterations in proteolytic capabilities have been associated with abnormalities in the aged eye lens, but in vivo tests of this hypothesis have been difficult to pursue. To simulate aging, we cultured cells from an 8-yr-old rabbit to early (population-doubling level 20 to 30) and late (population-doubling level > 125) passage. Long-lived (t_1/2>10 h) and short-lived (t_1/2<10 h) intracellular proteins were labeled with [³H]leucine, and the ability of the cells to mount a proteolytic response to the stress of serum withdrawal was determined. For early passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 62 and 39 h, respectively. For late-passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 58 and 43 h, respectively. The net increase in intracellular proteolysis in the absence of serum was 59 and 35% for early and late-passage cells, respectively. Thus, in vitro-aged rabbit lens epithelial cells mount only 60% the proteolytic response to serum removal shown in “younger” cells. The enhanced ability of early passage cells to respond to serum removal seems to involve lower homeostatic levels of proteolysis in the presence of serum and greater enhancement of proteolysis in the absence of serum. Less than 2% of the protein is in the pool of short-lived proteins. Rates of proteolysis of short-lived proteins in the presence and absence of serum were indistinguishable. With respect to basal proteolytic rates in the presence of serum and ability to mount a proteolytic response upon serum withdrawal, these rabbit lens epithelial cells are similar to bovine lens epithelial cells and fibroblasts. This work was supported in part by contract 53-3K06-5-10 U.S. Department of Agriculture, Washington, DC, Massachusetts Lions Eye Research FUnd, Inc., the Daniel and Florence Guggenheim Foundation, and a grant EY00362 from the National Eye Institute, Bethesda, MD.     

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards ¹²⁵I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [³H]phenylalanine, was reduced by only 10–17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (E_a = 18 kcal/mol) and short-lived proteins (E_a = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37°C in contrast to the breakdown of LDL which ceased below 20°C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.     

Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

Exposure of cultured rat hepatoma (HTC) cells to a 43 degrees C heat shock transiently accelerates the degradation of the long-lived fraction of cellular proteins. The rapid phase of proteolysis which lasts approximately 2 h after temperature step-up is followed by a slower phase of proteolysis. During the first 2 h after temperature step-up there is a wave of ubiquitin conjugation to cellular proteins which is accompanied by a fall in ubiquitin and ubiquitinated histone 2A (uH2A) levels. Upon continued incubation at 43 degrees C the levels of ubiquitin conjugates fall with a corresponding increase of ubiquitin and uH2A to initial levels. The burst of protein degradation and ubiquitin conjugation after temperature step-up is not affected by the inhibition of heat shock protein synthesis. Cells of the FM3A ts85 mutant, which have a thermolabile ubiquitin activating enzyme (E1), do not accelerate protein degradation in response to a 43 degrees C heat shock, whereas wild-type FM3A mouse cells do. This observation indicates that the ubiquitin system is involved in the degradation of heat-denatured proteins. Sequential temperature jump experiments show that the extent of proteolysis at temperatures up to 43 degrees C is related to the final temperature and not to the number of steps taken to attain it. Temperature step-up to 45 degrees C causes the inhibition of intracellular proteolysis. We propose the following explanation of the above observations. Heat shock causes the conformational change or denaturation of a subset of proteins stable at normal temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aging as a catabolic malfunction

Cellular degradative processes, which include lysosomal (autophagic) and proteasomal degradation, as well as catabolism of proteins by cytosolic and mitochondrial proteases, provide for a continuous turnover of cellular components, such as damaged or obsolete biomolecules and organelles. Inherent insufficiency of these degradative processes results in progressive accumulation within long-lived postmitotic cells of biological ‘garbage’ (waste material), such as various oxidized proteins, functionally effete mitochondria, and lipofuscin (age pigment), an intralysosomal, polymeric, undegradable material. There is increasing evidence that lipofuscin hampers lysosomal degradative capacity, thus promoting the aggravation of accumulated damage at old age. Being rich in redox-active iron, lipofuscin granules also may exacerbate oxidative stress levels in senescent cells. Thus, increasing the efficiency of cellular degradative pathways and preventing involvement of iron in oxidant-induced lysosomal and cellular damage may be potential strategies for anti-aging interventions.     

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.     

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Peptide sequences that target proteins for enhanced degradation during serum withdrawal

Fibroblasts increase the catabolism of certain intracellular proteins in response to serum withdrawal, and these proteins contain specific peptide regions that may be required for their increased degradation. We show that the increased degradation of microinjected ribonuclease A during serum withdrawal can be blocked by co-injection of a pentapeptide corresponding to residues 7-11 of ribonuclease A, Lys-Phe-Glu-Arg-Gln. Furthermore, similar peptide sequences appear to play a widespread role in targeting proteins for enhanced degradation. Affinity-purified antibodies raised against the pentapeptide are able to precipitate 20-35% of radiolabeled cytosolic proteins from fibroblasts. Such proteins are preferentially degraded when cells are deprived of serum while nonimmunoprecipitable proteins are degraded at the same rate in the presence and absence of serum. Immunoreactive cytosolic proteins also exist in rat liver and kidney, and these proteins are depleted when protein degradation rates are enhanced due to starvation. Several types of evidence suggest that the peptides recognized in cellular proteins are similar to Lys-Phe-Glu-Arg-Gln but are not this exact sequence. Analyses of amino acid sequences for four proteins whose degradative rates are enhanced in response to serum withdrawal and for four proteins that are degraded in a serum-independent manner indicate two possible peptide motifs related to Lys-Phe-Glu-Arg-Gln that may target cellular proteins for enhanced degradation. These results, combined with previous studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that these peptide regions target specific proteins to a lysosomal pathway of degradation during serum withdrawal.     

The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Proteins damaged by oxygen radicals are rapidly degraded in extracts of red blood cells

We have suggested that red blood cell proteolytic systems can degrade oxidatively damaged proteins, and that both damage and degradation are independent of lipid peroxidation (Davies, K. J. A., and Goldberg, A. L. (1987) J. Biol. Chem. 262, 8220-8226. These ideas have now been tested in cell-free extracts of rabbit erythrocytes and reticulocytes. Exposure to oxygen radicals or H2O2 increases the degradation of endogenous proteins in cell-free extracts, as in intact cells. Various radical-generating systems (acetaldehyde or xanthine + xanthine oxidase, ascorbic acid + iron, H2O2 + iron) and H2O2 alone enhanced the rates of proteolysis severalfold. Since these extracts were free of membrane lipids, protein damage and degradation must be independent of lipid peroxidation. An antioxidant buffer consisting of HEPES, glycerol, and dithiothreitol inhibited the increased proteolysis by 60-100%. Mannitol caused a 50-80% reduction in proteolysis suggesting that the hydroxyl radical (.OH), or a species with similar reactivity, may be the initiator of protein damage. When casein or bovine serum albumin were exposed to .OH (generated by H2O2 + Fe2+, or COCo radiation) these proteins were degraded up to 50 times faster than untreated proteins during subsequent incubations with red cell extracts. Mannitol inhibited this increase in proteolysis only if present during .OH exposure; mannitol did not affect the degradative system. Although ATP increased the degradation of untreated proteins 4- to 6-fold in reticulocyte extracts, it had little or no effect on the degradation of proteins exposed to .OH. ATP also did not stimulate hydrolysis of .OH-treated proteins in erythrocyte extracts. Leupeptin did not affect the degradative processes in either extract; thus lysosomal or Ca2+-activated thiol proteases were not involved. We propose that red cells contain a soluble, ATP-independent proteolytic pathway which may protect against the accumulation of proteins damaged by .OH or other active oxygen species.     

Role of the proteasome in membrane extraction of a short-lived ER-transmembrane protein.

Selective degradation of proteins at the endoplasmic reticulum (ER-associated degradation) is thought to proceed largely via the cytosolic ubiquitin-proteasome pathway. Recent data have indicated that the dislocation of short-lived integral-membrane proteins to the cytosolic proteolytic system may require components of the Sec61 translocon. Here we show that the proteasome itself can participate in the extraction of an ER-membrane protein from the lipid bilayer. In yeast mutants expressing functionally attenuated proteasomes, degradation of a short-lived doubly membrane-spanning protein proceeds rapidly through the N-terminal cytosolic domain of the substrate, but slows down considerably when continued degradation of the molecule requires membrane extraction. Thus, proteasomes engaged in ER degradation can directly process transmembrane proteins through a mechanism in which the dislocation of the substrate and its proteolysis are coupled. We therefore propose that the retrograde transport of short-lived substrates may be driven through the activity of the proteasome.