Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3665-3669, May 1990 Biochemistry Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for synthesis (ubiquitin-dependent proteolysis/arginyl-tRNA protein ) PILLARISETTI SIVARAM AND MURRAY P. DEUTSCHER Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06032 Communicated by Mary J. Osborn, March 1, 1990

ABSTRACT Arginyl-tRNA synthetase (arginine-tRNA li- interactions play an important role in holding aminoacyl- gase, EC 6.1.1.19) is found in extracts ofmammalian cells both tRNA synthetases in the complex (10-12) and that terminal as a free protein (Mr = 60,000) and as a component (Mr extensions on the individual , not required for catal- 72,000) of the high molecular weight aminoacyl-tRNA synthe- ysis, participate in these associations (13). tase complex (Mr > 106). Several pieces of evidence indicate For a few aminoacyl-tRNA synthetases (namely, those for that the low molecular weight free form is not a proteolytic arginine and aspartic acid), both high and low molecular degradation of the complex-bound but that it weight forms of the enzyme coexist in the same extract (14, preexists in vivo: (i) the endogenous free form differs in size 15). Arginyl-tRNA synthetase (arginine-tRNA , EC from the active proteolytic fragment generated in vitro, (ii) 6.1.1.19) is present in extracts ofrat liver as a free protein (Mr conditions expected to increase or decrease the amount of = 60,000) as well as a component (Mr 72,000) of the high proteolysis do not alter the ratio ofthe two forms ofthe enzyme, molecular weight complex (Mr > 106) (5, 16). Comparison of and (iu) the free form contains an NH2-terminal methionine the catalytic and immunological properties of the free and residue. A model is presented that provides a rationale for the complexed forms of this enzyme initially suggested that they existence of two forms of arginyl-tRNA synthetase in cells. In are closely related proteins (17), and peptide mapping indi- this model the complexed enzyme supplies arginyl-tRNA for cated that they are probably identical except for a Mr = protein synthesis, whereas the free enzyme provides arginyl- 12,000 NH2-terminal extension present on the complexed tRNA for the N112-terminal arginine modification of proteins form (13). by arginyl-tRNA:protein arginyltransferase. This latter pro- On the basis of a comparison of the endogenous free form cess targets certain proteins for removal by the ubiquitin- of arginyl-tRNA synthetase with a proteolytically derived dependent protein degradation pathway. The necessity for an fragment released from the complex in vitro, it was thought additional pool of arginyl-tRNA for the modifcation reaction that the free form might have arisen by a limited cleavage of leads to the conclusion that the arginyl-tRNA destined for the complex-bound protein by some endogenous protease protein synthesis (and/or protein modification) is channeled (13, 17). However, several pieces of evidence did not fit with Other evidence this idea (17), and the question of whether proteolysis occurs and unavailable for other processes. supporting during isolation or whether the free form preexists in the cell channeling in protein synthesis is discussed. was also left open. We have now examined this issue in more detail. Our It is becoming increasingly clear that many, if not all, of the resUlts indicate that the endogenous free form differs from the macromolecular components of cells are organized into mul- in vitro proteolytically derived enzyme, that the free form tienzyme complexes or associated with subcellular structures probably preexists in the cell, and that most likely it is a (see ref. 1 for a review). A major consequence of such distinct translation product. The existence of two forms of organization is that it can lead to compartmentalization or arginyl-tRNA synthetase in mammalian cells, coupled with channeling ofmetabolic pathways (i.e., the transfer ofmetab- the recently discovered role of arginyl-tRNA in ubiquitin- olites from one enzyme to another without equilibration with dependent protein degradation (18), leads us to propose a the total fluid of the cell). Channeled pathways presumably model that has important consequences with regard to the would be more efficient than free ones by increasing the local channeling of aminoacyl-tRNAs for protein biosynthesis. concentrations for a given amount of substrate, by minimizing destruction of unstable intermediates or loss due to side reactions, and by allowing for combined regulation of MATERIALS AND METHODS multiple activities. Materials. Sephacryl S-300 was obtained from Pharmacia- The protein biosynthetic machinery is among the most LKB Biotechnology. The protease inhibitors leupeptin, an- complex in the cell; it consists of a large number of protein tipain, pepstatin A, and phenylmethylsulfonyl fluoride were and nucleic acid components. Organization of these various purchased from Sigma. a2-Macroglobulin was from Boeh- components has been studied most extensively in the case of ringer Mannheim. [14C]Arginine was obtained from ICN. the aminoacyl-tRNA synthetases. In extracts of higher eu- Rabbit livers (from young, fasted animals) and rat livers (from karyotic cells, these are generally found in high adult, fasted animals) were purchased from Pel-Freez Bio- molecular weight multienzyme complexes, often containing logicals. Sprague-Dawley female rats (-200 g) were obtained other components as well (2, 3). The number of synthetases from Charles River Breeding Laboratories. Normal rat liver found in these complexes can vary depending on the method cells (clone 9) were obtained from the American Type Culture of isolation, but as many as eight or nine are routinely found Collection. They were maintained as monolayers in Ham's associated with each other (4-7), and even higher numbers nutrient mixture F-12 with 10o (vol/vol) fetal bovine serum. have been observed (8, 9). It is now known that hydrophobic Rabbit liver tRNA was prepared as described (19). Reagents for immunoblotting were products of Promega. All other The publication costs of this article were defrayed in part by page charge materials were as reported (20). payment. This article must therefore be hereby marked "advertisement" Preparation ofArginyl-tRNA Synthetases. Rat liver extracts in accordance with 18 U.S.C. §1734 solely to indicate this fact. for gel filtration were prepared from fresh liver by homoge- 3665 Downloaded by guest on October 2, 2021 3666 Biochemistry: Sivaram and Deutscher Proc. Natl. Acad. Sci. USA 87 (1990) nization and high-speed centrifugation (see the legend to Fig. 2). The low molecular weight free form of arginyl-tRNA synthetase was purified from frozen livers as described by Deutscher and Ni (16). The proteolyzed form of arginyl- tRNA synthetase was prepared from the high molecular weight complex purified through the Controlled-Pore glass (Electro-Nucleonics) column step followed by treatment with papain (17). Enzyme Assays. Aminoacyl-tRNA synthetase activity was determined by measurement of the incorporation of 14C- labeled amino acid into liver tRNA as described (20). One unit ofarginyl-tRNA synthetase represents the incorporation of 1 nmol of amino acid per min under standard conditions. Electrophoresis and Immunoblotting. SDS/PAGE was car- ried out with 8.5% acrylamide gels as reported (20). Protein blotting and immunodetection were performed according to .I ) .: the instructions in the Promega kit using IgG prepared against __r-- -. -.-, k )il the free form of arginyl-tRNA synthetase (13, 20). 1. 1\ P.;.

RESULTS AND DISCUSSION The Endogenous Free Form of Arginyl-tRNA Synthetase Differs from the Proteolyticafly Derived Enzyme. Arginyl- tRNA synthetase differs from most other synthetases in that it is routinely found in both free and complexed forms in FIG. 1. Electrophoresis and immunoblotting ofdifferent forms of extracts of eukaryotic cells (5, 14-16). In rat liver, the ratio arginyl-tRNA synthetase. The high molecular weight complex (25 /dl, of high to low molecular weight enzyme is about 2:1 (5, 16, 2 mg/ml) purified through Controlled-Pore glass (17) was incubated 17). It was originally thought that the free form might be with or without 15 jul of papain (0.25 mg/ml) at 37°C for 25 min in a derived from the final volume of 75 ,u. The reaction was stopped by the addition of 10 complexed enzyme by proteolytic cleavage. ,.l of leupeptin (10 mg/ml). Untreated or treated complex (40 1I) was First ofall, the two proteins were closely related structurally, used for the analysis. Lane 1, untreated complex; lane 2, papain- and second, in vitro treatment of the complex with various treated complex; lane 3, endogenous free form of arginyl-tRNA proteases released an active fragment of arginyl-tRNA syn- synthetase (20 ,g) (16). Samples were electrophoresed and immu- thetase that was the same size as the free form on gel filtration noblotted as described in Materials and Methods. (17). In addition, since active, lower molecular weight forms of lysyl- and methionyl-tRNA synthetases could also be As a further test to eliminate extraneous proteolysis of the released from the complex by in vitro limited proteolysis (21, complex as the source of the free form of arginyl-tRNA 22), it appeared that these enzymes might contain a specific synthetase, an extract of the supernatant resulting from protease-sensitive site between their catalytic domain and a centrifugation at 100,000 x g (S-100) was prepared from a domain required for complex formation. liver that was immediately placed in liquid N2 to stop possible However, more detailed analysis of the proteolytically degradative reactions. Partially frozen liver was homoge- derived arginyl-tRNA synthetase has revealed that it actually nized in a medium containing a mixture of protease inhibi- differs in size from the endogenous free form. SDS/PAGE tors. After centrifugation, gel infiltration ofthis material gave and immunoblotting of the two proteins showed that the Mr the usual pattern of arginyl-tRNA synthetase activity with a of the free form is -2000-3000 less than that of the prote- ratio of complexed form to free form of about 2:1 (Fig. 2). olytically derived fragment; both forms are substantially In another experiment, cultured liver cells were preincu- smaller than the complexed enzyme (Fig. 1). The size differ- bated for 90 min with a mixture of leupeptin (0.2 ,uM), ence between the two smaller forms may also account for the pepstatin A (0.1 AM), and phenylmethylsulfonyl fluoride (500 somewhat greater hydrophobicity previously observed for ,uM). Again, the ratio of low to high molecular weight forms the proteolyzed protein compared to the endogenous free was unaffected by this treatment, despite the fact that these form (17). Thus, although a lower molecular weight arginyl- protease inhibitors can act intracellularly to inhibit proteo- tRNA synthetase can be released from the complex by in lysis that might occur even prior to opening the cells (data not vitro proteolysis, it is clear from these data that a single highly shown). The finding that these extreme procedures also do protease-sensitive site cleaved by both exogenous and en- not change the proportion of free arginyl-tRNA synthetase dogenous proteases is not responsible for the production of lends strong support to the conclusion that this form of the both the in vivo and in vitro low molecular weight enzymes. enzyme preexists in the cell and is not generated by adven- As a consequence, the previously presumed identity of titious proteolysis subsequent to cell disruption. exogenous and endogenous cleavage sites no longer supports Low Molecular Weight Arginyl-tRNA Synthetase Is Proba- the idea that endogenous proteolysis is the origin of the free bly a Distinct Translation Product. NH2-terminal sequence form of the protein. analysis of the endogenous low molecular weight arginyl- Extraction Conditions Do Not Alter the Ratio of Complexed tRNA synthetase provides further evidence for the existence to Low Molecular Weight Arginyl-tRNA Synthetase. Addi- of this form of the enzyme in the cell and, in addition, tional evidence that argues against artifactual proteolysis for suggests that it is a separate translation product. The free the origin ofthe endogenous free enzyme is that extraction of form was purified as reported (16) and also by SDS/PAGE rat liver by using a variety of conditions does not affect the and then subjected to 20 cycles of gas-phase sequencing ratio ofthe complexed form to the free form. In earlier work, (Table 1). Interestingly, in contrast to many eukaryotic conditions as extreme as homogenizing livers in the presence proteins, the enzyme was found to be unblocked and, in of seven protease inhibitors or incubating the homogenate for addition, to begin with an NH2-terminal methionine residue. 1 hr at room temperature in the absence ofprotease inhibitors This finding strongly suggests that the low molecular weight resulted in no change in the ratio of the two forms of the form of the enzyme arises by translation. Since methionine enzyme (5, 17). residues represent only 3% ofthe amino acids in the complex- Downloaded by guest on October 2, 2021 Biochemistry: Sivaram and Deutscher Proc. Natl. Acad. Sci. USA 87 (1990) 3667 Table 1. NH2-terminal sequence analysis of the low molecular

2 weight free form of arginyl-tRNA synthetase

- Amino acid Is2 Cycle Yield, pmol 1 Met 97 m 2 lie 88 1- 3 Asn 22 4 Ile 36

4._ 5 Asn 14 6 Ser 18 D 7 Xaa 8 Leu 17 9 Gln 16 0 20 40 10 Glu 14 60 11 Leu 17 Fraction Number 12 Phe 19 13 Gly 17 FIG. 2. Gel filtration of a rat liver S-100 fraction prepared under 14 Xaa conditions to minimize proteolysis. The liver was removed from an anesthetized rat, immediately frozen in liquid N2, and left for 30 min. 15 Ala 16 The frozen liver was crumbled with a mortar and pestle, and the small 16 Ile 13 pieces were allowed to partially thaw in an ice-cold medium con- 17 Lys 10 taining 50 mM Hepes-KOH (pH 7.4), 165 mM potassium acetate (pH 18 Ala 10 7.0), 3 mM MgCI2, 3 mM glutathione, 0.2 mM EDTA, 0.4 mM 19 Ala 13 phenylmethylsulfonyl fluoride, leupeptin (0.05 ,ug/ml), a2-macro- 20 Tyr 16 globulin (0.05 ,g/ml), and 7% (vol/vol) glycerol. The sample was homogenized by hand and centrifuged, and 10 ml ofthe S-100 fraction Arginyl-tRNA synthetase was purified as described (16) followed was chromatographed on a column (495 ml) of Sephacryl S-300 (17). by further purification by SDS/PAGE on a 1o acrylamide gel. The Fractions of6 ml were and sample was transferred to an Immobilon membrane (Millipore); the collected, 15-Al portions were assayed for band corresponding to arginyl-tRNA synthetase was identified by arginyl-tRNA synthetase activity for 4 min at 37°C. immunoblotting and cut out for sequencing. Gas-phase sequencing was carried out on an Applied Biosystems model 470A sequencer bound enzyme (13), a proteolytic cleavage that generates an equipped with a model 120A phenylthiohydantoin analyzer for 20 NH2-terminal methionine would be extremely unlikely. cycles. The equivalent "180 pmol of pure arginyl-tRNA synthetase Additional evidence suggesting that the free arginyl-tRNA protein was loaded on the gel. Xaa, amino acid could not be synthetase arises by translation comes from a comparison of identified. its NH2-terminal sequence with the NH2 terminus of the Escherichia coli enzyme (23). As shown in Fig. 3, there is a alternate translation initiation events from two different high degree of homology between these two sequences. in-frame start codons (26, 27). Seven of 19 residues (37%) within the sequence are identical, Taken together, all the data presented here provide sub- and an additional 7 are related by a single nucleotide change stantial support for the conclusion that mammalian cells in their respective codons. Such a high level of sequence contain two forms of arginyl-tRNA synthetase, one associ- conservation between these widely separated organisms sug- ated with other aminoacyl-tRNA synthetases and one free, gests that this portion of the protein has been maintained and that the latter form arises from a distinct translation relatively intact during evolution and supports the idea that event. Although further work at the gene and mRNA levels the sequence from the liver enzyme is actually the NH2 will be necessary to conclusively prove these points, these terminus. conclusions fit well with the model presented below, which If the suggestion of a distinct translation event for the low provides a consistent explanation for this unusual situation. molecular weight enzyme is correct, it is likely that both Rationale for the Existence of Two Forms of Arginyl-tRNA forms of the enzyme, one of which contains over 100 extra Synthetase. For almost all other aminoacyl-tRNA syn- NH2-terminal residues but is otherwise identical to the free thetases, only a single form of each enzyme is found in enzyme (13), arise from a single gene. This could be accom- cytoplasmic extracts of mammalian cells. What then might plished either by synthesis of two mRNAs from different explain the presence of two arginyl-tRNA synthetases in transcription initiation sites or by a single mRNA with two these cells? One explanation, which reconciles a number of alternate initiation codons. Several examples are now known disparate observations about arginyl-tRNA metabolism, has in which two forms of an aminoacyl-tRNA synthetase are important consequences for our understanding of protein produced from a single gene (24, 25). In these cases, which biosynthesis. were observed in yeast, transcription is initiated from two It has been known for years that all eukaryotic cells contain sites, leading to two mRNAs: the longer one encodes the an arginyl-tRNA: protein transferase that catalyzes the post- mitochondrial form of the enzyme, and the 5' shortened one translational addition of arginine residues to the NH2 termini encodes the cytoplasmic protein. The extra NH2-terminal of certain acceptor proteins (28, 29). The enzyme utilizes all domains on the longer proteins are thought to target these isoacceptors of arginyl-tRNA as the donor, but only proteins forms to the mitochondria. Alternatively, there are also some with NH2 terminal glutamic, aspartic, or cysteine residues situations in which two proteins arise from a single mRNA by can serve as acceptors (29). Until recently, the function of

Rat liver Met Ile Asn Ile Asn Ser X Leu Gln Glu Leu Phe Gly X Ala Ile Lys Ala Ala Tyr I 11 11 I I 11 11 I 11 11 11 11 E.coli Met Asn Ile Gln Ala Leu Leu Ser Glu Lys Val Arg Gln Ala Met Ile Ala Ala Gly FIG. 3. Comparison ofthe NH2-terminal sequences ofthe rat liver low molecular weight synthetase and theE. coli arginyl-tRNA synthetase. The first 20 amino acids of the liver enzyme are aligned with the NH2-terminal 19 amino acids of the E. coli enzyme. Identities are denoted by double lines, and residues related by a single nucleotide change in the respective codons are denoted by a single line. Downloaded by guest on October 2, 2021 3668 Biochemistry: Sivaram and Deutscher Proc. Natl. Acad Sci. USA 87 (1990) this unusual enzyme was unknown. However, several studies elongation factor, to the ribosome, and back to the synthetase have now implicated this enzyme in the ubiquitin-mediated without mixing with the bulk cytoplasm (Fig. 4). If correct, proteolytic degradation of certain proteins (18, 30, 31). this would have important consequences for our understand- Proteins destined for degradation by this pathway are ing of protein synthesis in higher organisms. modified by conjugation with ubiquitin on E-NH2 groups of Channeling for Protein Synthesis. Despite extensive study lysine (32). An important determinant for reaction with of protein biosynthesis, relatively little attention has been ubiquitin is the identity of the targeted protein's NH2- given to an examination of channeling in this process. None- terminal amino acid (31). For proteins with an acidic NH2- theless, several pieces of evidence support the conclusion terminal residue, the presence of tRNA is required for that channeling does occur. Of particular relevance are the ubiquitin conjugation (33), and this requirement is a conse- numerous observations that components of the protein- quence of the addition of an arginine residue to the NH2 synthesizing apparatus are organized. Such organization termini of these proteins by arginyl-tRNA:protein transfer- would provide a structural basis for channeling. These orga- ase (18). Proteins with NH2-terminal arginine residues are nized structures include the complexes of aminoacyl-tRNA among those most rapidly degraded by the ubiquitin- synthetases described above, the association of aminoacyl- dependent system, and the arginine modification reaction tRNA synthetases with ribosomes (34-36) and elongation serves as a signal for the rapid addition of ubiquitin (18, 31). factor (37), and the binding of many protein synthesis com- It is now clear that in reticulocyte extracts the arginine ponents to the cytoskeletal framework of the cell (38-41). In modification reaction targets for degradation those proteins addition, there is evidence for a functional interaction be- containing NH2-terminal glutamic, aspartic, cysteine, gluta- tween aminoacyl-tRNA synthetases and initiation factors mine, or asparagine residues (31). Furthermore, the require- (42). ment for tRNA in this system can be accounted for com- A number of other studies have also shown that exoge- pletely by the arginyltransferase reaction (30, 31). nously supplied amino acids can enter the pathway ofprotein On the basis ofthese considerations, arginyl-tRNA may be synthesis without mixing with the free amino acid pool ofthe unique among mammalian aminoacyl-tRNAs because it is cell (43-46), implying that channeling may begin early in the needed for a process other than protein biosynthesis. The process. existence of two forms of arginyl-tRNA synthetase in mam- Two other studies also suggest that exogenously supplied malian cells may be directly related to this requirement for a tRNAs are not used for protein synthesis in intact systems. supply of arginyl-tRNA for two distinct processes. We pro- In one, tRNAPhe injected into Xenopus oocytes was a very pose that two arginyl-tRNA synthetases are necessary be- poor substrate for aminoacylation by the endogenous ami- cause there are two separate pools of arginyl-tRNA, one for noacyl-tRNA synthetase despite the fact that the same tRNA protein synthesis and one for the arginine modification re- was an active substrate for this enzyme in extracts of these action. If one or both of these pools were sequestered and cells (47). The conclusion drawn from this experiment was unavailable for other processes, the cell would require sep- that the endogenous aminoacyl-tRNA synthetase is compart- arate synthetases to keep each of the pools supplied with mentalized and not available to exogenous tRNA. In a second arginyl-tRNA. One conclusion that is suggested by such a study, a gently prepared protein-synthesizing system from model is that arginyl-tRNA, and presumably other ami- rabbit reticulocytes was shown to incorporate radioactive noacyl-tRNAs, are channeled for protein synthesis, transfer- amino acid with a linear time course, whereas incorporation ring directly from the aminoacyl-tRNA synthetase to the from labeled aminoacyl-tRNA proceeded only after a sub-

Arginine Arginine tRNA 9

AA-tRNA Synthetase Complex $ (72 kDa Arg-tRNA synthetase) C (bound tRNA) Arginyl-tRNA Synthetase y ( 60 kDa free form ) T 0 p L [ Arg-tRNA ] EFi A Arg-tRNA S M Ribosome Arg-tRNA-Protein transferase

N-terminal-Arg- modified Newly-synthesized protein protein

Ubiquitin-dependent degradation FIG. 4. Model to explain the existence of two forms of arginyl-tRNA synthetase. Arginine that enters the high molecular weight aminoacyl-tRNA synthetase complex is added to endogenous tRNA by the 72-kDa form of arginyl-tRNA synthetase. The arginyl-tRNA is directly transferred to elongation factor 1 (EF1) and the ribosome and incorporated into protein without mixing with the bulk cytoplasm. Arginine interacting with the 60-kDa free form of arginyl-tRNA synthetase is added to free tRNAArg to generate arginyl-tRNA that is used by arginyl-tRNA-protein transferase to modify the NH2 terminus of certain proteins destined for ubiquitin-dependent protein degradation. Downloaded by guest on October 2, 2021 Biochemistry: Sivaram and Deutscher Proc. Natl. Acad. Sci. USA 87 (1990) 3669 stantial lag (48). The conclusion from this work was that the 16. Deutscher, M. P. & Ni, R. C. (1982) J. Biol. Chem. 257, aminoacyl-tRNA was first deacylated and that the free amino 6003-6006. use of the exogenous 17. Vellekamp, G., Sihag, R. K. & Deutscher, M. P. (1985) J. Biol. acid was then incorporated, bypassing Chem. 260, 9843-9847. tRNA. Both of these studies are consistent with the idea that 18. Ferber, S. & Ciechanover, A. (1987) Nature (London) 326, endogenous aminoacyl-tRNAs and synthetases are seques- 808-811. tered and that exogenous macromolecules do not enter the 19. Deutscher, M. P. (1972) J. Biol. Chem. 247, 459-468. protein-synthesizing apparatus. Obviously, this is not true for 20. Sivaram, P., Vellekamp, G. & Deutscher, M. P. (1988) J. Biol. the many disrupted systems studied over the years in which Chem. 263, 18891-188%. in 21. Kellerman, O., Viel, C. & Waller, J.-P. (1978) Eur. J. Biochem. exogenously supplied aminoacyl-tRNAs can function pro- 88, 197-204. tein synthesis. 22. Mirande, M., Cirakoglu, B. & Waller, J.-P. (1983) Eur. J. The data and model presented here raise many interesting Biochem. 131, 163-170. questions about protein synthesis in vivo and in vitro. In 23. Eriani, G., Dirheimer, G. & Gangloff, J. (1989) Nucleic Acids particular, what is the nature of the organization of the Res. 17, 5725-5736. protein-synthesizing machinery and what is its location 24. Chatton, B., Walter, P., Ebel, J.-P., Lacroute, F. & Fasiolo, F. vitro (1988) J. Biol. Chem. 263, 52-57. within the cell, and second, do in protein-synthesizing 25. Natsoulis, G., Hilger, F. & Fink, G. R. (1986) Cell 46, 235-243. systems accurately reflect the in vivo situation when the 26. Conneely, 0. M., Kettelberger, D. M., Tsai, M.-J., Schrader, organization has been disrupted and when large excesses of W. T. & O'Malley, B. W. (1989) J. Biol. Chem. 264, 14062- exogenous tRNA or aminoacyl-tRNA are added to the sys- 14064. tem? A detailed understanding of protein synthesis will need 27. Spence, A. M., Sheppard, P. C., Davie, J. R., Matuo, Y., to consider how organization of the various components and Nishi, N., McKeehan, W. L., Dodd, J. G. & Matusik, R. J. the intermediates might influence the (1989) Proc. Natl. Acad. Sci. USA 86, 7843-7847. how channeling of 28. Kaji, H., Novelli, G. D. & Kaji, A. (1963) Biochim. Biophys. process in vivo. Acta 76, 474-477. 29. Deutch, C. E., Scarpulla, R. C. & Soffer, R. L. (1978) Curr. We thank Dr. Gary Vellekamp for some initial studies on this Top. Cell. Regul. 13, 1-28. problem and George Korza and Dr. Juris Ozols for the amino acid 30. Ciechanover, A., Ferber, S., Ganoth, D., Elias, S., Hershko, sequence analyses. The technical assistance of Martin Worrall is A. & Arfin, S. (1988) J. Biol. Chem. 263, 11155-11167. greatly appreciated. This work was supported by Grant GM16317 31. Gonda, D. K., Bachmair, A., Wunning, I., Tobias, J. W., from the National Institutes of Health. Lane, W. S. & Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712. 1. Srere, P. A. (1987) Annu. Rev. Biochem. 56, 89-124. 32. Rechsteiner, M. (1987) Annu. Rev. Cell Biol. 3, 1-30. 2. Deutscher, M. P. (1984) J. Cell Biol. 99, 373-377. 33. Ferber, S. & Ciechanover, A. (1986) J. Biol. Chem. 261, 3. Yang, D. C. H., Garcia, J. V., Johnson, Y. D. & Wahab, S. 3128-3134. (1985) Curr. Top. Cell. Regul. 26, 325-335. 34. Irvin, J. D. & Hardesty, B. (1972) Biochemistry 11, 1915-1920. 4. Mirande, M., LeCorre, D. & Waller, J.-P. (1985) Eur. J. 35. Moline, G., Hampel, A. & Enger, M. D. (1974) Biochem. J. 143, 191-195. Biochem. 147, 281-289. 36. Graf, H. (1976) Biochim. Biophys. Acta 425, 175-184. 5. Cirakaglu, B. & Waller, J.-P. (1985) Biochim. Biophys. Acta 37. Bec, G., Kerjan, P., Zha, X. D. & Waller, J.-P. (1989) J. Biol. 829, 173-179. Chem. 264, 21131-21137. 6. Godar, D. E., Godar, D. E., Garcia, V., Jacabo, A., Aebi, U. 38. Nielson, P., Goelz, S. & Trachsel, H. (1983) CellBiol. Int. Rep. & Yang, D. C. H. (1988) Biochemistry 27, 6921-6928. 7, 245-254. 7. Norcum, M. T. (1989) J. Biol. Chem. 264, 15043-15051. 39. Howe, J. G. & Hershey, J. W. B. (1984) Cell 37, 85-93. 8. Bandyopadhyay, A. K. & Deutscher, M. P. (1971) J. Mol. Biol. 40. Bonneau, A.-M., Darveau, A. & Sonenberg, N. (1985) J. Cell 60, 113-122. Biol. 100, 1209-1218. 9. Walker, E. J., Trecy, G. B. & Jeffrey, P. D. (1983) Biochem- 41. Orrelles, D. A., Fey, E. G. & Penman, S. (1986) Mol. Cell. istry 22, 1934-1941. Biol. 6, 1650-1662. 10. Dang, C. V. & Yang, D. C. H. (1979) J. Biol. Chem. 254, 42. Pollard, J. W., Galpine, A. R. & Clemens, M. J. (1989) Eur. J. 5350-5356. Biochem. 182, 1-9. 11. Sihag, R. K. & Deutscher, M. P. (1983) J. Biol. Chem. 258, 43. vanVenrooij, W. J., Moonen, H. & vanLoon-Klaassen, L. 11846-11850. (1974) Eur. J. Biochem. 50, 297-304. 12. Cirakoglu, B., Mirande, M. & Waller, J.-P. (1985) FEBS Lett. 44. Airhart, J., Vidrieh, A. & Khairallah, E. A. (1974) Biochem. J. 183, 185-190. 140, 539-548. 13. Vellekamp, G. & Deutscher, M. P. (1987) J. Biol. Chem. 262, 45. Hod, Y. & Hershko, A. (1976) J. Biol. Chem. 251, 4458-4467. 9927-9930. 46. Gehrke, L. & Ilan, J. (1983) Proc. Natl. Acad. Sci. USA 80, 14. Som, K. & Hardesty, B. (1975) Arch. Biochem. Biophys. 166, 3274-3278. 507-517. 47. Gatica, M., Allende, C. C. & Allende, J. E. (1980) Arch. 15. Ritter, P. O., Enger, M. D. & Hampel, A. E. (1979) Biochim. Biochem. Biophys. 202, 653-656. Biophys. Acta 562, 377-385. 48. Hradec, J. & Dusek, Z. (1978) Biochem. J. 172, 1-7. Downloaded by guest on October 2, 2021