Intermediate-sized filaments in Drosophila tissue culture cells

In using a monoclonal antibody against a major cytoplasmic protein of 46,000 mol wt, we have characterized an intermediate-sized (10 nm) filamentous cytoskeleton in Drosophila melanogaster tissue culture cells. Indirect immunofluorescence, immunoelectron microscopy, and protein blotting show that this cytoskeleton exhibits features typical of the vertebrate vimentin cytoskeleton, including the diameter and appearance of filaments, sensitivity to 10(-6) M colcemid, and insolubility in buffers containing 1% Triton X-100. The antibody cross- reacts with vimentin and desmin from baby hamster kidney cells and stains a vimentin cytoskeleton in the vertebrate Chinese hamster ovary cell line. We, therefore, conclude that the 46,000-mol wt Drosophila protein is homologous to vertebrate vimentin. Three minor, higher- molecular-weight polypeptides are also detected in the Drosophila cells that react with the antibody. At least two of these are members of a family of proteins with properties resembling those of the 46,000-mol wt intermediate filament protein.     

Intracellular appearance of a glycoprotein in VSV-infected BHK cells lacking the membrane-anchoring oligopeptide of the viral G-protein.

Infection of BHK 21 cells by vesicular stomatitis virus (VSV) results in the intracellular synthesis of the five viral proteins which are easily detectable in polyacrylamide gels after short labeling periods with [35S]methionine. In addition, a 6th prominent radioactive protein band appears intracellularly in VSV-infected BHK cells. This additional polypeptide is also coded by the viral genome, because it is immunoprecipitated by antibodies against viral particles and more specifically by antibodies against purified G-protein. We propose to call this derivative of the G-protein Gsi-protein (short intracellular G-protein). It is associated with intracellular membranes and has an apparent mol. wt. of 58 000. Both G- and Gsi-protein have the same kinetics of appearance in the cell. The ratio of G-:Gsi-protein in BHK 21 cells is approximately 85:15. The mol. wt. difference of approximately 6000 daltons between G- and Gsi-protein is not due to variations in the degree of glycosylation because trypsin digestions of both [3H]mannose-labeled glycoproteins gave rise to identical glycopeptide patterns. Incubation of microsomes with trypsin demonstrates that Gsi-protein is protected in its full length by intracellular membranes. Gsi-protein is lacking an extended carboxy-terminal region of the viral G-protein sequence because it is not modified by palmitic acid and is not immunprecipitated by specific antibodies against a C-terminal peptide of the G-protein. Limited proteolysis by endoproteinase arg C indicates that the structure of Gsi-protein is very similar to the shedded form of the G-protein which has been previously described in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening for Drosophila proteins with distinct expression patterns during development by use of monoclonal antibodies

Kc 167 is a cell line established from Drosophila embryonic hemocytes and has been shown to express many extracellular matrix (ECM) and other proteins important during development. We have screened monoclonal antibodies (mAbs) raised against heparin affinity purified proteins from conditioned medium of Kc 167 cells to identify novel proteins with important roles for development. One mAb recognized a protein expressed with temporary and tissue specific patterns during Drosophila embryogenesis and larval development. This approach is an alternative to screening of Expression Sequence Tag (EST) clones by in situ hybridization to initiate reverse genetics. In addition, a number of mAbs recognizing ECM proteins were also identified. These mAbs will be useful for biochemical and cell biological analyses of Drosophila ECM proteins.     

Immunological analysis of chemoattractive proteins from earthworm to garter snakes.

1. Two sets of polyclonal antibodies to two highly purified prey-derived snake-attractive proteins, a low molecular weight (3000) protein and a 20,000 mol. wt protein, were generated in rabbits. 2. They are immunospecific for their respective purified immunogens and do not cross-react with each other. 3. Eight prey-derived proteins that elicit attack by garter snakes (Thamnophis sirtalis) from earthworms (Lumbricus terrestris) were analyzed with these antibodies, and can be assigned to three distinct groups on the basis of their antigenic properties. 4. Unfolding or denaturation of the low molecular weight protein did not alter its antigenic activity to its polyclonal antibodies, suggesting the antigenic epitopes contain contiguous amino acid sequences. 5. In contrast, unfolding of the 20,000 mol. wt protein resulted in a loss of its binding with antibodies, suggesting that the epitope of this protein contains noncontiguous amino acid sequences. 6. The snake-attractivity of the 20,000 mol. wt protein could not be neutralized by reacting it with its antiserum, suggesting that the antigenic determinant (the epitope) of the antigen is not an integral part of the attractive domain of the ES20 protein. 7. In contrast, the attractivity of the purified low molecular weight protein could be neutralized by the polyclonal antibodies.     

Different keratin polypeptides in epidermis and other epithelia of human skin: a specific cytokeratin of molecular weight 46,000 in epithelia of the pilosebaceous tract and basal cell epitheliomas

Cytokeratin polypeptides of human epidermis, of epithelia microdissected from various zones of the pilosebaceous tract (outer root-sheath of hair follicle, sebaceous gland), and of eccrine sweat-glands have been separated by one- and two-dimensional gel electrophoresis and characterized by binding of cytokeratin antibodies and by peptide mapping. The epithelium of the pilosebaceous tract has three major keratin polypeptides in common with interfollicular epidermis (two basic components of mol wts 58,000 and 56,000 and one acidic polypeptide of mol wt 50,000); however, it lacks basic keratin polypeptides in the mol wt range of 64,000-68,000 and two acidic keratin-polypeptides of mol wts 56,000 and 56,500 and contains an additional characteristic acidic cytokeratin of mol wt 46,000. Another cytokeratin polypeptide of mol wt 48,000 that is prominent in hair-follicle epithelium is also found in nonfollicular epidermis of foot sole. Both epidermis and pilosebaceous tract are different from eccrine sweat-gland epithelium, which also contains two major cytokeratins of mol wts 52,500 and 54,000 (isoelectric at pH 5.8-6.1) and a more acidic cytokeratin of mol wt 40,000. A striking similarity between the cytokeratins of human basal-cell epitheliomas and those of the pilosebaceous tract has been found: all three major cytokeratins (mol wts 58,000; 50,000; 46,000) of the tumor cells are also expressed in hair-follicle epithelium. The cytokeratin of mol wt 46,000, which is the most prominent acidic cytokeratin in this tumor, is related, by immunological and peptide map criteria, to the acidic keratin-polypeptides of mol wts 48,000 and 50,000, but represents a distinct keratin that is also found in other human tumor cells such as in solid adamantinomas and in cultured HeLa cells. The results show that the various epithelia present in skin, albeit in physical and ontogenic continuity, can be distinguished by their specific cytokeratin-polypeptide patterns and that the cytoskeleton of basal-cell epitheliomas is related to that of cells of the pilosebaceous tract.     

Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Synthesis of frog virus 3 proteins occurs on intermediate filament-bound polyribosomes

Immunogold labeling and biochemical methods were used to localize the site of viral protein synthesis in frog virus 3 (FV3)-infected baby hamster kidney (BHK) cells. Immunogold labeling studies of Triton-extracted (cytoskeletons), FV3-infected BHK cells with antivimentin antibodies showed that the major components of the detergent-resistant residue are the intermediate filaments (IF) and polyribosomes. Double immunogold labeling studies with anti-FV3 and antivimentin antibodies revealed that FV3 proteins are associated with polyribosomes that are bound to the IF network. Biochemical studies of the cytoskeletons prepared from FV3-infected cells showed that a substantial fraction of all newly synthesized FV3 proteins associate with the cytoskeleton and that the association is not disrupted by colchicine (microtubule-disrupting drug) or cytochalasin B (microfilament-disrupting drug). Together the above studies suggest that IF may provide the scaffold for the attachment of polyribosomes that are active in viral protein synthesis.     

Characterization of molecules involved in protein translocation using a specific antibody

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.     

Patterns of proteins synthesized by non-proliferating murine L cells

When L cells were plated at high density (2 × 10⁵/cm²), proliferation ceased within 3 days, but the cells remained viable and capable of reinitiating DNA synthesis for at least 16 days. SDS-polyacrylamide gel electrophoresis of [³⁵S]methionine labeled polypeptides at various intervals after plating revealed distinct changes in the relative abundance of major cellular proteins beginning 9 days after DNA synthesis ceased. An 84 000 mol. wt polypeptide increased markedly in amount while a polypeptide of 94 000 mol. wt disappeared. Autoradiograms following pulse-labeling showed that these changes were due to increased synthesis of the 84 000 mol. wt polypeptide and decreased synthesis of the 94 000 mol. wt polypeptide. Increased synthesis of a 109 000 mol. wt polypeptide occurred without a concomitant change in its relative abundance. These alterations in the pattern of proteins synthesized revert to normal after feeding with serum-free medium even though DNA synthesis does not resume. Therefore, it appears that even though no abrupt changes in the synthesis of major cellular proteins occurred upon cessation of proliferation, eventually a distinct adaptive pattern of protein synthesis develops in response to the changing culture conditions.     

Differential distribution of microtubule-associated proteins MAP-1 and MAP-2 in neurons of rat brain and association of MAP-1 with microtubules of neuroblastoma cells (clone N2A).

To study the individual location of the microtubule proteins MAP-1 and MAP-2 in neuronal tissues and cells, antisera to electrophoretically purified MAP-1 and MAP-2 components were raised in rabbits. When frozen sections through rat brain were examined by immunofluorescence microscopy the antibodies to MAP-1 strongly stained a variety of nerve cells including dendrites and myelinated axons in the cerebrum and cerebellum. Antibodies to MAP-2 showed similar staining patterns, except that myelinated axons were unstained. These results were confirmed by immunoelectron microscopy of frozen sections through cerebellum using the peroxidase technique. Thereby, the association of MAP-1 with microtubules was also clearly demonstrated. When cultured mouse neuroblastoma N2A cells were examined by immunofluorescence microscopy the antiserum to MAP-1 brightly stained filamentous structures resembling microtubules, whereas relatively weak and diffuse staining of the cytoplasm was observed with the antiserum to MAP-2. In agreement with the immunolocalization, MAP-1, but not MAP-2, was found as a prominent component of microtubules proteins polymerized in vitro by taxol from soluble N2A cell extracts. Together these results indicate that neuronal microtubules are preferentially associated with distinct high mol. wt. polypeptides. Therefore, they support the concept that different complements of associated proteins determine distinct functions of microtubules.     

Comparison by electrophoresis of proteins characteristic of the lateral line and skin of Xenopus laevis

In experiments aimed at determining acousticolateralis marker proteins, fractions of lateral-line organs and skin of Xenopus laevis were analyzed by one- and two-dimensional polyacrylamide-gel electrophoresis. A protein fraction of approximately 44K mol. wt (K = 1000 daltons) and isoelectric pH 6.3, consisting of at least two components, was enhanced in lateral-line neuromast tissue (containing hair cells) and was decreased in tactile organs and skin (lacking hair cells). This "neuromast-marker-protein" fraction had a mol. wt close to that of actin but was shown to be different from actin. Two other major proteins, at mol. wts 16 and 28K, were present in gels of skin and absent in gels of lateral-line tissue. These proteins were shown to be due to secretion of the amphibian granular glands and were designated "negative marker proteins".     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

Evolutionary trends of neurofilament proteins in fish.

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.     

Ecdysterone induction of actin synthesis and polymerization in a Drosophila melanogaster cultured cell line

Actin pools have been evaluated in Drosophila melanogaster Kc 0% cells, through an actin assay based on differential inhibition of DNase I by globular (G) and filamentous (F) actin. Total actin represents about 4 % of total proteins and 54 % is G-actin. In ecdysterone treated cells (0.1 μM), the total actin content increases up to 9 % of total proteins after 3 days of treatment. Ecdysterone induces increase of G-actin as well as F-actin. Increase of both actins, detectable after only 24 hrs of treatment, is roughly parallel during the first two days of treatment. For longer hormonal treatment, actin polymerization is more important than accumulation of G-actin. Indirect immunofluorescence microscopy with antibodies to exogeneous DNase I suggests that actin is widely distributed in the whole cytoplasm before and after ecdysterone treatment. These results suggest that ecdysterone induces actin synthesis and polymerization in Drosophila melanogaster cells.     

Synthesis and localization of myeloperoxidase protein in transfected BHK cells

Processing and localization of myeloperoxidase was studied in nonmyeloid cells. For this purpose BHK cells were transfected with human myeloperoxidase cDNA. In the transfected cells a protein with mol wt of 85,000 was found, which reacted with the specific anti-human myeloperoxidase antiserum. In size and in sensitivity to endo-beta-N-acetylglucosaminidase H this protein resembled the myeloperoxidase precursor synthesized in human promyelocytes. Unlike in the promyelocytes, in BHK cells the 85,000-Da protein was not converted to 60,000- and 14,000-Da polypeptides of the mature enzyme. In Percoll gradients the protein was found predominantly in the light membrane fractions. Microscopic examination revealed a conspicuous immune reaction over the endoplasmic reticulum and nuclear membranes and a moderate labeling over lysosome-like organelles. Pulse-chase experiments indicated that the protein was slowly released from the endoplasmic reticulum; after 1 day the protein was found in similar amounts in cells and in the medium. The secreted protein contained at least one endo-beta-N-acetylglucosaminidase-resistant oligosaccharide. It is suggested that normal intracellular segregation of myeloperoxidase depends on a signal or component, which is not or incompletely expressed in BHK cells.     

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.     

Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin.

Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.