Chloroplast envelope proteins are encoded by the chloroplast genome of Chlamydomonas reinhardtii.

To characterize envelope proteins encoded by the chloroplast genome, envelopes were isolated from Chlamydomonas reinhardtii cells labeled with [35S] sulfate while blocking synthesis by cytoplasmic ribosomes. One and two-dimensional gel electrophoresis of envelopes and fluorography revealed four highly labeled proteins. Two with masses of 29 and 30 kDa and pI 5.5 were absent from the stroma and thylakoid fractions, while the others at 54 kDa, pI 5.2 and 61 kDa, pI 5.4 were detected there in smaller amounts. The 29- and 30-kDa proteins were associated with outer envelope membranes separated from inner envelope membranes after chloroplast lysis in hypertonic solution. A 32-kDa protein not labeled by [35S]sulfate was found exclusively in the inner membrane fraction, suggesting the existence of a phosphate translocator in C. reinhardtii. To identify envelope proteins exposed on the chloroplast surface, isolated active chloroplasts were surface-labeled with 125I and lactoperoxidase. The 54-kDa, pI 5.2 protein as well as a protein corresponding to either of the 29- or 30-kDa proteins described above were among the labeled components. These results show that envelope proteins of C. reinhardtii are encoded by the chloroplast genome and two are located on the outer envelope membranes.     

The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.     

Tic20 and Tic22 Are New Components of the Protein Import Apparatus at the Chloroplast Inner Envelope Membrane

Two components of the chloroplast envelope, Tic20 and Tic22, were previously identified as candidates for components of the general protein import machinery by their ability to covalently cross-link to nuclear-encoded preproteins trapped at an intermediate stage in import across the envelope (Kouranov, A., and D.J. Schnell. 1997. J. Cell Biol. 139:1677–1685). We have determined the primary structures of Tic20 and Tic22 and investigated their localization and association within the chloroplast envelope. Tic20 is a 20-kD integral membrane component of the inner envelope membrane. In contrast, Tic22 is a 22-kD protein that is located in the intermembrane space between the outer and inner envelope membranes and is peripherally associated with the outer face of the inner membrane. Tic20, Tic22, and a third inner membrane import component, Tic110, associate with import components of the outer envelope membrane. Preprotein import intermediates quantitatively associate with this outer/inner membrane supercomplex, providing evidence that the complex corresponds to envelope contact sites that mediate direct transport of preproteins from the cytoplasm to the stromal compartment. On the basis of these results, we propose that Tic20 and Tic22 are core components of the protein translocon of the inner envelope membrane of chloroplasts.     

Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts

Translocation of proteins across membranes is essential for the biogenesis of each cell and is achieved by proteinaceous complexes. We analyzed the translocation complex of the intermembrane space from chloroplasts and identified a 12-kDa protein associated with the Toc machinery. Toc12 is an outer envelope protein exposing a soluble domain into the intermembrane space. Toc12 contains a J-domain and stimulates the ATPase activity of DnaK. The conformational stability and the ability to stimulate Hsp70 are dependent on a disulfide bridge within the loop region of the J-domain, suggesting a redox-regulated activation of the chaperone. Toc12 is associated with Toc64 and Tic22. Its J-domain recruits the Hsp70 of outer envelope membrane to the intermembrane space translocon and facilitates its interaction to the preprotein.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

Calcium regulation of chloroplast protein import

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.     

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163–171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained antigenicity. The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF₁), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, a 34-kDa protein (OEP34 or IAP34), the channel-forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor (OEP86 or IAP86) were tested for their localization. The previous localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.Abbreviations FCS fetal calf serum - IAP import intermediate associated protein - IEP inner envelope protein - OEP outer envelope protein (numbers signifying the relative molecular mass in kilodaltons) - PBS phosphate buffered saline - PVP polyvinyl pyrrolidone - Rubisco ribulose-1,5-biophosphate carboxylase/oxygenase     

Chloroplast phosphoproteins: phosphorylation of a 12-kDa stromal protein by the redox-controlled kinase of thylakoid membranes

Activation of the redox-controlled protein kinase of thylakoid membranes is detectable in vivo by measuring radioisotope incorporation into the light-harvesting Chl a/b protein and four photosystem II proteins (8.3, 32, 34, and 44 kDa). In normal barley leaves, the kinase is active under both aerobic and anaerobic (N2) conditions, but in the Chl b-less chlorina f2 mutant it is active only under anaerobic conditions. The responsiveness of this enzyme in the mutant to changes in the gas phase has been exploited to distinguish its protein substrates from those of other leaf protein kinases. Most of the soluble phosphoproteins of normal and mutant leaves (including a conspicuously labeled 67-kDa polypeptide) are labeled equally under both aerobic and anaerobic conditions, indicating that they are not substrates of the redox-controlled protein kinase. The major exception is a 12-kDa phosphoprotein, which is labeled in the mutant only under anaerobic conditions. The 67- and 12-kDa phosphoproteins are located in the chloroplast and are labeled when isolated organelles are incubated with [32P]orthophosphate in the light. When thylakoids and stroma are prepared from chloroplasts and are incubated with [gamma-32P]ATP in vitro, the 12-kDa protein is phosphorylated in the thylakoid preparation and then released from the membranes into the medium. The electron transport inhibitor diuron blocks activation of the redox-controlled kinase and prevents phosphorylation of the 12-kDa protein, which is thus the first example of a soluble protein to be phosphorylated by the thylakoid-bound protein kinase. The 67-kDa protein is phosphorylated by a distinct stromal kinase whose activity is not sensitive to diuron.     

Toc64--a preprotein-receptor at the outer membrane with bipartide function

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.     

Membrane topography of the subunits of ubiquinol-cytochrome-c oxidoreductase of Saccharomyces cerevisiae. The 14-kDa and the 11-kDa subunits face opposite sides of the mitochondrial inner membrane

The topology of the subunits of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been determined using a digitonin/proteinase K assay. With this assay we were able selectively to disrupt the mitochondrial membranes and to identify the subunits which became proteinase-K sensitive after disruption of either the outer or both outer and inner membranes. This approach confirmed previous indications for the localization of the core I protein, cytochrome c1, cytochrome b, the FeS protein and the 17-kDa subunit, while it also provided direct evidence for the site of accessibility to proteinase K of the 14-kDa and 11-kDa subunits. The 14-kDa subunit faces the mitochondrial matrix and the 11-kDa subunit faces the intermembrane space.     

Tic22 is targeted to the intermembrane space of chloroplasts by a novel pathway.

Tic22 previously was identified as a component of the general import machinery that functions in the import of nuclear-encoded proteins into the chloroplast. Tic22 is peripherally associated with the outer face of the inner chloroplast envelope membrane, making it the first known resident of the intermembrane space of the envelope. We have investigated the import of Tic22 into isolated chloroplasts to define the requirements for targeting of proteins to the intermembrane space. Tic22 is nuclear-endoded and synthesized as a preprotein with a 50-amino acid N-terminal presequence. The analysis of deletion mutants and chimerical proteins indicates that the precursor of Tic22 (preTic22) presequence is necessary and sufficient for targeting to the intermembrane space. Import of preTic22 was stimulated by ATP and required the presence of protease-sensitive components on the chloroplast surface. PreTic22 import was not competed by an excess of an authentic stromal preprotein, indicating that targeting to the intermembrane space does not involve the general import pathway utilized by stromal preproteins. On the basis of these observations, we conclude that preTic22 is targeted to the intermembrane space of chloroplasts by a novel import pathway that is distinct from known pathways that target proteins to other chloroplast subcompartments.     

Lipid phosphorylation in chloroplast envelopes. Evidence for galactolipid CTP-dependent kinase activities

Lipid phosphorylation takes place within the chloroplast envelope. In addition to phosphatidic acid, phosphatidylinositol phosphate, and their corresponding lyso-derivatives, we found that two novel lipids underwent phosphorylation in envelopes, particularly in the presence of carrier-free [gamma-(32)P]ATP. These two lipids incorporated radioactive phosphate in chloroplasts in the presence of [gamma-(32)P]ATP or [(32)P]P(i) and light. Interestingly, these two lipids were preferentially phosphorylated in envelope membranes in the presence [gamma-(32)P]CTP, as the phosphoryl donor, or [gamma-(32)P]ATP, when supplemented with CDP and nucleoside diphosphate kinase II. The lipid kinase activity involved in this reaction was specifically inhibited in the presence of cytosine 5'-O-(thiotriphosphate) (CTPgammaS) and sensitive to CTP chase, thereby showing that both lipids are phosphorylated by an envelope CTP-dependent lipid kinase. The lipids were identified as phosphorylated galactolipids by using an acid hydrolysis procedure that generated galactose 6-phosphate. CTPgammaS did not affect the import of the small ribulose-bisphosphate carboxylase/oxygenase subunit into chloroplasts, the possible physiological role of this novel CTP-dependent galactolipid kinase activity in the chloroplast envelope is discussed.     

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.     

In situ incorporation of Fatty acids into lipids of the outer and inner envelope membranes of pea chloroplasts

When incubated with [1-¹⁴C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, ¹⁴C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg²⁺-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their ¹⁴C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The ¹⁴C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.     

Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum.

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [gamma-32P]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino-acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali-stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.     

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity

Taking advantage of the unique topology of oxidase assembly 1 (Oxa1) protein, a mitochondrial inner membrane protein with N (intermembrane space)-C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity, and in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial intermembrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a single molecule, can serve as a versatile submitochondrial localization marker that doubles as a membrane integrity marker.     

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.     

Phosphoproteins and protein-kinase activity in isolated envelopes of pea (Pisum sativum L.) chloroplasts

A protein kinase was found in envelope membranes of purified pea (Pisum sativum L.) chloroplasts. Separation of the two envelope membranes showed that most of the enzyme activity was localized in the outer envelope. The kinase was activated by Mg²⁺ and inhibited by ADP and pyrophosphate. It showed no response to changes in pH in the physiological range (pH 7-8) or conventional protein substrates. Up to ten phosphorylated proteins could be detected in the envelope-membrane fraction. The molecular weights of these proteins, as determined by polyacrylamide-gel electrophoresis were: two proteins higher than 145 kDa, 97, 86, 62, 55, 46, 34 and 14 kDa. The 86-kDa band being the most pronounced. Experiments with separated inner and outer envelopes showed that most labeled proteins are also localized in the outer-envelope fraction. The results indicate a major function of the outer envelope in the communication between the chloroplast and the parent cell.     

Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion

Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.     

Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors

Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF-stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+-requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed.