Alternative Processing of Arabidopsis Hsp70 Precursors during Protein Import into Chloroplasts

During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.     

Transport of proteins into chloroplasts. Import and maturation of precursors to the 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex

The 33-, 23-, and 16-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized as precursors in the cytoplasm and transported into the thylakoid lumen of higher plant chloroplasts. In this report we have analyzed the import and maturation of these precursors, using reconstituted protein import assays and partially purified preparations of the processing peptidases involved. Precursors of the 33- and 23-kDa proteins from Spinacia and Triticum aestivum are processed by a stromal peptidase to intermediate forms; polypeptides of similar size are observed during the transport of these precursors and possibly that of the 16-kDa protein, into isolated chloroplasts. Complete maturation of the 33- and 23-kDa proteins is carried out by a thylakoidal peptidase shown previously to be involved in plastocyanin biogenesis. The data support an import mechanism involving successive cleavages by the stromal and thylakoidal processing peptidases.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

The Hsp90 family of proteins in Arabidopsis thaliana

The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.     

A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.     

Mutations in the processing site of the precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit: effects on import,processing, assembly and stability

The small subunit (SSU) of Rubisco is synthesized in the cytosol in a precursor form. Upon import into the chloroplast, it is proteolytically processed at a Cys-Met bond to yield the mature form of the protein. To assess the importance of the Met residue for recognition and processing by the stromal peptidase, we substituted this residue with either Thr, Arg or Asp. The mutant precursor proteins were imported into isolated chloroplasts, and the products of the import reactions were analyzed. Mutants containing Thr or Arg residues at the putative processing site were processed to a single peptide, comigrating with the wild-type protein. N-terminal radio-sequencing revealed that these mutants were processed at the Cys-Thr and the Cys-Arg bond, respectively. After import of the Asp-containing mutant, four processed forms of the protein were observed. Analysis of the most abundant one, co-migrating with the wild-type protein, demonstrated that this species was also a product of correct processing, at the Cys-Asp bond. All the correctly processed peptides were found to be associated with the holoenzyme of Rubisco, and remained stable within the chloroplast, like the wild-type protein. The results of this study, together with previous ones, suggest that proper recognition and processing of the SSU precursor are more affected by residues N-terminal to the processing site than by the residue on the C-terminal side of this site.     

Evolution of Chloroplast J Proteins

Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.     

ATP Requirement for Chloroplast Protein Import Is Set by the Km for ATP Hydrolysis of Stromal Hsp70 in Physcomitrella patens

The 70-kD family of heat shock proteins (Hsp70s) is involved in a number of seemingly disparate cellular functions, including folding of nascent proteins, breakup of misfolded protein aggregates, and translocation of proteins across membranes. They act through the binding and release of substrate proteins, accompanied by hydrolysis of ATP. Chloroplast stromal Hsp70 plays a crucial role in the import of proteins into plastids. Mutations of an ATP binding domain Thr were previously reported to result in an increase in the K_m for ATP and a decrease in the enzyme’s k_cat. To ask which chloroplast stromal chaperone, Hsp70 or Hsp93, both of which are ATPases, dominates the energetics of the motor responsible for protein import, we made transgenic moss (Physcomitrella patens) harboring the K_m-altering mutation in the essential stromal Hsp70-2 and measured the effect on the amount of ATP required for protein import into chloroplasts. Here, we report that increasing the K_m for ATP hydrolysis of Hsp70 translated into an increased K_m for ATP usage by chloroplasts for protein import. This thus directly demonstrates that the ATP-derived energy long known to be required for chloroplast protein import is delivered via the Hsp70 chaperones and that the chaperone’s ATPase activity dominates the energetics of the reaction.     

Pea Chloroplast DnaJ-J8 and Toc12 Are Encoded by the Same Gene and Localized in the Stroma

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as higher molecular mass precursors with an N-terminal extension known as the transit peptide. Precursor proteins are imported into chloroplasts through a translocon complex located at the chloroplast envelope. Translocon components associated with the outer membrane are called Toc (for translocon of the outer envelope membrane of chloroplast) proteins, and those associated with the inner membrane are called Tic (for translocon of the inner envelope membrane of chloroplast) proteins. Cleavage of the transit peptide from the precursor by a specific stromal processing peptidase during translocation results in the production of the lower molecular mass mature protein. Various translocon components have been assigned functions in the basic steps of the import process (for review, see Inaba and Schnell, 2008; Jarvis, 2008; Li and Chiu, 2010). For example, Toc159 (the no. indicates the calculated molecular mass of the protein) and Toc34 are receptors for the transit peptides, and Toc75 is the protein-translocating channel across the outer membrane. Toc64, on the other hand, has a dual function: it serves as a docking site for the cytosolic Hsp90 through its cytosolic domain and as a scaffold for translocon components located in the intermembrane space through its intermembrane space domain (Qbadou et al., 2007).Protein import into chloroplasts involves at least two distinct ATP-consuming steps. The first step is called “early import intermediate” or “docking,” in which less than 100 μm ATP is required and precursors are translocated across the outer membrane and come into contact with translocon components in the inner membrane (Olsen et al., 1989; Kouranov and Schnell, 1997; Inaba et al., 2003; Inoue and Akita, 2008). It has been shown that the ATP is used in the intermembrane space (Olsen and Keegstra, 1992), most likely by a yet unidentified intermembrane space Hsp70 called imsHsp70 or Hsp70-IAP (ims for “intermembrane space” and IAP for “import intermediate-associated protein”; Marshall et al., 1990; Schnell et al., 1994; Qbadou et al., 2007). The second ATP-consuming step is the complete translocation of precursors across the two envelope membranes into the stroma. This step requires about 1 mm ATP. The ATP is most likely used by the stromal Hsp93 and chloroplast Hsc70 associated with the translocon to drive protein translocation into the stroma (Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010).Hsp70 family proteins are involved in many cellular processes, including protein folding, protein translocation across membranes, and regulation of protein degradation. Hsp70 proteins are often recruited to perform a certain function by specifically localized J domain-containing proteins. The J domain-containing proteins interact with Hsp70 when Hsp70 is bound to ATP and stimulate ATP hydrolysis by Hsp70. The specific J domain-containing cochaperone that recruits the stromal chloroplast Hsc70 to the inner envelope membrane to assist in protein translocation has not been identified. The specific J domain-containing cochaperone for imsHsp70 for its function in protein import into chloroplasts is proposed to be a protein named Toc12 (Becker et al., 2004).Toc12 was identified as a novel J domain-containing protein from pea (Pisum sativum) chloroplasts. It belongs to the type III J domain proteins containing only the J domain without the Gly- and Phe-rich domain (G/F domain) and the zinc-finger domain originally found in Escherichia coli DnaJ. It has been shown that the protein is synthesized at its mature size of 103 amino acids without a cleavable transit peptide. After import, the protein has been shown to anchor in the outer membrane by its N-terminal part, which has been suggested to form a β-barrel-type domain. Its C-terminal part, composed of the J domain, has been shown to localize in the intermembrane space. Toc12 has been shown to associate with imsHsp70. Toc12 and imsHsp70 interact with the intermembrane space domain of Toc64, which in turn associates with another intermembrane space translocon component, Tic22. It is proposed that the Toc12-imsHsp70-Toc64-Tic22 complex mediates protein translocation across the intermembrane space through specific precursor binding and ATP hydrolysis (Becker et al., 2004; Qbadou et al., 2007). However, the existence of imsHsp70 has only been shown on immunoblots by its reactivity to the monoclonal antibody SPA820 raised against human Hsp70. Its encoding gene has never been identified. The Arabidopsis (Arabidopsis thaliana) Hsp70 gene family has 14 members. Only two of them are localized in chloroplasts, and both have been shown to locate in the stroma (Ratnayake et al., 2008; Su and Li, 2008). A recent study has further shown that the major protein recognized by the SPA820 antibody in pea chloroplasts is located in the stroma, indicating that imsHsp70 is most likely a stromal protein (Ratnayake et al., 2008).Most translocon components were originally identified from pea chloroplasts. While all translocon components identified from pea have easily recognizable Arabidopsis homologs, Toc12 seems to be an exception. The Arabidopsis gene suggested to be the pea TOC12 homolog, At1g80920 (Inoue, 2007; Jarvis, 2008), encodes a protein that is much larger than pea Toc12 and is annotated as J8 (referred to as AtJ8 herein). The entire pea Toc12 has a high sequence similarity to the N-terminal two-thirds of AtJ8. AtJ8 contains an extra C-terminal domain of 60 amino acids that is highly conserved among J8 proteins from other higher plants. However, in contrast to pea Toc12, AtJ8 is predicted to locate in the stroma (Miernyk, 2001; www.arabidopsis.org). Indeed, a fusion protein consisting of the first 80 amino acids of AtJ8 fused at the N terminus of GFP was imported into the chloroplast stroma, and approximately 46 amino acids from the N terminus were processed after import (Lee et al., 2008), indicating that the first 46 amino acids of AtJ8 function as a cleavable stroma-targeting transit peptide. A T-DNA insertion in the AtJ8 gene that causes the truncation of the last three amino acids results in no visible phenotype. However, detailed analyses indicate that the mutant has lower CO₂ assimilation and Rubisco activity than the wild type (Chen et al., 2010).We are interested in identifying J domain-containing proteins interacting with stromal Hsp70. As part of the initial effort, we investigated the suborganellar location of J8 and examined the relationship between Toc12 and J8. We found that, in pea, there are at least two genes encoding J8, which we named PsJ8a and PsJ8b. TOC12 represents part of PsJ8b. Toc12, AtJ8, and the two PsJ8 proteins could be imported into chloroplasts and processed to stromally localized soluble mature proteins. Four alleles of AtJ8 mutants were analyzed, but none of them showed any defect in the import of various chloroplast precursor proteins.     

Precursors with altered affinity for Hsp70 in their transit peptides are efficiently imported into chloroplasts

Protein import into chloroplasts is postulated to occur with the involvement of molecular chaperones. We have determined that the transit peptide of ferredoxin-NADP(H) reductase precursor binds preferentially to an Hsp70 from chloroplast stroma. To investigate the role of Hsp70 molecular chaperones in chloroplast protein import, we analyzed the import into pea chloroplasts of preproteins with decreased Hsp70 binding affinity in their transit peptides. Our results indicate that the precursor with the lowest affinity for Hsp70 molecular chaperones in its transit peptide was imported to chloroplasts with similar apparent Km as the wild type precursor and a 2-fold increase in Vmax. Thus, a strong interaction between chloroplast stromal Hsp70 and the transit peptide seems not to be essential for protein import. These results indicate that in chloroplasts the main unfolding force during protein import may be applied by molecular chaperones other than Hsp70s. Although stromal Hsp70s undoubtedly participate in chloroplast biogenesis, the role of these molecular chaperones in chloroplast protein translocation differs from the one proposed in the mechanisms postulated up to date.     

Molecular chaperones involved in chloroplast protein import.

Transport of cytoplasmically synthesized precursor proteins into chloroplasts, like the protein transport systems of mitochondria and the endoplasmic reticulum, appears to require the action of molecular chaperones. These molecules are likely to be the sites of the ATP hydrolysis required for precursor proteins to bind to and be translocated across the two membranes of the chloroplast envelope. Over the past decade, several different chaperones have been identified, based mainly on their association with precursor proteins and/or components of the chloroplast import complex, as putative factors mediating chloroplast protein import. These factors include cytoplasmic, chloroplast envelope-associated and stromal members of the Hsp70 family of chaperones, as well as stromal Hsp100 and Hsp60 chaperones and a cytoplasmic 14-3-3 protein. While many of the findings regarding the action of chaperones during chloroplast protein import parallel those seen for mitochondrial and endoplasmic reticulum protein transport, the chloroplast import system also has unique aspects, including its hypothesized use of an Hsp100 chaperone to drive translocation into the organelle interior. Many questions concerning the specific functions of chaperones during protein import into chloroplasts still remain that future studies, both biochemical and genetic, will need to address.     

A Stromal Heat Shock Protein 70 System Functions in Protein Import into Chloroplasts in the Moss Physcomitrella patens

Heat shock protein 70s (Hsp70s) are encoded by a multigene family and are located in different cellular compartments. They have broad-ranging functions, including involvement in protein trafficking, prevention of protein aggregation, and assistance in protein folding. Hsp70s work together with their cochaperones, J domain proteins and nucleotide exchange factors (e.g., GrpEs), in a functional cycle of substrate binding and release accompanied by ATP hydrolysis. We have taken advantage of the gene targeting capability of the moss Physcomitrella patens to investigate the functions of chloroplast Hsp70s. We identified four Hsp70 genes and two GrpE cochaperone homolog genes (CGE) in moss that encode chloroplast proteins. Disruption of one of the Hsp70 genes, that for Hsp70-2, caused lethality, and protein import into heat-shocked chloroplasts isolated from temperature-sensitive hsp70-2 mutants was appreciably impaired. Whereas the double cge null mutant was not viable, we recovered a cge1 null/cge2 knock down mutant in which Hsp70-2 was upregulated. Chloroplasts isolated from this mutant demonstrated a defect in protein import. In addition, two different precursors staged as early import intermediates could be immunoprecipitated with an Hsp70-2–specific antibody. This immunoprecipitate also contained Hsp93 and Tic40, indicating that it represents a precursor still in the Toc/Tic translocon. Together, these data indicate that a stromal Hsp70 system plays a crucial role in protein import into chloroplasts.     

Toward a unified model of the action of CLP/HSP100 chaperones in chloroplasts

In chloroplasts, Hsp70 and Hsp100 chaperones have been long suspected to be the motors that provide the necessary energy for the import of precursor proteins destined to the organelle. The chaperones associate with the import translocon and meet the transit peptides as they emerge through the channel. After decades of active research, recent findings demonstrated that Hsp100 chaperones recognize transit peptides both in vitro and in vivo. Moreover, Hsp70 also plays a part in precursor import. The updated model of protein translocation into chloroplasts now presents new questions about the role of the chaperones in the process.     

Protein targeting across the three membranes of the Euglena chloroplast envelope.

A system has been developed for the import in vitro of precursor proteins into Euglena chloroplasts, which have three envelope membranes. Preparation of functional chloroplasts with intact envelope membranes has been optimized. Import of the precursor (50 kDa) for the tetrapyrrole biosynthesis enzyme porphobilinogen deaminase (PBGD), and processing to the mature size (40 kDa), occurred at 25 degrees C in the light and the presence of ATP, with an estimated efficiency of 62%. Pretreatment of the chloroplasts with proteases abolished this import, suggesting the involvement of specific protein receptors. The presequence of PBGD was found to be cleaved by Escherichia coli leader peptidase to an intermediate form (46 kDa). A construct in which the first 30 residues of the presequence (presumed to be the region removed by leader peptidase) had been deleted was no longer imported. Neither prePBGD nor the truncated precursor were imported into pea chloroplasts, although both bound to the pea chloroplast envelope. Conversely, a chimeric construct, in which the mature PBGD protein was fused downstream of the transit peptide for pea ferredoxin-NADP reductase, was efficiently imported into pea chloroplasts and processed to the mature size. However, this was not imported into Euglena chloroplasts, although again it bound to them. These results provide preliminary evidence for the possibility of two functional domains within the Euglena PBGD presequence. The implications of these findings with respect to the evolution of Euglena chloroplasts are discussed.     

Import, targeting, and processing of a plant polyphenol oxidase.

A tomato (Lycopersicon esculentum L.) gene encoding a precursor of polyphenol oxidase (PPO) was transcribed and translated in vitro. The import, targeting, and processing of the [35S]methionine-labeled precursor protein (pPPO) were studied in isolated chloroplasts. The protein was routed to the thylakoid lumen in two steps. The 67-kD precursor was first imported into the stroma in an ATP-dependent step. It was processed to a 62-kD intermediate by a stromal peptidase. Translocation into the lumen was light dependent and involved processing of the 62-kD to the 59-kD mature form. The mature polypeptide was soluble in the lumen and not bound to thylakoids. This two-step targeting pattern was observed in plastids from a variety of plants including pea (Pisum sativum L.), tomato, and maize (Zea mays L.). The ratio between the intermediate and mature forms observed depended on the plant species, leaf age, growth conditions, and illumination regime to which the plants had been subjected. Cu2+ was not required for pPPO import or processing. Furthermore, low concentrations of Cu2+ (1-5 microM) markedly inhibited the first import step. Tentoxin specifically inhibited pPPO import, leaving the precursor bound to the envelope membrane. The two-step routing of pPPO into chloroplasts, typical of thylakoid lumen proteins, is consistent with the two-domain structure of the transit peptide and appears to be a feature of all plant PPO genes isolated so far. No evidence was found for unorthodox routing mechanisms, which have been suggested to be involved in the import of plant PPOs. The two-step routing may account for some of the multiplicity of PPO observed in vivo.     

Transport of proteins into chloroplasts. Requirements for the efficient import of two lumenal oxygen-evolving complex proteins into isolated thylakoids

The 33- and 23-kDa proteins of the photosynthetic oxygen-evolving complex are synthesized in the cytosol and targeted into the thylakoid lumen by bipartite presequences. In this report, we describe conditions for the efficient import of each of these proteins by isolated pea thylakoids. Import of the 33-kDa protein requires both light and stromal extract. The probable function of the stromal extract is to provide stromal processing peptidase to remove the first "envelope transit" signal of the presequence. Import of the 23-kDa protein is also driven by light, but stromal extract is not required for import; furthermore, efficient import is still observed if the precursor is modified to completely block cleavage by residual stromal processing peptidase activity. The intermediate form of the 23-kDa protein, which is generated by incubation of the precursor protein with stromal processing peptidase, is also efficiently imported. The results indicate that the thylakoidal protein transport system can import both the precursor and intermediate forms of the 23-kDa protein, but probably only the intermediate form of the 33-kDa protein.