Protein Folding in the Plant Cell

Whole-genome sequencing projects have drastically changed the landscape of biological research. The lexicon of contemporary biology contains a plethora of new terms, including: genomics, research pertaining to the genome; proteomics, description of the protein complement of an organism; and bioinformatics, the collection and interpretation of biological information (primarily nucleic acid and amino acid sequence data) (Bouchez and Höfte, 1998). However, obtaining sequence information is not an end unto itself. It is essential that the products of these genes be identified and their function and physiological significance discovered (Bork et al., 1998;Saier, 1998). In complex eukaryotes such as flowering plants, as many as 5 × 104 genes can be selectively expressed in individual cells. It is the products of these genes, the proteins, that determine the fate and function of the cells. Protein function is determined by how the protein folds to form a specific three-dimensional structure. The way that a protein folds is determined by the free energy of the constituent amino acid residues (Levitt et al., 1997). As much as 50% of the primary amino acid sequence is necessary just to define the three-dimensional structure of a typical protein (Dobson et al., 1998). The classic in vitro studies of Anfinsen, which resulted in his receipt of the 1972 Nobel prize in chemistry, demonstrated that the primary amino acid sequence of a protein can contain all of the information necessary to direct the folding of a polypeptide chain to the correct final structure (for review, see Anfinsen, 1973). However, the conditions of temperature and pH, the salt concentration, and especially the total protein concentration found in vivo tend to promote a plethora of side reactions that compete with the single pathway that will lead to the correct final structure. Unfolded and partially folded proteins tend to aggregate when present at the concentrations found in vivo, which are estimated to be as high as 340 mg mL −1 in Escherichia coli. Molecular chaperones, proteins that prevent inappropriate association or aggregation of exposed hydrophobic surfaces of unfolded or partially folded proteins and direct them into productive folding, transport, or degradation pathways, function to minimize protein aggregation and can promote dissociation of aggregates that have formed (Boston et al., 1996; Miernyk, 1997; Netzer and Hartl, 1998; Sigler et al., 1998). Protein-folding catalysts, conventional enzymes that accelerate the rate-limiting steps in protein folding, allowing folding intermediates to avoid aggregation and non-productive interactions with other proteins, also assist cellular proteins to avoid aggregation by accelerating the rate of correct folding (Schmid, 1993; Boston et al., 1996; Huppa and Ploegh, 1998). The molten globule state is an intermediate stage where a protein is “partly unfolded,” and it is thought that proteins are in the molten globule state when recognized by chaperones or for membrane translocation. The Stress70 chaperone machine prevents thermal aggregation of the model protein malate dehydrogenase. Malate dehydrogenase (300 nm) was incubated at 45°C with no chaperone (○), with recombinant Arabidopsis Stress70 (▿), Stress70 plus the chaperone activating protein AtJ2 (▵), or Stress70 plus AtJ2 plus the nucleotide exchange factor AtE1 (⋄). Light scattering, measured at 320 nm, increased as the enzyme was unfolded during heat treatment. Data are the means ± se of three measurements. The 100-kD stress protein is found in all organisms, with the actual size ranging from 84 to 104 kD. There are two major subclasses: class 1 proteins (A, B, C, and D) have two ATP-binding sites, and class 2 proteins (M, N, X, and Y) have a single ATP-binding site (Schirmer et al., 1996). Class 1 Stress100/Clp proteins have a coiled-coil secondary structure separating the ATP-binding domains (Nieto-Sotelo et al., 1999). Transmission electron microscopy of Stress100 reveals ring-shaped particles with a 6-fold rotational symmetry. Side views show two rings stacked together (a dodecamer). The HSP100 proteins have been extensively studied in E. colias subunits of an ATP-dependent protease (caseino-lytic protease [Clp]) (Hoskins et al., 1998). Clp consists of two distinct subunits: ClpP is the actual protease, while the ClpA/B/C/X chaperone subunits designate target specificity. The ClpP sequence is unrelated to ClpA/B/C/X, and ClpP is not a chaperone. Either as subunits of the homomeric ring structure or as subunits of the protease, HSP100/Clp employs ATP hydrolysis to promote changes in protein folding and assembly. Thus, the HSP100/Clp proteins constitute a class of molecular chaperones (Boston et al., 1996; Hoskins et al., 1998). In plant cells HSP100/Clp is both cytoplasmic and organellar (within plastids and/or mitochondria). HSP100/Clp is expressed in developmental and organ-specific patterns and is up-regulated by a variety of environmental stress conditions (heat, cold, high salt, and heavy metals) (Schirmer et al., 1994). Structures of selected molecular chaperones. A, Ribbon diagram derived from the 1.8-Å x-ray structure of the tetragonal form of the N-terminal domain of Saccharomyces cerevisiae Stress90 complexed with the specific inhibitor geldanamycin. This inhibitor binds to the ATP-binding site. B, Ribbon diagram derived from the 1.7-Å x-ray structure of the 44-kD N-terminal ATP-binding domain of bovine Stress70. C, Ribbon diagram derived from the NMR structure of the J-domain of the human DnaJ homolog HdJ1. D through F, Structures of E. coli GroE reconstructed from cryo-electron microscopy. D, GroEL; E, GroEL plus 30 mm ATP; F, GroEL-ATP-GroES (http://bioc09.uthscsa.edu/ approximately seale/chap/em1.html). HSP90 can function independently as a chaperone; however, it also acts in concert with a group of other proteins that together comprise the foldosome, or cytoplasmic chaperone heterocomplex (CCH). The CCH has been studied most extensively in mammalian cells, where it plays an important role in signal transduction via interaction with steroid hormone receptors and protein kinases including the Src and Raf components of the MAP kinase system (Buchner, 1999). HSP90 is associated with at least six partner proteins complexed with the hormone-free receptor. Formation of this complex is essential for subsequent hormone binding. After binding of the ligand and dissociation of the CCH, the activated hormone-bound receptor functions as a transcription factor. In the absence of ligands, the receptors interact with HSP70 to start a new cycle. Two important factors have led to the characterization of CCH function: (a) the CCH can be assembled in vitro from isolated components, and (b) this assembly can be prevented by geldanamycin, a specific inhibitor of Stress90 function (Buchner, 1999). HSP90 partner proteins in the CCH include: Stress70, Hip (HSP70 interacting protein; p48), Hop (HSP70/HSP90 organizing protein; p60 or Sti1p), a DnaJ homolog, the folding catalyst prolyl-isomerase (PPI), and p23 (Sba1p in yeast). An additional component, p50 (Cdc37), has only been detected in complexes of the CCH with protein kinases. Although the CCH has been best characterized in mammalian systems, a complex that is very similar in both composition and function can also be found in both yeast and filamentous fungi (e.g. Brunt et al., 1998). Plant cells also contain a CCH capable of activating the mammalian glucocorticoid receptor in vitro. HSP70, HSP90, and an FKBP-type prolyl-isomerase have been identified as components of the wheat CCH (Reddy et al., 1998). The maize CCH also contains a DnaJ homolog and a cyclophilin-type prolyl-isomerase (J.A. Miernyk, unpublished data). Plant and yeast homologs of the mammalian steroid hormone receptors have not yet been reported, and the native targets for CCH function in these cells are at this time unknown. The 70-kD stress proteins comprise a ubiquitous set of highly conserved molecular chaperones that range in actual size from 68 to 110 kD (Vierling, 1991; Boston et al., 1996; Miernyk, 1997). Some family members are constitutively expressed and are often referred to as HSC70 (70-kD heat shock cognate). Other family members are expressed only when the organism is challenged by environmental stresses such as temperature extremes, anoxia, heavy metals, and predation. These family members are generally referred to as HSP70 (70-kD heat shock protein), even, for example, when they are induced by cold shock. No differences in actual chaperone function have been described between the constitutive and stress-induced proteins, and hereafter they will be discussed collectively as Stress70. Several excellent recent reviews cover the molecular details of stress protein induction in plants (e.g.Schöffl et al., 1998), which are outside the scope of this review. Specific species of the Stress70 proteins are found in all subcellular compartments (Boston et al., 1996; Miernyk, 1997). While plant cytoplasmic Stress70 proteins are more closely related to their mammalian isologs, the mitochondrial and plastidic proteins are more similar to their prokaryotic counterparts. The rough ER luminal resident form of Stress70 is variously referred to as BiP (from early studies on its function as the IgG-binding protein of mammalian cells), GRP78 (78-kD Glc-regulated protein), and in yeast, KAR2 (karyogamy). Schematic presentation of the Stress70 chaperone machine showing the interactions among the central ATP-dependent chaperone, the chaperone-activating protein/DnaJ, and the nucleotide exchange factor/GrpE. The 44-kD N-terminal ATPase domain of Stress70 is indicated as a gray rectangle; the peptide-binding domain is indicated in black. A newly synthesized polypeptide can be recognized and bound by the ATP-ligated form of Stress70 while still nascent or immediately after release from the ribosome. This binary complex is recognized by the chaperone-activating protein (blue trapezoid) that binds and stimulates ATP hydrolysis. The ADP-ligated form of Stress70 has a lower affinity for the polypeptide, which is then released from the machine. If the polypeptide folds incorrectly, it is prone to aggregation. Similarly, a mature correctly folded protein will aggregate when unfolded/denatured. The ADP-ligated form of Stress70 is recognized by the nucleotide exchange factor (orange triangle) that promotes the exchange of ADP for ATP, allowing another cycle by the machine. A typical protein would require several cycles of binding and release before reaching the final correctly folded conformation. The polypeptide chain is not folded by the machine, but rather “held” in a conformation that allows expression of the folding information present in the primary sequence. While both the primary sequence and structural organization of the Stress70 proteins are highly conserved, results from recent studies suggest that the Stress70 reaction cycle might be significantly different in prokaryotes and the eukaryotic cytoplasm. The eubacterial paradigm begins with DnaJ binding to an unfolded peptide and this binary complex subsequently interacts with ATP-ligated DnaK. When ATP occupies the nucleotide site of DnaK, the chaperone is in the open state and can effectively bind the extended peptide (Fig. 2B). DnaJ promotes the hydrolysis of ATP by DnaK, and the concomitant conformational change drives the release of a more structurally organized portion of the polypeptide chain. GrpE subsequently facilitates the exchange of ADP for ATP. This cycle repeats until a stable structure is achieved. In the mammalian Stress70 cycle, the unfolded peptide reacts directly with HSP70 (Fig. 3). Next, the DnaJ homolog binds to the polypeptide/HSP70 complex and stimulates the ATPase activity. The HSP70 complex then adopts the more stable ADP-ligated state. This state is further stabilized by association with Hip, the HSP70 interacting protein. In the absence of a nucleotide exchange factor, the cycle ends with dissociation of of the ATP and release of the polypeptide from the open form of Stress70. In to Hip and which are in the on HSP90, are at least two other proteins that bind to the mammalian Stress70 complex and can chaperone activity. In with the these have been and the also as or is a of Stress70 chaperone activity. the also as or is a is also a stress protein and HSP70 chaperone by dissociation of the The but until model of the mammalian Stress70 chaperone machine a more to with a recent that Hop interacts with and the protein folding of the chaperone proteins that are related to GroE by primary sequence or by nucleotide exchange et al., 1998). This would an of with Hop two structurally unrelated chaperone is the Stress70 chaperone machine in the of plant cells. The HSP70 proteins and DnaJ homologs of plant cells are structurally similar to their or mammalian (Boston et al., 1996; Miernyk, 1997). While GrpE is a nucleotide exchange factor for the prokaryotic Stress70 chaperone machine, it is that the mammalian cytoplasmic machine functions such a A Arabidopsis protein that stimulates exchange by a plant complex has been characterized and Miernyk, unpublished This protein be the has no sequence with Hip, or only other plant component, a Hop homolog has been or Thus, it that the plant cytoplasmic Stress70 chaperone machine has in with both and mammalian and as The comprise the family of molecular chaperones (Boston et al., 1996; Netzer and Hartl, 1998; Sigler et al., 1998). are that a that folding proteins from the cellular (Fig. are two distinct of The group is which in the of and the and plastids of eukaryotic cells, where it is referred to as (Boston et al., 1996). the and the eukaryotic cytoplasmic variously referred to as or ring of both require energy derived from ATP hydrolysis to protein folding, and the of However, details at the molecular et al., 1998). a partner or in while a distinct protein is not of the group In group protein folding, an in which is bound to only acts as the polypeptide (Fig. to the ring not by After polypeptide a of ATP hydrolysis in the ring release of (Fig. After binds to the where the polypeptide is In this form the polypeptide is a in an central ATP then binds to the polypeptide folding (Fig. ATP hydrolysis drives release and allows the polypeptide to the central This cycle is until the polypeptide the final native structure and 1998; Sigler et al., 1998). group are found the plastids and of plant cells, and it was studies of the as it was that the molecular chaperone was and extended et al., The and reaction cycles of the plant mitochondrial are highly to of The reaction cycle of the is also to that of the prokaryotic counterparts. There however, structural contain two distinct of GroEL and It are distinct and in vivo or are of of and The of distinct subunits distinct and distinct protein target however, this has not yet been of that the protein was at kD. The an open that consists of two to this (Boston et al., 1996). It has been that the two of the protein might different in The form of is found only in eukaryotes (Boston et al., 1996). It is that is of the that the after the that to In mammalian cells, a structure that is very similar in to The is different subunits have been characterized has been best characterized as a chaperone for and in mammalian cells (Boston et al., 1996; Sigler et al., 1998). In to the organellar is in plant cells. The (e.g. or has been from Arabidopsis and and the amino acid are similar to of their mammalian and yeast counterparts. In plant cells the subunits are of and have not yet been of specific chaperone activity. The heat shock proteins are ubiquitous among and a important class of molecular chaperones in plant cells (Vierling, The a conserved domain with the mammalian that in vivo the are found not as in complexes of to kD. the of species of they to as rather as This different functions for the different when present in the cellular In the can both the of proteins and prevent protein aggregation (Boston et al., 1996). on these it is that the can also in vivo as a of molecular chaperone. In to members of the other of molecular chaperones, the of the is ATP In plants, six have been family proteins distinct cellular including the rough and et al., 1996). specific are expressed during of plant (Boston et al., 1996). is a rough chaperone that is also a protein et al., 1997; and 1998). is a membrane protein with a cytoplasmic domain and a rough ER to the is the a sequence that in and chaperone binds ATP, no ATPase has been reported, and ATP binding promotes a to et al., 1997). interacts with a range of newly synthesized proteins, then from these proteins folding to or further assembly et al., 1997). Thus, is a of the rough ER system that allows only proteins that have a final native conformation to on through the pathway (Boston et al., 1996). is a protein of the rough is a protein et al., 1998). has been found associated with other proteins in the rough ER and has similar and target to that of and in the rough ER as chaperones in the folding of proteins et al., 1997; and 1998). of the with the of the rough ER and Ploegh, 1998), protein folding is a complex molecular chaperones and folding of the interactions in for proteins to the correct native structure. While binds to all folding with the species immediately to or with et al., 1997). Thus, and binding by to interaction with the rough folding and promote correct protein folding in the rough ER by and and degradation of or folded In to molecular chaperones, the folding are conventional Protein-folding accelerate the reactions in protein folding that might be rate the of the rate-limiting cellular proteins would be in intermediate and most folding intermediates are to aggregation and to non-productive interactions with other proteins (Dobson et al., 1998). The two are protein and Protein is a of the of proteins, and contains two of the Protein facilitates folding through its to or in the of an or such as and Ploegh, 1998). by the to the most stable et al., In eukaryotic cells, is the of the rough This is a for protein folding in of pH, and high concentrations and Ploegh, 1998; and 1998). to the on are by the by In to the enzyme are variously referred to and and 1997). The from the of to bind The are that bind the A, while are that bind the the in protein folding by accelerating the of peptide can be the rate-limiting in protein folding, and this reaction is by (Schmid, After the correct form of is stabilized by the polypeptide secondary or structure. While most proteins are in the are present the rough the and the mitochondrial (Boston et al., 1996; et al., 1999). The between chaperones and folding have in The folding bind to their target proteins, and in in vitro it has been that this binding can prevent aggregation. the folding also be has been to essential residues in or and the proteins still chaperone in vitro. However, such results be with it was that a form of that no in vitro with an when with an unfolded polypeptide as the et al., 1997). The between chaperones and folding that the accelerate the rate of target polypeptide The of proteins in the their It is important to which molecular chaperones and folding assist the newly synthesized cytoplasmic proteins to their final native state. This is a in plant cells that are complex in their of cytoplasmic chaperones. Some proteins, especially that are and are to in the or only with the of a folding Other proteins are recognized by the Stress70 chaperone machine and bound while still There are three for this the polypeptide might to the correct final structure and be released from the chaperone, it might be in an unfolded conformation until to the protein associated with an organellar or the polypeptide might be to a different chaperone for further folding, or the other cytoplasmic chaperones, the the CCH, and the are the best the most the actual native target proteins for of the chaperone The of the or as it is referred is to no kD and Hartl, 1998; Sigler et al., 1998). A structural model of the CCH has not yet been however, in the HSP70 and HSP90 components are capable of the folding of proteins kD. In mammalian cells the only CCH target identified to are receptors and protein kinases and 1997). The are thought to bind target on the surfaces and be capable of in the folding of size protein (Vierling, 1991; Boston et al., 1996). the most HSP100/Clp are thought to the of proteins (Schirmer et al., 1996; Hoskins et al., 1998), but a role in the of protein folding be Protein by HSP100/Clp is by the Stress70 machine and/or The of the interactions has not yet been While protein folding is generally to be is in vitro for between chaperones and Hartl, 1998). are in which more two of the chaperone to be in the folding of a single target protein the protein has been during its to be associated with HSP70, the and Molecular chaperones have a interaction with proteins to the ER or of other of the These proteins are into a in the ER membrane that is with the and 1999). in a luminal The and for polypeptide into the ER are by the resident Stress70 chaperone, which acts as a et al., 1999). BiP interact with the polypeptide to minimize The polypeptide complex interacts with the J-domain of an membrane protein in which the ATPase function of the chaperone 1997). the ER polypeptide folding begins As in the non-productive folding is by interaction with the Stress70 chaperone machine and 1998). BiP is essential for protein and folding in the ER et al., In to variously interacting with the specific ER homologs of Stress70 and the found the rough ER and Ploegh, has to the and chaperones and the catalyst et al., 1997; et al., 1998; and 1998; and 1998). While all of the Stress70 proteins are to (e.g. et al., BiP is in that it is (Boston et al., 1996). Specific have not been for of the however, it has been that the of and with physiological state or in functions et al., organellar proteins are and synthesized as in the cytoplasm. As they will interact with cytoplasmic chaperones to membrane translocation. After membrane protein folding other subcellular of a plant similar to in the or and specific of HSP70, HSP90, the and the have been found in and (Boston et al., 1996; et al., 1996; and 1997; Miernyk, 1997). have to an of the by several of the chaperones in protein a presentation is by in two by and by is a chaperone for proteins that will be by is a mammalian ATP-dependent protein that chaperones mitochondrial from yeast and from are chaperones for and and chaperone in This but the of these chaperones is that no homolog has yet been identified in Thus, it that many different molecular chaperones most of the early studies on molecular chaperones and folding have been in mammalian and and model target It is important that the between these studies and actual in vivo then can be to have the of the A in this has been et While this system employs a model protein as the folding and the of molecular chaperones in this can be studied in vivo in further to and the targets of specific in vivo a to the of plant to and in the of protein folding in plant cells, and E. for information to and in for their was in the of the