Functional determinants in transit sequences: import and partial maturation by vascular plant chloroplasts of the ribulose-1,5- bisphosphate carboxylase small subunit of Chlamydomonas

The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.     

Chloroplast protein import : quantitative analysis of precursor binding

The first step of chloroplast protein import is binding of a precursor protein to the surface of the organelle. Precursor binding for the small subunit of ribulose-1,5-bisphosphate carboxylase to isolated pea chloroplasts was investigated using a receptor-ligand binding assay. Translocation of precursors was blocked by conducting the binding assays at 0°C. Binding of precursor was judged to be receptor mediated by the following criteria: (a) precursor binding was saturable at between 1500 and 3500 molecules per chloroplast; (b) binding is a high affinity interaction with a dissociation constant of 6 to 10 nanomoles; (c) binding is physiologically productive since most of the bound precursors could be imported from the bound state; and (d) precursor binding was sensitive to both protease and the sulfhydryl modifying reagent N-ethylmaleimide. The effects of these two reagents differed in that protease reduced the total number of binding sites from the surface of chloroplasts but had little effect on binding affinity, whereas N-ethylmaleimide reduced the binding affinity but had little or no effect on receptor density.     

Synthetic analogues of a transit peptide inhibit binding or translocation of chloroplastic precursor proteins

Although amino-terminal transit peptides of chloroplastic precursor proteins are known to be necessary and sufficient for import into chloroplasts, the mechanism by which they mediate this process is not understood. Another important question is whether different precursors share a common transport apparatus. We used 20-residue synthetic peptides corresponding to regions of the transit peptide of the precursor to the small subunit of ribulose bisphosphate carboxylase (prSS) as competitive inhibitors for the binding and translocation of precursor proteins into chloroplasts. Synthetic peptides with sequences corresponding to either end of the transit peptide had little to no effect on binding of prSS to chloroplasts, but significantly inhibited its translocation. Synthetic peptides corresponding to the central region of the transit peptide inhibited binding of prSS to chloroplasts. Each of the peptides inhibited binding or translocation of precursors to light-harvesting chlorophyll a/b protein, ferredoxin, and plastocyanin in the same manner and to a similar extent as prSS transport was inhibited. The results presented in this paper suggest that the central regions of the transit peptide of prSS mediate binding to the chloroplastic surface, whereas the ends of this transit peptide are more important for translocation across the envelope. Furthermore, all of the precursors tested appear to share components in the transport apparatus even though they are sorted to different chloroplastic compartments.     

Uptake and processing of the precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds

Intact leucoplasts from the endosperm of developing castor oil seed were isolated by Percoll density gradient centrifugation. The precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase from pea was synthesized in vitro from hybrid-selected mRNA. Leucoplasts imported this precursor by an ATP-requiring mechanism similar to that described in chloroplasts (AR Grossman et al. 1980 Nature 285: 625-628). The small subunit precursor was processed to a molecular weight that was identical with that of the mature pea small subunit. These results show that leucoplasts, though specialized for fatty acid biosynthesis and not photosynthesis, have a mechanism of protein import similar to that of chloroplasts.     

The N-terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts.

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N-terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino-acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein-mediated system, and purified it by reverse-phase HPLC. Its identity was confirmed by N-terminal amino-acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono- and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5-bisphosphate carboxylase/oxygenase.     

Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope

We have used synthetic peptides representing segments of the signal sequences of preferredoxin (pFd) and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) to study interactions with the signal sequence recognition system at the chloroplast surface. Peptides representing the COOH-terminal 30 amino acids of the pFd and pS signal peptides were able to completely and reversibly inhibit the import of their homologous precursors into isolated chloroplasts at a 2.5 microM concentration. Import was blocked at the level of precursor binding to the chloroplast. This inhibition of precursor binding and import was not due to disruption of chloroplast integrity as incubation of isolated chloroplasts with the peptides did not cause measurable perturbation of the envelope membranes. The peptides also were able to block the import of the heterologous precursor protein, suggesting that pS and pFd share a common signal sequence recognition system. Visualization of the bound peptides at the chloroplast surface by indirect immunofluorescence microscopy using antipeptide antibodies gave a marked punctate staining pattern. This pattern is consistent with the localization of chloroplast import receptor(s) at contact zones between the inner and outer envelope membranes.     

Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts

Precursor forms of chloroplast proteins synthesized in cell-free translation systems can be imported posttranslationally into isolated, intact chloroplasts. Radiochemically pure precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and to the light-harvesting chlorophyll a/b protein have been prepared by in vitro translation of hybrid-selected mRNA and used to study this import process. If chloroplasts are pretreated with the uncoupler nigericin, import does not occur, but the precursors bind to the chloroplast surface. Reincubation of the precursor-chloroplast complex in the presence of ATP results in import of bound precursors. The binding appears to be mediated by proteins of the outer chloroplast envelope membrane because pretreatment of chloroplasts with protease inhibits their ability to bind as well as to import precursors. These results indicate that at least a portion of the observed binding is to functional receptor proteins involved in the import process.     

OEP61 is a chaperone receptor at the plastid outer envelope

Chloroplast precursor proteins encoded in the nucleus depend on their targeting sequences for delivery to chloroplasts. There exist different routes to the chloroplast outer envelope, but a common theme is the involvement of molecular chaperones. Hsp90 (heat-shock protein 90) delivers precursors via its receptor Toc64, which transfers precursors to the core translocase in the outer envelope. In the present paper, we identify an uncharacterized protein in Arabidopsis thaliana OEP61 which shares common features with Toc64, and potentially provides an alternative route to the chloroplasts. Sequence analysis indicates that OEP61 possesses a clamp-type TPR (tetratricopeptide repeat) domain capable of binding molecular chaperones, and a C-terminal TMD (transmembrane domain). Phylogenetic comparisons show sequence similarities between the TPR domain of OEP61 and those of the Toc64 family. Expression of mRNA and protein was detected in all plant tissues, and localization at the chloroplast outer envelope was demonstrated by a combination of microscopy and in vitro import assays. Binding assays show that OEP61 interacts specifically with Hsp70 (heat-shock protein 70) via its TPR clamp domain. Furthermore, OEP61 selectively recognizes chloroplast precursors via their targeting sequences, and a soluble form of OEP61 inhibits chloroplast targeting. We therefore propose that OEP61 is a novel chaperone receptor at the chloroplast outer envelope, mediating Hsp70-dependent protein targeting to chloroplasts.     

Import of proteins into chloroplasts. Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates

Many of the thylakoid membrane proteins of plant and algal chloroplasts are synthesized in the cytosol as soluble, higher molecular weight precursors. These precursors are post-translationally imported into chloroplasts, incorporated into the thylakoids, and proteolytically processed to mature size. In the present study, the process by which precursors are incorporated into thylakoids was reconstituted in chloroplast lysates using the precursor to the light-harvesting chlorophyll a/b protein (preLHCP) as a model. PreLHCP inserted into thylakoid membranes, but not envelope membranes, if ATP was present in the reaction mixture. Correct integration into the bilayer was verified by previously documented criteria. Integration could also be reconstituted with purified thylakoid membranes if reaction mixtures were supplemented with a soluble extract of chloroplasts. Several other thylakoid precursor proteins in addition to preLHCP, but no stromal precursor proteins, were incorporated into thylakoids under the described assay conditions. These results suggest that the observed in vitro activity represents in vivo events during the biogenesis of thylakoid proteins.     

Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.     

The LDL receptor locus in familial hypercholesterolemia: multiple mutations disrupt transport and processing of a membrane receptor

The receptor for low-density lipoprotein (LDL) is synthesized as a 120 kd precursor that undergoes a 40 kd posttranslational increase in apparent molecular weight en route to the cell surface. We describe seven mutations that disrupt synthesis, processing and transport of the receptor in fibroblasts from 77 subjects with the clinical diagnosis of homozygous familial hypercholesterolemia. One mutation obliterates synthesis of immunoprecipitable precursor. Three mutations specify precursors (100, 120 and 135 kd) that fail to undergo normal processing and fail to reach the cell surface. The other three mutations specify precursors (100, 120, and 170 kd) that undergo a normal 40 kd increase in molecular weight and reach the surface, but do not bind LDL normally. Pedigree studies show that each mutation segregates as an allele at the LDL receptor locus. These data imply that signals for transport of receptors from endoplasmic reticulum to the cell surface are contained within the amino acid sequences of the receptors, and that mutations affecting these sequences can disrupt receptor transport.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Toc64, a new component of the protein translocon of chloroplasts

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.     

Preparation of chlamydomonas chloroplasts for the in vitro import of polypeptide precursors

To study the import of polypeptide precursors we have adapted and compared two procedures for the isolation of competent chloroplasts from the green unicellular alga, Chlamydomonas reinhardtii: silicasol gradient centrifugation and elutriation. The chloroplasts actively import the precursor of the small subunit of ribulose bisphosphate carboxylase-oxygenase in vitro.     

Effect of precursor protein phosphorylation on import into isolated chloroplasts from Chlamydomonas.

In higher plants, chloroplast-destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14-3-3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.     

Processing of a wheat light-harvesting chlorophyll a/b protein precursor by a soluble enzyme from higher plant chloroplasts

A processing activity has been identified in higher plant chloroplasts that cleaves the precursor of the light-harvesting chlorophyll a/b- binding protein (LHCP). A wheat LHCP gene previously characterized (Lamppa, G.K., G. Morelli, and N.-H. Chua, 1985. Mol. Cell Biol. 5:1370- 1378) was used to synthesize RNA and subsequently the labeled precursor polypeptide in vitro. Incubation of the LHCP precursors with a soluble extract from lysed chloroplasts, after removal of the thylakoids and membrane vesicles, resulted in the release of a single 25-kD peptide. In contrast, when the LHCP precursors were used in an import reaction with intact pea or wheat chloroplasts, two forms (25 and 26 kD) of mature LHCP were found. The peptide released by the processing activity in the organelle-free assay comigrated with the lower molecular mass form of mature LHCP produced during import. Properties of the processing activity suggest that it is an endopeptidase. Chloroplasts from both pea and wheat, two divergent higher plants, contain the processing enzyme, suggesting its physiological importance in LHCP assembly into the thylakoids. We discuss the implications of LHCP precursor processing by a soluble enzyme that may be in the stromal compartment.     

A constituent of the chloroplast import complex represents a new type of GTP-binding protein

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATR. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [α-³²P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.     

The C terminus of a chloroplast precursor modulates its interaction with the translocation apparatus and PIRAC.

The import of proteins into chloroplasts involves a cleavable, N-terminal targeting sequence known as the transit peptide. Although the transit peptide is both necessary and sufficient to direct precursor import into chloroplasts, the mature domain of some precursors has been shown to modulate targeting and translocation efficiency. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SS-tp), and three C-terminal deletion mutants (Delta52, Delta67, and Delta74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by competitive import of (35)S-prSSU. Both IC(50) and K(i) values consistently suggest that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Non-competitive import studies demonstrated that prSSU and Delta52 were properly processed and accumulated within the chloroplast, whereas Delta67 and Delta74 were rapidly degraded via a plastid-localized protease. The ability of prSSU-derived proteins to induce inactivation of the protein-import-related anion channel was also evaluated. Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not effect the mean duration of channel closure. Possible mechanisms by which C-terminal residues of prSSU modulate chloroplast targeting are discussed.     

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.