A component of the chloroplast protein import apparatus functions in bacteria

Toc36 is a family of 44-kDa envelope polypeptides previously identified as components of the chloroplast protein import apparatus by virtue of their close physical proximity to translocating proteins. An indication of their function thus remains at large. A heterologous in vivo approach for studying the function of Toc36 was developed in this study by introducing a member of Toc36 into E. coli to assess its effect on bacterial protein translocation. The presence of Toc36 enhances the translocation of two bacterial periplasmic proteins in a manner resembling the chloroplast system. Translocation of the two bacterial periplasmic proteins was less sensitive to sodium azide, resembling more the azide-insensitive nature of the chloroplast protein import process. Mutated Toc36 proteins were not capable of causing the same effect as that observed for unaltered Toc36. Toc36 was also capable of complementing bacterial strains with temperature-sensitive secA mutations that affected protein translocation. The combined results provide evidence that Toc36 plays a central role in the chloroplast protein translocation process.     

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.     

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares approximately 22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.     

The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog

The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems. However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium Synechocystis sp. PCC 6803. We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins. We provide evidence that the cyanobacterial protein is also localized in the outer membrane. From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs. synToc75 is essential, as homozygous null mutants were not recovered after directed mutagenesis. Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known. However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.     

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).     

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.     

Transport of proteins into the various subcompartments of mitochondria

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targeting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targeting pathways to this subcompartment are known to exist.     

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.     

Multiple pathways for protein transport into or across the thylakoid membrane

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.     

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.     

Characterization of the import pathway of the F(A)d subunit of mitochondrial ATP synthase into isolated plant mitochondria

The synthetic precursor of the F(A)d subunit of mitochondrial ATP synthase was imported into isolated soybean cotyledon mitochondria. Import of the F(A)d precursor was accompanied by processing to a lower molecular weight mature form. The F(A)d precursor displayed the following import characteristics not seen before with plant mitochondria: efficient import in the absence of external ATP and import of wheat germ-translated precursor. Pretreatment of the F(A)d precursor with NEM did not inhibit import. Taken together with the lack of a requirement for external ATP, this indicates that this precursor does not require extramitochondrial ATP-dependent factors for import. Binding studies indicated that the F(A)d precursor bound to a proteinaceous component of the mitochondrial outer membrane. Inhibitor studies indicated that processing was most likely via the general mitochondrial processing peptidase. The results suggest that import of this subunit occurs via a pathway different from the general import pathway described for the majority of precursor proteins.     

Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.     

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F1 beta subunit of the ATP synthase (pF1 beta) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondrial than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu2+ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF1 beta with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1 beta into intact mitochondria. Import of pF1 beta through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu2+ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.     

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.     

A 50-picosiemens anion channel of the chloroplast envelope is involved in chloroplast protein import

Single channel recordings were used to investigate the changes on the pea chloroplast envelope during protein import. In the inside-out patch configuration a 50-picosiemens (pS) anion channel of the chloroplast envelope membrane was identified. The open time probability of the channel was decreased by the addition of the wild type precursor protein of ferredoxin (wt-prefd) to the pipette-filling solution in the presence of 0.5 mM ATP. In the absence of ATP or in the presence of 50 microM ATP, wt-prefd did not affect the open time probability of the channel. A deletion mutant of prefd, Delta6-14-prefd, which is inactive in in vitro import, was also unable to affect the open time probability of the 50-pS anion channel. In the presence of 100 microM ATP, wt-prefd decreased the open time probability of the channel to a lesser extent, as did the transit peptide alone. It is concluded that the 50-pS anion channel could be part of the protein import machinery of the inner membrane. In addition the precursor protein under import conditions induced burst-like increases of the envelope conductivity. The implication of both responses for the chloroplast protein import process are discussed.     

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.     

The role of lipids in plastid protein transport

The elaborate compartmentalization of plant cells requires multiple mechanisms of protein targeting and trafficking. In addition to the organelles found in all eukaryotes, the plant cell contains a semi-autonomous organelle, the plastid. The plastid is not only the most active site of protein transport in the cell, but with its three membranes and three aqueous compartments, it also represents the most topologically complex organelle in the cell. The chloroplast contains both a protein import system in the envelope and multiple protein export systems in the thylakoid. Although significant advances have identified several proteinaceous components of the protein import and export apparatuses, the lipids found within plastid membranes are also emerging as important players in the targeting, insertion, and assembly of proteins in plastid membranes. The apparent affinity of chloroplast transit peptides for chloroplast lipids and the tendency for unsaturated MGDG to adopt a hexagonal II phase organization are discussed as possible mechanisms for initiating the binding and/or translocation of precursors to plastid membranes. Other important roles for lipids in plastid biogenesis are addressed, including the spontaneous insertion of proteins into the outer envelope and thylakoid, the role of cubic lipid structures in targeting and assembly of proteins to the prolamellar body, and the repair process of D1 after photoinhibition. The current progress in the identification of the genes and their associated mutations in galactolipid biosynthesis is discussed. Finally, the potential role of plastid-derived tubules in facilitating macromolecular transport between plastids and other cellular organelles is discussed.     

Dual signal peptides mediate the signal recognition particle/Sec-independent insertion of a thylakoid membrane polyprotein, PsbY

The nuclear psbY gene (formerly ycf32) encodes two distinct single-spanning chloroplast thylakoid membrane proteins in Arabidopsis thaliana. After import into the chloroplast, the precursor protein is processed to a polyprotein in which each "mature" protein is preceded by an additional hydrophobic region; we show that these regions function as signal peptides that are cleaved after insertion into the thylakoid membrane. Inhibition of the first or second signal cleavage reaction by enlargement of the -1 residues leads in each case to the accumulation of a thylakoid-integrated intermediate containing three hydrophobic regions after import into chloroplasts; a double mutant is converted to a protein containing all four hydrophobic regions. We propose that the overall insertion process involves (i) insertion as a double-loop structure, (ii) two cleavages by the thylakoidal processing peptidase on the lumenal face of the membrane, and (iii) cleavage by an unknown peptidase on the stromal face on the membrane between the first mature protein and the second signal peptide. We also show that this polyprotein can insert into the thylakoid membrane in the absence of stromal factors, nucleoside triphosphates, or a functional Sec apparatus; this effectively shows for the first time that a multispanning protein can insert posttranslationally without the aid of signal recognition particle, SecA, or the membrane-bound Sec machinery.