Sec-independent insertion of thylakoid membrane proteins. Analysis of insertion forces and identification of a loop intermediate involving the signal peptide

A group of membrane proteins are synthesized with cleavable signal sequences but inserted into the thylakoid membrane by an unusual Sec/SRP-independent mechanism. In this report we describe a key intermediate in the insertion of one such protein, photosystem II subunit W (PSII-W). A single mutation in the terminal cleavage site partially blocks processing and leads to the formation of an intermediate-size protein in the thylakoid membrane during chloroplast import assays. This protein is in the form of a loop structure: the N and C termini are exposed on the stromal face, whereas the cleavage site has been translocated into the lumen. In this respect the insertion of this protein resembles that of M13 procoat, which also adopts a loop structure during insertion, and we present preliminary evidence that a similar mechanism is used by another thylakoid protein, PSII-X. However, whereas the negatively charged region of procoat is translocated by an apparently electrophoretic mechanism using the DeltamuH+, the corresponding region of PSII-W is equally acidic but insertion is DeltamuH+ independent. We furthermore show that neutralization of this region has no apparent effect on the insertion process. We propose that a central element in this insertion mechanism is a loop structure whose formation is driven by hydrophobic interactions.     

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.     

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 signal peptides in targeting of proteins in cyanobacteria

Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen.     

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.     

A mutation in the Escherichia coli secY gene that produces distinct effects on inner membrane protein insertion and protein export

E. coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature. Here we describe experiments designed to explain the conditional phenotype associated with this allele. We found that combining the secY40 mutation with defects in the signal recognition particle targeting pathway led to synthetic lethality. Since the signal recognition particle is required for the insertion of inner membrane proteins (IMPs) into the cytoplasmic membrane but not for protein export, this observation prompted us to examine the effect of the secY40 mutation on IMP biogenesis. The membrane insertion of all IMPs that we tested was impaired at both permissive and nonpermissive temperatures in secY40 cells grown in either rich or minimal medium. The magnitude of the insertion defects was greatest in cells grown at low temperature in rich medium, conditions in which the growth defect was most pronounced. Consistent with previous reports, we could not detect protein export defects in secY40 cells grown in minimal medium. Upon growth in rich medium, only slight protein export defects were observed. Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains. Furthermore, these results provide the first evidence that the protein export and membrane protein insertion functions of the translocon are genetically separable.     

Chloroplast SRP54 interacts with a specific subset of thylakoid precursor proteins

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23- and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.     

Membrane integration of Na,K-ATPase alpha-subunits and beta-subunit assembly

The control of membrane insertion of polytopic proteins is still poorly understood. We carried out in vivo translation/insertion experiments in Xenopus oocytes with combined wild type or mutant membrane segments of the alpha-subunit of the heterodimeric Na, K-ATPase linked to a glycosylation reporter sequence. We confirm that the four N-terminal hydrophobic segments of the alpha-subunit behave as alternating signal anchor/stop transfer motifs necessary for two lipid-inserted membrane pairs. For the six C-terminal membrane segments, however, proper packing depends on specific sequence information and association with the beta-subunit. M5 is a very inefficient signal anchor sequence due to the presence of prolines and polar amino acids. Its correct membrane insertion is probably mediated by posttranslational hairpin formation with M6, which is favored by a proline pair in the connecting loop. M7 has partial signal anchor function, which may be mediated by the presence of glycine and glutamine residues. The formation of a transmembrane M7/M8 pair requires the association of the beta-subunit, which induces a conformational change in the connecting extracytoplasmic loop that favors M7/M8 packing. The formation of the M9/M10 pair appears to be predominantly mediated by the efficient stop transfer function of M10. Mutations that provide signal anchor function to M5, M7, and M9 abolish or impede the transport activity of the enzyme. These data illustrate the importance of specific amino acids near or within hydrophobic regions as well as of subunit oligomerization for correct topographical alignment that is necessary for proper folding and/or activity of oligomeric membrane proteins.     

SUPPRESSOR OF VARIEGATION4,a New var2 Suppressor Locus,Encodes a Pioneer Protein that Is Required for Chloroplast Biogenesis

VAR2 is an integral thylakoid membrane protein and a member of the versatile FtsH class of metalloproteases in prokaryotes and eukaryotes. Recessive mutations in the VAR2 locus give rise to variegated plants (var2) that contain white sectors with abnormal plastids and green sectors with normal-appearing chloroplasts. In a continuing effort to isolate second-site suppressors of var2 variegation,we characterize in this report ems2505,a suppressor strain that has a vi-rescent phenotype due to a missense mutation in At4g28590,the gene for a pioneer protein. We designated this gene SVR4 (for SUPPRESSOR OF VARIEGATI0N4) and the mutant allele in ems2505 as svr4-1. We demonstrate that SVR4 is located in chloroplasts and that svr4-1 single mutants are normal with respect to chloroplast anatomy and thylakoid membrane protein accumulation. However,they are modestly impaired in several aspects of photochemistry and have enhanced non-photochemical quenching (NPQ) capacity. A T-DNA insertion allele of SVR4,svr4-2,is seedling-lethal due to an early blockage of chloroplast development. We conclude that SVR4 is essential for chloroplast biogenesis,and hypothesize that SVR4 mediates some aspect of thylakoid structure or function that controls NPQ. We propose that in the suppressor strain,photoinhibitory pressure caused by a lack of VAR2 is ameliorated early in chloroplast development by enhanced NPQ capacity caused by reduced SVR4 activity. This would result in an increase in the number of chloroplasts that are able to surmount a threshold necessary to avoid photo-damage and thereby develop into functional chloroplasts.     

Energy-transducing thylakoid membranes remain highly impermeable to ions during protein translocation

We investigated the operation of a posttranslational protein translocation pathway to determine whether ions are excluded from the translocase during protein transport. The membrane capacitance during protein translocation across chloroplast thylakoid membranes was monitored via electric-field-indicating carotenoid electrochromic bandshift measurements. Evidence is presented that shows that the membrane ion conductance is not increased during the complete cycle of binding, transport, and substrate release by the DeltapH-dependent translocase; i.e., the membrane remains ion-tight during protein translocation. We further demonstrate that a synthetic targeting peptide that directs proteins across this membrane does not gate translocation pores. We conclude that protein transport across the thylakoid membrane does not compromise its ability to maintain ion gradients and is, thus, unlikely to affect its functions in energy transduction.     

A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and DeltapH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.     

The thylakoid lumen of chloroplasts. Isolation and characterization

The chloroplast compartment enclosed by the thylakoid membrane, the "lumen," is poorly characterized. The major aims of this work were to design a procedure for the isolation of the thylakoid lumen which could be generally used to characterize lumenal proteins. The preparation was a stepwise procedure in which thylakoid membranes were isolated from intact chloroplasts. Loosely associated thylakoid surface proteins were removed, and following Yeda press fragmentation the lumenal content was recovered in the supernatant following centrifugation. The purity and yield of lumenal proteins were determined using appropriate marker proteins specific for the different chloroplast compartments. Quantitative immunoblot analyses showed that the recovery of soluble lumenal proteins was 60-65% (as judged by the presence of plastocyanin), whereas contamination with stromal enzymes was less than 1% (ribulose-bisphosphate carboxylase) and negligible for thylakoid integral membrane proteins (D1 protein). Approximately 25 polypeptides were recovered in the lumenal fraction, of which several were identified for the first time. Enzymatic measurements and/or amino-terminal sequencing revealed the presence of proteolytic activities, violaxanthin de-epoxidase, polyphenol oxidase, peroxidase, as well as a novel prolyl cis/trans-isomerase.     

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.     

Old and new pathways of protein export in chloroplasts and bacteria

Targeting of chloroplast proteins to the thylakoid membrane is analogous to bacterial secretion, and much of what we know has been learned from secretory mechanisms in Escherichia coli. However, chloroplasts also use a delta pH-dependent pathway to target thylakoid proteins, at least some of which are folded before transport. Previously, this pathway seemed to have no cognate in bacteria, but recent results have shown that the HCF106 gene in maize encodes a component of this pathway and has bacterial homologues. This delta pH-dependent pathway might be an ancient conserved mechanism for protein translocation that evolved before the endosymbiotic origin of plastids and mitochondria.     

Topogenesis of a microsomal cytochrome P450 and induction of endoplasmic reticulum membrane proliferation in Saccharomyces cerevisiae

Cytochrome P450 52A3 (P450Cm1) is one of the membrane proteins known to trigger by its high-level expression a marked proliferation of the endoplasmic reticulum, (ER). To gain insight into the relationship between the expression of a membrane protein and the induction of ER proliferation we have characterized the membrane topology of P450Cm1 and identified the structural determinants required for ER targeting, formation of correct membrane orientation, and ER retention. We show that all these features are interrelated and determined by sequence elements within the NH2-terminal region of P450Cm1. Using several approaches--a protease protection assay followed by probing with peptide-specific antibodies, immunolabeling of the intact membrane-bound P450 protein, and expression of fusion proteins in Saccharomyces cerevisiae--membrane topology was defined as follows: residues 2-16 are located in the ER lumen, only the first hydrophobic segment (residues 17-34) spans the membrane, a second hydrophobic segment (48-66) is exposed at the cytoplasmic side, and the remaining part (67-523) forms a large cytosolic domain. Fused to a cytosolic reporter protein, the first 44-amino-acid sequence of P450Cm1 was sufficient to mediate ER targeting, wild-type membrane orientation, and retention in the ER. Similar to wild-type P450Cm1, various fusion proteins were able to induce distinctly organized structures of proliferated ER provided that they were either permanently retained in the ER or accumulated in this compartment due to a delay in further transportation. Thus, we conclude that membrane insertion of the first hydrophobic segment is sufficient to deliver a signal for increased membrane formation.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.     

An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria

Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded in Escherichia coli by an unassigned reading frame, yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-arginine translocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.     

Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2

Cytochrome P4501A1 is a hepatic, microsomal membrane-bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from beta-naphthoflavone-induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30-amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33-44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.     

Formation of the male pronuclear lamina in Drosophila melanogaster

E. coli homologs of the signal recognition particle (SRP) and its receptor are essential for viability, but their role in protein export is unclear. To elucidate their function, we devised a genome-wide screen to identify genes that encode SRP substrates. Inhibition of the SRP pathway sharply blocked the membrane insertion of several polytopic inner membrane proteins (IMPs) that were predicted to be SRP substrates, but had a smaller effect on the insertion of other IMPs and no significant effect on preprotein translocation. Our results suggest that whereas most E. coli preproteins and some IMPs can utilize SRP-independent targeting pathways effectively, the structural features of a subset of IMPs have required the conservation of an SRP-based targeting machinery.