Essential function in chloroplast recognition of the ferredoxin transit peptide processing region

Summary Plant ferredoxin is a nuclear-encoded chloroplast protein that is synthesized in the cytoplasm as a transit peptide-containing precursor molecule. To identify functional regions in the pre-ferredoxin transit peptide we constructed mutants with deletions of increasing length from the processing site toward the amino-terminus of the precursor. The mutant proteins were tested in an in vitro chloroplast binding and import assay. Deletion of the amino acids adjacent to the processing site completely abolishes binding and import. This region contains a sequence motif that is conserved among different precursor species. By constructing and testing mutants in the amino-terminal region of the mature part of the precursor protein, we found that this region of the molecule can greatly influence the import reaction.     

Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.     

Non-canonical transit peptide for import into the chloroplast

The large majority of plastid proteins are nuclear-encoded and, thus, must be imported within these organelles. Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region. In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope. Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane. Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein. These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein. These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.     

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

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

Determinants for removal and degradation of transit peptides of chloroplast precursor proteins

The stromal processing peptidase (SPP) cleaves a large diversity of chloroplast precursor proteins, removing an N-terminal transit peptide. We predicted previously that this key step of the import pathway is mediated by features of the transit peptide that determine precursor binding and cleavage followed by transit peptide conversion to a degradable substrate. Here we performed competition experiments using synthesized oligopeptides of the transit peptide of ferredoxin precursor to investigate the mechanism of these processes. We found that binding and processing of ferredoxin precursor depend on specific interactions of SPP with the region consisting of the C-terminal 12 residues of the transit peptide. Analysis of four other precursors suggests that processing depends on the same region, although their transit peptides are highly divergent in primary sequence and length. Upon processing, SPP terminates its interaction with the transit peptide by a second cleavage, converting it to a subfragment form. From the competition experiments we deduce that SPP releases a subfragment consisting of the transit peptide without its original C terminus. Interestingly, examination of the ATP-dependent metallopeptidase activity responsible for degradation of transit peptide subfragments suggests that it may recognize other unrelated peptides and, hence, act separately from SPP as a novel stromal oligopeptidase.     

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins

The regulated turnover of endoplasmic reticulum (ER)–resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture–based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover.     

Biophysical characterization of a transit peptide directing chloroplast protein import.

We have investigated the biophysical properties of a 35 amino acid peptide representing the entire length of a chloroplastic targeting sequence. The peptide, termed gamma-tp, corresponds in sequence to the transit peptide of the gamma subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii. We found that gamma-tp blocks the import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase into isolated pea chloroplasts (KI approximately 5 microM), suggesting that it interacts with higher plant plastids in a physiological manner. We also found the gamma-tp to have a high affinity for nonpolar environments, but not to cause a general disruption of membrane integrity. Hydrophobic moment analysis suggests that the gamma-tp can adopt an amphipathic beta structure. However, circular dichroism measurements indicate that the peptide is largely a random coil, in both the presence and absence of sodium laurylsulfate micelles. In the absence of a recognizable secondary structural targeting motif, we asked whether the presence of a transit peptide on a chloroplast protein increases the protein's overall affinity for nonpolar environments. Phase-partition experiments with Triton X-114 suggest that this is not the case. These results are discussed in relation to the mechanism of protein targeting to chloroplasts.     

atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203-207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1-13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93-105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.     

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

In vivo and in vitro interaction of DnaK and a chloroplast transit peptide

Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S-SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide-dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.     

Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.     

Signal peptide peptidase cleavage of GB virus B core protein is required for productive infection in vivo

Chronic infection by hepatitis C virus (HCV) is a leading cause of liver disease for which better therapies are urgently needed. Because a clearer understanding of the viral life cycle may suggest novel anti-viral approaches, we studied the role of host signal peptide peptidase (SPP) in viral infection. This intramembrane protease cleaves within a C-terminal signal sequence in the viral core protein, but the molecular determinants of cleavage and whether it is required for infection in vivo are unknown. To answer these questions, we studied SPP processing in GB virus B (GBV-B) infection. GBV-B is the closest phylogenetic relative of HCV and offers an accurate surrogate model for HCV infection. We demonstrate that SPP also processes GBV-B core protein and that a serine residue in the hydrophobic region of the signal sequence (present also in HCV) is critical for efficient SPP cleavage. The small size of the serine side chain combined with its ability to form intra- and interhelical hydrogen bonds likely contributes to recognition of the signal sequence as a substrate for SPP. By introducing mutations with differing effects on SPP processing into an infectious GBV-B molecular clone, we demonstrate that SPP processing of the core protein is required for productive infection in primates. These results broaden our understanding of the mechanism and requirements for SPP cleavage and reveal a functional role in vivo for intramembrane proteolysis in host-pathogen interactions. Moreover, they identify SPP as a potential therapeutic target for reducing the impact of HCV infection.     

Maturation of hepatitis C virus core protein by signal peptide peptidase is required for virus production

Complete maturation of hepatitis C virus (HCV) core protein requires coordinate cleavage by signal peptidase and an intramembrane protease, signal peptide peptidase. We show that reducing the intracellular levels of signal peptide peptidase lowers the titer of infectious virus released from cells, indicating that it plays an important role in virus production. Proteolysis by the enzyme at a signal peptide between core and the E1 glycoprotein is needed to permit targeting of core to lipid droplets. From mutagenesis studies, introducing mutations into the core-E1 signal peptide delayed the appearance of signal peptide peptidase-processed core until between 48 and 72 h after the beginning of the infectious cycle. Accumulation of mature core at these times coincided with its localization to lipid droplets and a rise in titer of infectious HCV. Therefore, processing of core by signal peptide peptidase is a critical event in the virus life cycle. To study the stage in virus production that may be blocked by interfering with intramembrane cleavage of core, we examined the distribution of viral RNA in cells harboring the core-E1 signal peptide mutant. Results revealed that colocalization of core with HCV RNA required processing of the protein by signal peptide peptidase. Our findings provide new insights into the sequence requirements for proteolysis by signal peptide peptidase. Moreover, they offer compelling evidence for a function for an intramembrane protease to facilitate the association of core with viral genomes, thereby creating putative sites for assembly of nascent virus particles.     

Amino-terminal and hydrophobic regions of the Chlamydomonas reinhardtii plastocyanin transit peptide are required for efficient protein accumulation in vivo

Nucleus-encoded chloroplast proteins of vascular plants are synthesized as precursors and targeted to the chloroplast by stroma-targeting domains in N-terminal transit peptides. Transit peptides in Chlamydomonas reinhardtii are considerably shorter than those in vascular plants, and their stroma-targeting domains have similarities to both mitochondrial and chloroplast targeting sequences. To examine Chlamydomonas transit peptide function in vivo, deletions were introduced into the transit peptide coding region of the petE gene, which encodes the thylakoid lumen protein plastocyanin (PC). The mutant petE genes were introduced into a plastocyanin-deficient Chlamydomonas strain, and transformants that accumulated petE mRNA were analyzed for PC accumulation. The most profound defects were observed with deletions at the N-terminus and those that extended into the hydrophobic region in the C-terminal half of the transit peptide. PC precursors were detected among pulse-labeled proteins in transformants with N-terminal deletions, suggesting that these precursors cannot be imported and are degraded in the cytosol. Intermediate PC species were observed in a transformant deleted for part of the hydrophobic region, suggesting that this protein is defective in lumen translocation and/or processing. Thus, despite its shorter length, the bipartite nature of the Chlamydomonas PC transit peptide appears similar to that of lumen-targeted proteins in vascular plants. Analysis of the synthesis, stability, and accumulation of PC species in transformants bearing deletions in the stroma-targeting domain suggests that specific regions probably have distinct roles in vivo. Abbreviations: cyt, cytochrome; ECL, enhanced chemiluminescence; LSU, large subunit; PC, plastocyanin; TP, transit peptide     

SEC11 is required for signal peptide processing and yeast cell growth

Among the collection of temperature-sensitive secretion mutants of Saccharomyces cerevisiae, sec11 mutant cells are uniquely defective in signal peptide processing of at least two different secretory proteins. At 37 degrees C, the restrictive growth temperature, sec11 cells accumulate core-glycosylated forms of invertase and acid phosphatase, each retaining an intact signal peptide. In contrast, other sec mutant strains in which transport of core-glycosylated molecules from the endoplasmic reticulum is blocked show no defect in signal peptide cleavage. A DNA fragment that complements the sec11-7 mutation has been cloned. Genetic analysis indicates that the complementing clone contains the authentic SEC11 gene, and that a null mutation at the SEC11 locus is lethal. The DNA sequence of SEC11 predicts a basic protein (estimated pI of 9.5) of 167 amino acids including an NH2-terminal hydrophobic region that may function as a signal and/or membrane anchor domain. One potential N-glycosylation site is found in the 18.8-kD (Sec 11p) predicted protein. The mass of the SEC11 protein is very close to that found for two of the subunits of the canine and hen oviduct signal peptidases. Furthermore, the chromatographic behavior of the hen oviduct enzyme indicates an overall basic charge comparable to the predicted pI of the Sec11p.     

Structural features in the NH2-terminal region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase

The 20-amino acid signal peptide of human pre (delta pro)apolipoprotein A-II contains the tripartite domain structure typical of eukaryotic prepeptides, i.e. a positively charged NH2-terminal (n) region, a hydrophobic core (h) region, and a COOH-terminal polar domain (c region). This signal sequence has multiple potential sites for cotranslational processing making it an attractive model for assessing the consequences of systematic structural alterations on the site selected for signal peptidase cleavage. We previously analyzed 40 mutant derivatives of this model preprotein using an in vitro translation/canine microsome processing assay. The results showed that the position of the boundary between the h and c regions and properties of the -1 residue are critical in defining the site of cotranslational cleavage. To investigate whether structural features in the NH2-terminal region of signal peptides play a role in cleavage specificity, we have now inserted various amino acids between the positively charged n region (NH2-Met-Lys) and the h region of a "parental" pre(delta pro)apoA-II mutant that has roughly equal cleavage between Gly18 decreases and Gly20 decreases. Movement of the n/h boundary toward the NH2 terminus results in a dramatic shift in cleavage to Gly18 decreases. Replacement of the Lys2 residue with hydrophilic, negatively charged residues preserves the original sites of cleavage. Replacement with a hydrophobic residue causes cleavage to shift "upstream." Simultaneous alteration of the position of n/h and h/c boundaries has an additive effect on the site of signal peptidase cleavage. None of these mutations produced a marked decrease in the efficiency of in vitro cotranslational translocation or cleavage. However, in sequence contexts having poor signal function, introduction of hydrophobic residues between the n and h regions markedly improved the efficiency of translocation/processing. We conclude that the position of the n/h boundary as well as positioning of the h/c boundary affects the site of cleavage chosen by signal peptidase.     

Structural characteristics of Heparan sulfate required for the binding with the virus processing Enzyme Furin

Glycoconjugate Journal - Furin is one of the nine-member proprotein convertase family. Furin cleaves proteins with polybasic residues, which includes many viral glycoproteins such as SARS-Cov-2...     

Three-dimensional structure of the signal peptide peptidase

Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. Here, we analyzed the structure of human SPP by electron microscopy and reconstructed the three-dimensional structure at a resolution of 22 Å. Enzymatically active SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 × 85 × 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. Intriguingly, the N-terminal region of SPP is sufficient for the tetrameric assembly. Moreover, overexpression of the N-terminal region inhibited the formation of the endogenous SPP tetramer and the proteolytic activity within cells. These data suggest that the homotetramer is the functional unit of SPP and that its N-terminal region, which works as the structural scaffold, has a novel modulatory function for the intramembrane-cleaving activity of SPP.