Dual targeting of organellar seryl-tRNA synthetase to maize mitochondria and chloroplasts

Aminoacyl-tRNA synthetases (AARSs) play a critical role in translation and are thus required in three plant protein-synthesizing compartments: cytosol, mitochondria and plastids. A systematic study had previously shown extensive sharing of organellar AARSs from Arabidopsis thaliana, mostly between mitochondria and chloroplasts. However, distribution of AARSs from monocot species, such as maize, has never been experimentally investigated. Here we demonstrate dual targeting of maize seryl-tRNA synthetase, SerZMo, into both mitochondria and chloroplasts using combination of complementary methods, including in vitro import assay, transient expression analysis of green fluorescent protein (GFP) fusions and immunodetection. We also show that SerZMo dual localization is established by the virtue of an ambiguous targeting peptide. Full-length SerZMo protein fused to GFP is targeted to chloroplast stromules, indicating that SerZMo protein performs its function in plastid stroma. The deletion mutant lacking N-terminal region of the ambiguous SerZMo targeting peptide was neither targeted into mitochondria nor chloroplasts, indicating the importance of this region in both mitochondrial and chloroplastic import.     

Evolution of organellar proton-ATPases.

Proton ATPases function in biological energy conversion in every known living cell. Their ubiquity and antiquity make them a prime source for evolutionary studies. There are two related families of H(+)-ATPases; while the family of F-ATPases function in eubacteria chloroplasts and mitochondria, the family of V-ATPases are present in archaebacteria and the vacuolar system of eukaryotic cells. Sequence analysis of several subunits of V- and F-ATPases revealed several of the important steps in their evolution. Moreover, these studies shed light on the evolution of the various organelles of eukaryotes and suggested some events in the evolution of the three kingdoms of eubacteria, archaebacteria and eukaryotes.     

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.     

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.     

Protein N-acylation overrides differing targeting signals

In a bioinformatics based screen for chloroplast-localized protein kinases we noticed that available protein targeting predictors falsely predicted chloroplast localization. This seems to be due to interference with N-terminal protein acylation, which is of particular importance for protein kinases. Their N-myristoylation was found to be highly overrepresented in the proteome, whereas myristoylation motifs are almost absent in known chloroplast proteins. However, only abolishing their myristoylation was not sufficient to target those kinases to chloroplasts and resulted in nuclear accumulation instead. In contrast, inhibition of N-myristoylation of a calcium-dependent protein kinase was sufficient to alter its localization from the plasma membrane to chloroplasts and chloroplast localization of ferredoxin-NADP+ reductase and Rubisco activase could be efficiently suppressed by artificial introduction of myristoylation and palmitoylation sites.     

The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting

We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.     

Peroxisomal protein targeting and identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. Recently, it has been demonstrated that peroxisomes contain a number of enzymes involved in cholesterol biogenesis that previously were considered to be cytosolic or located in the endoplasmic reticulum. Peroxisomes have been shown to contain acetoacetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, phosphomevalonate decarboxylase, isopentenyl diphosphate isomerase and FPP synthase. Moreover, the activities of these enzymes are also significantly decreased in liver tissue and fibroblast cells obtained from patients with peroxisomal deficiency diseases. In addition, the cholesterol biosynthetic capacity is severely impaired in cultured skin fibroblasts obtained from patients with peroxisomal deficiency diseases. These findings support the proposal that peroxisomes play an essential role in isoprenoid biosynthesis. This paper presents a review of peroxisomal protein targeting and of recent studies demonstrating the localization of cholesterol biosynthetic enzymes in peroxisomes and the identification of peroxisomal targeting signals in these proteins.     

Global organellar proteomics

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.     

Redundant mitochondrial targeting signals in yeast adenylate kinase

Yeast adenylate kinase (Aky2p, Adk1p) occurs simultaneously in cytoplasm and mitochondrial intermembrane space. It has no cleavable mitochondrial targeting sequence, and the signal for mitochondrial import and submitochondrial sorting is largely unknown. The extreme N terminus of Aky2p is able to direct cytoplasmic passengers to mitochondria. However, an Aky2 mutant lacking this sequence is imported with about the same efficiency as the wild type. To identify possible import-relevant information in the interior, parts of Aky2p were exchanged by homologous in vitro recombination for the respective segments of the purely cytoplasmic isozyme, Ura6p. Import studies revealed an internal region of about 40 amino acids, which was sufficient to direct the chimera to mitochondria but not for correct submitochondrial sorting. The respective Ura6p hybrid was arrested in the mitochondrial membrane at a position where it was inaccessible to protease but was released by alkaline extraction, suggesting that it had entered an import channel and passed the initial steps of recognition and uptake. Site-specific mutations within the presumptive address-specifying segment identified the amphipathic helix 5. A Ura6 mutant protein in which helix 5 had been replaced with the respective sequence from Aky2p was imported, and this address sequence cooperates with the N terminus in the respective double mutant in a synergistic fashion.     

Bimodal protein targeting through activation of cryptic mitochondrial targeting signals by an inducible cytosolic endoprotease

Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals.     

Using nuclear targeting signals to enhance non-viral gene transfer

Summary Gene therapy involves the introduction of DNA-encoding therapeutic gene products into appropriate cells of an affected individual. The limitations of the approach relate largely to the poor efficiency of the delivery of the therapeutic DNA to the nucleus. This review examines recent work in the area of non-viral gene transfer, building on developments in the field of nuclear protein import and their application in the field of non-viral gene transfer. In particular, advances in the area of enhancing DNA targeting to the nucleus are discussed, including the use of modular nuclear targeting signals recognised by the cellular nuclear import machinery and DNA condensing agents to facilitate passage through the nuclear pore. Optimising nuclear DNA delivery through these and other strategies should assist greatly in rendering gene therapy a viable and realistic possibility for treating disease.     

Multiple distinct targeting signals in integral peroxisomal membrane proteins

Peroxisomal proteins are synthesized on free polysomes and then transported from the cytoplasm to peroxisomes. This process is mediated by two short well-defined targeting signals in peroxisomal matrix proteins, but a well-defined targeting signal has not yet been described for peroxisomal membrane proteins (PMPs). One assumption in virtually all prior studies of PMP targeting is that a given protein contains one, and only one, distinct targeting signal. Here, we show that the metabolite transporter PMP34, an integral PMP, contains at least two nonoverlapping sets of targeting information, either of which is sufficient for insertion into the peroxisome membrane. We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information. These results challenge a major assumption of most PMP targeting studies. In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34. Together, these results raise the interesting possibility that PMP import may require novel mechanisms to ensure the solubility of integral PMPs before their insertion in the peroxisome membrane, and that PEX19 may play a central role in this process.     

Cytoplasmic targeting signals in transmembrane invariant surface glycoproteins of trypanosomes

Protein targeting mechanisms in flagellated protozoan parasites have received considerable interest because of a huge bias in these organisms toward the glycosylphosphatidylinositol anchor as a mechanism for the membrane attachment of cell surface macromolecules. In this study, the trafficking of invariant surface glycoprotein 65 (ISG65), a family of type I transmembrane proteins, was examined. Analysis of the C-terminal domains of ISG65 family members demonstrated a high level of conservation and, in particular, the presence of three lysine residues contained within the cytoplasmic tails of all ISG65s. ISG65 was expressed on the cell surface, in agreement with earlier work, but an intracellular pool of ISG65 was also detected within a Rab5A early endosome. Transplantation of the C-terminal 74 amino acids of ISG65 (encompassing the 23 C-terminal residues of the extracellular domain, the transmembrane peptide, and the cytoplasmic domain) onto the N-terminal domain of BiP (BiPN) was sufficient to target the chimera to the same internal compartments as native ISG65. Further, site-directed mutagenesis indicated that the cytoplasmic tail was required for endoplasmic reticulum exit and that at least two of the cytoplasmic domain lysine residues are needed for endosomal targeting, as removal of all three led to surface expression. Kinetic measurements demonstrate that the BiPN fusion protein (containing the ISG65 C terminus) has a short half-life, indicating rapid turnover. In contrast, BiPN fusion proteins containing a glycosylphosphatidylinositol anchor instead of the ISG65 C-terminal region are stably expressed on the surface, confirming the requirement for the ISG65 sequence for endosomal targeting. We suggest that the lack of surface expression of the BiPN-ISG65 fusion protein is likely due to more efficient internalization compared with ISG65. Taken together, these data demonstrate the presence of a lysine-dependent endocytosis signal in the ISG65 family.     

Mitochondrial targeting signals and mature peptides of 3-methylcrotonyl-CoA carboxylase

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog. The amino-termini containing 39 (MCCalpha) or 20 amino acids (MCCbeta) were both necessary and sufficient for targeting. Structural requirements for mitochondrial import were defined by site-directed mutagenesis. Our studies provide the prerequisite to understand the impact of specific mutations on the clinical phenotype of MCC deficiency.