In Polytomella sp. mitochondria, biogenesis of the heterodimeric COX2 subunit of cytochrome c oxidase requires two different import pathways

In the vast majority of eukaryotic organisms, the mitochondrial cox2 gene encodes subunit II of cytochrome c oxidase (COX2). However, in some lineages including legumes and chlorophycean algae, the cox2 gene migrated to the nucleus. Furthermore, in chlorophycean algae, this gene was split in two different units. Thereby the COX2 subunit is encoded by two independent nuclear genes, cox2a and cox2b, and mitochondria have to import the cytosol-synthesized COX2A and COX2B subunits and assemble them into the cytochrome c oxidase complex. In the chlorophycean algae Chlamydomonas reinhardtii and Polytomella sp., the COX2A precursor exhibits a long (130-140 residues), cleavable mitochondrial targeting sequence (MTS). In contrast, COX2B lacks an MTS, suggesting that mitochondria use different mechanisms to import each subunit. Here, we explored the in vitro import processes of both, the Polytomella sp. COX2A precursor and the COX2B protein. We used isolated, import-competent mitochondria from this colorless alga. Our results suggest that COX2B is imported directly into the intermembrane space, while COX2A seems to follow an energy-dependent import pathway, through which it finally integrates into the inner mitochondrial membrane. In addition, the MTS of the COX2A precursor is eliminated. This is the first time that the in vitro import of split COX2 subunits into mitochondria has been achieved.     

Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of Trypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria in vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a T. brucei mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.     

Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.

BACKGROUND: At M phase, cyclin B1 is phosphorylated in the cytoplasmic retention sequence (CRS), which is required for nuclear export. During interphase, cyclin B1 shuttles between the nucleus and the cytoplasm because constitutive nuclear import is counteracted by rapid nuclear export. In M phase, cyclin B moves rapidly into the nucleus coincident with its phosphorylation, an overall movement that might be caused simply by a decrease in its nuclear export. However, the questions of whether CRS phosphorylation is required for cyclin B1 translocation in mitosis and whether a reduction in nuclear export is sufficient to explain its rapid relocalisation have not been addressed. RESULTS: We have used two forms of green fluorescent protein to analyse simultaneously the translocation of wild-type cyclin B1 and a phosphorylation mutant of cyclin B1 in mitosis, and correlated this with an in vitro nuclear import assay. We show that cyclin B1 rapidly translocates into the nucleus approximately 10 minutes before breakdown of the nuclear envelope, and that this movement requires the CRS phosphorylation sites. A cyclin B1 mutant that cannot be phosphorylated enters the nucleus after the wild-type protein. Phosphorylation of the CRS creates a nuclear import signal that enhances cyclin B1 import in vitro and in vivo, in a manner distinct from the previously described import of cyclin B1 mediated by importin beta. CONCLUSIONS: We show that phosphorylation of human cyclin B1 is required for its rapid translocation to the nucleus towards the end of prophase. Phosphorylation enhances cyclin B1 nuclear import by creating a nuclear import signal. The phosphorylation of the CRS is therefore a critical step in the control of mitosis.     

Adaptations required for mitochondrial import following mitochondrial to nucleus gene transfer of ribosomal protein S10

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphipathic alpha-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal.     

Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex

The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements. The majority of these proteins are encoded in the cell's nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome. A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane. One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins. Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis. Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein. Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins. Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts). The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here.     

Mitochondrial protein p32 can accumulate in the nucleus

Human p32 was first isolated associated with the splicing factor ASF/SF-2. The p32 protein is translated as pre-protein from which a mitochondrial import signal is cleaved off to create the mature p32. The majority of p32 is consequently found in the mitochondria. In this study we investigated extramitochondrial p32. An increased nuclear localisation of endogenous p32 was demonstrated as a response to leptomycin B or actinomycin D treatment of cells. Mature p32 gene and deletion mutants were cloned into enhanced green fluorescence protein reporter plasmids. On transfection, EGFP-p32 protein was mainly localised to the cytoplasm and to a lesser extent to the nucleus of transfected COS cells. Upon treatment with actinomycin D or leptomycin B, the EGFP-p32 protein accumulated in the nucleus. Deletion analysis indicated which regions of EGFP-p32 are involved in nuclear export and nuclear import.     

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.     

Characterization of a novel transferable CRM-1-independent nuclear export signal in a herpesvirus tegument protein that shuttles between the nucleus and cytoplasm

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.     

Mitochondrial preprotein translocases as dynamic molecular machines

Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments--outer membrane, intermembrane space (IMS), inner membrane and matrix--requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria.     

Splicing before import - an intein in a mitochondrially targeted preprotein folds and is catalytically active in the cytoplasm in vivo

Nuclear-encoded mitochondrial proteins are cytoplasmically synthesized and imported into the organelle. The intein-containing RecA protein of Mycobacterium tuberculosis, with or without the CoxIVp mitochondrial targeting signal (MTS), was used to determine where a protein targeted to mitochondria folds and becomes catalytically active. Analysis of fractions from Saccharomyces cerevisiae cells expressing RecA without the MTS revealed that RecA and intein proteins remained cytoplasmic. With the MTS, most of RecA was directed to mitochondria, while most of the intein remained in the cytoplasm. The intein therefore folds into a catalytically active state in the cytoplasm prior to RecA import into mitochondria.     

Human APE2 protein is mostly localized in the nuclei and to some extent in the mitochondria, while nuclear APE2 is partly associated with proliferating cell nuclear antigen

In human cells APE1 is the major AP endonuclease and it has been reported to have no functional mitochondrial targeting sequence (MTS). We found that APE2 protein possesses a putative MTS. When its N-terminal 15 amino acid residues were fused to the N-terminus of green fluorescent protein and transiently expressed in HeLa cells the fusion protein was localized in the mitochondria. By electron microscopic immunocytochemistry we detected authentic APE2 protein in mitochondria from HeLa cells. Western blotting of the subcellular fraction of HeLa cells revealed most of the APE2 protein to be localized in the nuclei. We found a putative proliferating cell nuclear antigen (PCNA)-binding motif in the C-terminal region of APE2 and showed this motif to be functional by immunoprecipitation and in vitro pull-down binding assays. Laser scanning immunofluorescence microscopy of HeLa cells demonstrated both APE2 and PCNA to form foci in the nucleus and also to be co-localized in some of the foci. The incubation of HeLa cells in HAT medium containing deoxyuridine significantly increased the number of foci in which both molecules were co-localized. Our results suggest that APE2 participates in both nuclear and mitochondrial BER and also that nuclear APE2 functions in the PCNA-dependent BER pathway.     

Nuclear import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

Central Role of the Oxygen-dependent Degradation Domain of Drosophila HIFα/Sima in Oxygen-dependent Nuclear Export

The Drosophila HIFα homologue, Sima, is localized mainly in the cytoplasm in normoxia and accumulates in the nucleus upon hypoxic exposure. We have characterized the mechanism governing Sima oxygen-dependent subcellular localization and found that Sima shuttles continuously between the nucleus and the cytoplasm. We have previously shown that nuclear import depends on an atypical bipartite nuclear localization signal mapping next to the C-terminus of the protein. We show here that nuclear export is mediated in part by a CRM1-dependent nuclear export signal localized in the oxygen-dependent degradation domain (ODDD). CRM1-dependent nuclear export requires both oxygen-dependent hydroxylation of a specific prolyl residue (Pro850) in the ODDD, and the activity of the von Hippel Lindau tumor suppressor factor. At high oxygen tension rapid nuclear export of Sima occurs, whereas in hypoxia, Sima nuclear export is largely inhibited. HIFα/Sima nucleo-cytoplasmic localization is the result of a dynamic equilibrium between nuclear import and nuclear export, and nuclear export is modulated by oxygen tension.     

A human nuclear shuttling protein that interacts with human immunodeficiency virus type 1 matrix is packaged into virions

Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55(Gag) and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4(+) T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55(Gag) and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.     

Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.     

Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.