Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.

A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal

The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal–ventral axis in the Drosophila embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal–Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube–Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.

The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.

The Saccharomyces cerevisiae Homologue of Human Wiskott–Aldrich Syndrome Protein Las17p Interacts with the Arp2/3 Complex

Yeast Las17 protein is homologous to the Wiskott–Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Δ ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Δ cells showed that receptor-mediated internalization of α factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.

RanGTP Targets p97 to RanBP2, a Filamentous Protein Localized at the Cytoplasmic Periphery of the Nuclear Pore Complex

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of ∼36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.

The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein α subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein β subunit (Gβ5L). RGS9 and Gβ5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein γ-subunit like domain that can mediate their association with Gβ5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307–13312). We report an example of such a complex whose cellular localization and function are clearly defined.

DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1⋅IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⋅IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⋅IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.