The N-terminal 209-aa domain of high molecular- weight 4.1R isoforms abrogates 4.1R targeting to the nucleus

An extensive repertoire of protein 4.1R isoforms is predominantly generated by alternative pre-mRNA splicing and differential usage of two translation initiation sites. The usage of the most upstream ATG (ATG-1) generates isoforms containing N-terminal extensions of up to 209 aa compared with those translated from the downstream ATG (ATG-2). To characterize nonerythroid 4.1R proteins translated from ATG-1 and analyze their intracellular localization, we cloned 4.1R cDNAs containing this translation initiation site. Six different clones were isolated from the nucleated human MOLT-4 T-cell line by reverse transcriptase–PCR techniques. Transient expression of the six ATG-1-translated 4.1R isoforms tagged with a c-Myc epitope revealed that all of them predominantly distributed to the plasma membrane and the endoplasmic reticulum. Staining of MOLT-4 cell plasma membranes but not nuclei was also observed by immunofluorescence microscopy by using an antibody specific to the N-terminal extension. Consistent with this, the antibody reacted with a major endogenous protein of ≈145 kDa present in nonnuclear but absent from nuclear fractions prepared from MOLT-4 cells. Because these data suggested that ATG-1-translated 4.1R isoforms were predominantly excluded from the nucleus, we fused the 209-aa domain to nuclear 4.1R isoforms encoded from ATG-2 and observed that this domain inhibited their nuclear targeting. All these results indicate that the N-terminal domain of ATG-1-translated 4.1R isoforms plays a pivotal role in differential targeting of proteins 4.1R.

An HP1-like protein is missing from transcriptionally silent micronuclei of Tetrahymena

We report the identification and cloning of a 28-kDa polypeptide (p28) in Tetrahymena macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from Drosophila. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.

Syndapin I, a Synaptic Dynamin-binding Protein that Associates with the Neural Wiskott-Aldrich Syndrome Protein

The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to the Trans-Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.

Effect of the protein import machinery at the mitochondrial surface on precursor stability

Many biological processes require proteins to undergo conformational changes at the surface of membranes. For example, some precursor proteins unfold at the surface of mitochondria and chloroplasts before translocation into the organelles, and toxins such as colicin A unfold to the molten globule state at bacterial surfaces before inserting into the cell membrane. It is commonly thought that the membrane surfaces and the associated protein machinery destabilize the substrate proteins and that this effect is required for membrane insertion or translocation. One of the best characterized translocation processes is protein import into mitochondria. By measuring the contributions of individual interactions within a model protein to its stability at the mitochondrial surface and in free solution, we show here that the mitochondrial surface neither induces the molten globule state in this protein nor preferentially destabilizes any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Because it is not possible to measure absolute protein stability at the surface of mitochondria, we determined the stability of a tightly associated protein–protein complex at the mitochondrial import site as a model of the stability of a protein. We found the binding constants of the protein–protein complex at the mitochondrial surface and in free solution to be identical. Our results demonstrate that the mitochondrial surface does not destabilize importing precursor proteins in its vicinity.

Regulation of the pAD1 sex pheromone response of Enterococcus faecalis by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein

The Enterococcus faecalis conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of traE1. In vitro studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.

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.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Role of the F-box protein Skp2 in lymphomagenesis

The F-box protein Skp2 (S-phase kinase-associated protein 2) positively regulates the G1-S transition by controlling the stability of several G1 regulators, such as the cell cycle inhibitor p27. We show here that Skp2 expression correlates directly with grade of malignancy and inversely with p27 levels in human lymphomas. To directly evaluate the potential of Skp2 to deregulate growth in vivo, we generated transgenic mice expressing Skp2 targeted to the T-lymphoid lineage as well as double transgenic mice coexpressing Skp2 and activated N-Ras. A strong cooperative effect between these two transgenes induced T cell lymphomas with shorter latency and higher penetrance, leading to significantly decreased survival when compared with control and single transgenic animals. Furthermore, lymphomas of Nras single transgenic animals often expressed higher levels of endogenous Skp2 than tumors of double transgenic mice. This study provides evidence of a role for an F-box protein in oncogenesis and establishes SKP2 as a protooncogene causally involved in the pathogenesis of lymphomas.

Division of Mitochondria Requires a Novel DNM1-interacting Protein, Net2p

Mitochondria are dynamic organelles that undergo frequent division and fusion, but the molecular mechanisms of these two events are not well understood. Dnm1p, a mitochondria-associated, dynamin-related GTPase was previously shown to mediate mitochondrial fission. Recently, a genome-wide yeast two-hybrid screen identified an uncharacterized protein that interacts with Dnm1p. Cells disrupted in this new gene, which we call NET2, contain a single mitochondrion that consists of a network formed by interconnected tubules, similar to the phenotype of dnm1Δ cells. NET2 encodes a mitochondria-associated protein with a predicted coiled-coil region and six WD-40 repeats. Immunofluorescence microscopy indicates that Net2p is located in distinct, dot-like structures along the mitochondrial surface, many of which colocalize with the Dnm1 protein. Fluorescence and immunoelectron microscopy shows that Dnm1p and Net2p preferentially colocalize at constriction sites along mitochondrial tubules. Our results suggest that Net2p is a new component of the mitochondrial division machinery.

The HMG-domain protein BAP111 is important for the function of the BRM chromatin-remodeling complex in vivo

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes is how they interact with DNA, particularly in the large genomes of higher eukaryotes. Here, we report the characterization of BAP111, a BRM-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the BRM complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, we found that the majority of BAP111 protein in embryos is associated with the BRM complex. Furthermore, heterozygosity for BAP111 enhanced the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo.

Desmuslin, an intermediate filament protein that interacts with α-dystrobrevin and desmin

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, α1-syntrophin, and the sarcoglycan complex. The precise role of α-dystrobrevin in skeletal muscle has not yet been determined. To study α-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and α-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.

The in vivo neuromodulatory effects of the herbal medicine ginkgo biloba

Extracts of Ginkgo biloba leaves are consumed as dietary supplements to counteract chronic, age-related neurological disorders. We have applied high-density oligonucleotide microarrays to define the transcriptional effects in the cortex and hippocampus of mice whose diets were supplemented with the herbal extract. Gene expression analysis focused on the mRNAs that showed a more than 3-fold change in their expression. In the cortex, mRNAs for neuronal tyrosine/threonine phosphatase 1, and microtubule-associated τ were significantly enhanced. Hyperphosphorylated τ is the major constituent of the neurofibrillary tangles in the brains of Alzheimer's disease patients. The expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2, calcium and chloride channels, prolactin, and growth hormone (GH), all of which are associated with brain function, were also up-regulated. In the hippocampus, only transthyretin mRNA was upregulated. Transthyretin plays a role in hormone transport in the brain and possibly a neuroprotective role by amyloid-β sequestration. This study reveals that diets supplemented with Ginkgo biloba extract have notable neuromodulatory effects in vivo and illustrates the utility of genome-wide expression monitoring to investigate the biological actions of complex extracts.

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP− mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-γ production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP− mice produced increased IFN-γ and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.