A role for heterodimerization in nuclear localization of a homeodomain protein

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (β-glucuronidase) gene and showing that β-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

Preferential localization of self-stimulation sites in striosomes/patches in the rat striatum

Histological sections of the mammalian striatum reveal a “matrix” that is histochemically distinguishable from patches, or “striosomes”. The latter are cross sections of a compartment that consists primarily of tube-shaped structures radiating through the matrix. As a test of the hypothesis that the function of the striosome/patch compartment includes the mediation of behaviors related to reward, the present study examined electrical self-stimulation of the caudoputamen in rats with electrodes in either of the two compartments. Rats acquired and maintained bar-pressing responses that were contingent on stimulation through electrodes making contact with striosomes/patches more reliably than animals with electrodes terminating exclusively in the matrix. The results provide in vivo evidence that the striosome/patch compartment is functionally differentiated from the matrix compartment: Stimulation centered in or around the striosome/patch compartment but not in the matrix led to rapid acquisition of a new behavior.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

Nuclear localization of prostaglandin E2 receptors

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites such as Toxoplasma gondii contain a primitive plastid, the apicoplast, whose genome consists of a 35-kb circular DNA related to the plastid DNA of plants. Plants synthesize fatty acids in their plastids. The first committed step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase (ACC). This enzyme is encoded in the nucleus, synthesized in the cytosol, and transported into the plastid. In the present work, two genes encoding ACC from T. gondii were cloned and the gene structure was determined. Both ORFs encode multidomain proteins, each with an N-terminal extension, compared with the cytosolic ACCs from plants. The N-terminal extension of one isozyme, ACC1, was shown to target green fluorescent protein to the apicoplast of T. gondii. In addition, the apicoplast contains a biotinylated protein, consistent with the assertion that ACC1 is localized there. The second ACC in T. gondii appears to be cytosolic. T. gondii mitochondria also contain a biotinylated protein, probably pyruvate carboxylase. These results confirm the essential nature of the apicoplast and explain the inhibition of parasite growth in cultured cells by herbicides targeting ACC.

Localization and regulation of GLUTx1 glucose transporter in the hippocampus of streptozotocin diabetic rats

We describe the localization of the recently identified glucose transporter GLUTx1 and the regulation of GLUTx1 in the hippocampus of diabetic and control rats. GLUTx1 mRNA and protein exhibit a unique distribution when compared with other glucose transporter isoforms expressed in the rat hippocampus. In particular, GLUTx1 mRNA was detected in hippocampal pyramidal neurons and granule neurons of the dentate gyrus as well as in nonprincipal neurons. With immunohistochemistry, GLUTx1 protein expression is limited to neuronal cell bodies and the most proximal dendrites, unlike GLUT3 expression that is observed throughout the neuropil. Immunoblot analysis of hippocampal membrane fractions revealed that GLUTx1 protein expression is primarily localized to the intracellular compartment and exhibits limited association with the plasma membrane. In streptozotocin diabetic rats compared with vehicle-treated controls, quantitative autoradiography showed increased GLUTx1 mRNA levels in pyramidal neurons and granule neurons; up-regulation of GLUTx1 mRNA also was found in nonprincipal cells, as shown by single-cell emulsion autoradiography. In contrast, diabetic and control rats expressed similar levels of hippocampal GLUTx1 protein. These results indicate that GLUTx1 mRNA and protein have a unique expression pattern in rat hippocampus and suggest that streptozotocin diabetes increases steady-state mRNA levels in the absence of concomitant increases in GLUTx1 protein expression.

Expression and Differential Intracellular Localization of Two Major Forms of Human 8-Oxoguanine DNA Glycosylase Encoded by Alternatively Spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1–1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1–1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1–2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1–2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1–2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1–1a depends on the NLS at its C terminus.

Ric1p and the Ypt6p GTPase Function in a Common Pathway Required for Localization of Trans-Golgi Network Membrane Proteins

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Δ) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Δ cells also harboring end3Δ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Δ. Further comparison of ric1Δ and ypt6Δ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Δ and ypt6Δ cells. SLY1–20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments

Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, β-catenin, and α-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.

The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants

In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses.

An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization

Paxillin acts as an adaptor protein in integrin signaling. We have shown that paxillin exists in a relatively large cytoplasmic pool, including perinuclear areas, in addition to focal complexes formed at the cell periphery and focal adhesions formed underneath the cell. Several ADP-ribosylation factor (ARF) GTPase-activating proteins (GAPs; ARFGAPs) have been shown to associate with paxillin. We report here that Git2-short/KIAA0148 exhibits properties of a paxillin-associated ARFGAP and appears to be colocalized with paxillin, primarily at perinuclear areas. A fraction of Git2-short was also localized to actin-rich structures at the cell periphery. Unlike paxillin, however, Git2-short did not accumulate at focal adhesions underneath the cell. Git2-short is a short isoform of Git2, which is highly homologous to p95PKL, another paxillin-binding protein, and showed a weaker binding affinity toward paxillin than that of Git2. The ARFGAP activities of Git2 and Git2-short have been previously demonstrated in vitro, and we provided evidence that at least one ARF isoform, ARF1, is an intracellular substrate for the GAP activity of Git2-short. We also showed that Git2-short could antagonize several known ARF1-mediated phenotypes: overexpression of Git2-short, but not its GAP-inactive mutant, caused the redistribution of Golgi protein β-COP and reduced the amounts of paxillin-containing focal adhesions and actin stress fibers. Perinuclear localization of paxillin, which was sensitive to ARF inactivation, was also affected by Git2-short overexpression. On the other hand, paxillin localization to focal complexes at the cell periphery was unaffected or even augmented by Git2-short overexpression. Therefore, an ARFGAP protein weakly interacting with paxillin, Git2-short, exhibits pleiotropic functions involving the regulation of Golgi organization, actin cytoskeletal organization, and subcellular localization of paxillin, all of which need to be coordinately regulated during integrin-mediated cell adhesion and intracellular signaling.