Immunoblot Analysis Of Subcellular Fractions Research Articles

Characterization of Nrf1b, a Novel Isoform of the Nuclear Factor-Erythroid-2 Related Transcription Factor-1 That Activates Antioxidant Response Element-Regulated Genes

Nuclear factor E2-related factor 1 (Nrf1) is a basic leucine zipper transcription factor that plays an important role in the activation of cytoprotective genes through the antioxidant response elements. The previously characterized long isoform of Nrf1 (Nrf1a) is targeted to the endoplasmic reticulum and accumulates in the nucleus in response to activating signals. Here we characterized a novel Nrf1 protein isoform (Nrf1b) generated through an alternative promoter and first exon that lacks the ER targeting domain of Nrf1a. The 5′-flanking region of Nrf1b directed high levels of luciferase reporter expression in cells. RT-PCR and Western blotting showed Nrf1b is widely expressed in various cell lines and mouse tissues. Immunoblot analysis of subcellular fractions and imaging of green fluorescence protein (GFP)-tagged Nrf1b demonstrate Nrf1b is constitutively localized to the nucleus. Nrf1b can activate GAL4-dependent transcription when fused to the heterologous GAL4 DNA-binding domain. Gel-shift and coimmunoprecipitation experiments demonstrate that Nrf1b forms a complex with MafG, and expression of Nrf1b activates the expression of antioxidant response element containing reporters and genes in cells. These results suggest Nrf1b is targeted to the nucleus where it activates ARE-driven genes and may play a role in modulating antioxidant response elements.

Hint2 Is Expressed in the Mitochondria of H295R Cells and Is Involved in Steroidogenesis

Hint2 belongs to the superfamily of histidine triad hydrolase enzymes. Recently, it has been shown to influence the mitochondria-dependent apoptosis occurring in hepatocytes, but its mechanism of action is still obscure. Here, we demonstrate that Hint2 is expressed in the mitochondria of H295R cells and in normal adrenals, and that this protein is involved in steroidogenesis. The presence of Hint2 in H295R cells was revealed by RT-PCR and by immunoblot analysis of subcellular fractions. The protein appeared associated with mitochondrial membranes, probably facing the interior of the organelle. Hint2 overexpression in H295R cells had no effect on pregnenolone secretion elicited by angiotensin II or K+, whereas protein silencing with specific small interfering RNA resulted in a marked reduction of the steroidogenic response. The duration of the mitochondrial calcium signal induced by angiotensin II was also reduced upon Hint2 down-regulation with small interfering RNA, but not affected after its overexpression, suggesting that under basal conditions, Hint2 is optimally expressed, and not rate limiting in steroidogenesis. Moreover, Hint2 also appeared involved in Ca2+-independent pathways leading to steroid formation. Indeed, pregnenolone formation in response to either forskolin or a hydroxyl analog of cholesterol was markedly reduced after Hint2 silencing. Calcium-dependent and calcium-independent actions of Hint2 on steroidogenesis could be related to its ability to maintain a favorable mitochondrial potential. In conclusion, these data suggest that, in H295R cells, Hint2 is required for an optimal steroidogenic response, possibly because of a particular signalling function exerted within the mitochondria and that still remains to determine at the molecular level.

Viral targeting of DEAD box protein 3 reveals its role in TBK1/IKKɛ-mediated IRF activation

Viruses are detected by different classes of pattern recognition receptors (PRRs), such as Toll-like receptors and RIG-like helicases. Engagement of PRRs leads to activation of interferon (IFN)-regulatory factor 3 (IRF3) and IRF7 through IKKepsilon and TBK1 and consequently IFN-beta induction. Vaccinia virus (VACV) encodes proteins that manipulate host signalling, sometimes by targeting uncharacterised proteins. Here, we describe a novel VACV protein, K7, which can inhibit PRR-induced IFN-beta induction by preventing TBK1/IKKepsilon-mediated IRF activation. We identified DEAD box protein 3 (DDX3) as a host target of K7. Expression of DDX3 enhanced Ifnb promoter induction by TBK1/IKKepsilon, whereas knockdown of DDX3 inhibited this, and virus- or dsRNA-induced IRF3 activation. Further, dominant-negative DDX3 inhibited virus-, dsRNA- and cytosolic DNA-stimulated Ccl5 promoter induction, which is also TBK1/IKKepsilon dependent. Both K7 binding and enhancement of Ifnb induction mapped to the N-terminus of DDX3. Furthermore, virus infection induced an association between DDX3 and IKKepsilon. Therefore, this study shows for the first time the involvement of a DEAD box helicase in TBK1/IKKepsilon-mediated IRF activation and Ifnb promoter induction.

Role of FoxO1 activation in MDR1 expression in adriamycin-resistant breast cancer cells

The development of multidrug resistance 1 (MDR1) can be mediated by a number of different mechanisms but elevated gene expression of MDR1 (P-glycoprotein) has often been a major cause of chemoresistance in many cancer cells. Therefore, the present study aimed to investigate the role of forkhead box-containing protein, O subfamily (FoxO), transcription factors in regulating the MDR1 gene expression. The proximal promoter region of the human MDR1 contained a putative FoxO-binding site, which partially overlapped with the enhancer/enhancer-binding protein beta-binding region. Gel shift and immunoblot analysis of subcellular fractions revealed that nuclear levels of FoxO1 and its DNA-binding activity were selectively enhanced in MCF-7/ADR cells, which was reversed by a FoxO1 antibody. Reporter gene assays showed that the transcription of MDR1 gene is stimulated by FoxO1 overexpression. Moreover, both MDR1 expression and doxorubicin resistance in MCF-7/ADR cells were reversed by FoxO1 small interfering RNA (siRNA). The MDR1 expression in MCF-7/ADR cells was also inhibited by insulin, a functional FoxO1 inactivator. In conclusion, FoxO1 is a novel transcriptional activator of MDR1 and is crucial for MDR1 induction in MCF-7/ADR cells.

DYRK2 Is Targeted to the Nucleus and Controls p53 via Ser46 Phosphorylation in the Apoptotic Response to DNA Damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.

The PLAC1 protein localizes to membranous compartments in the apical region of the syncytiotrophoblast

PLAC1 is a trophoblast-specific gene that maps to a locus on the X-chromosome important to placental development. We have previously shown that PLAC1 gene expression is linked to trophoblast differentiation. The objective of this study was to define the localization of the PLAC1 polypeptide as a prerequisite to understanding its function. Polyclonal antibodies specific for the putative PLAC1 polypeptide were generated. The subcellular localization of PLAC1 in the trophoblast was examined by immunohistochemical analysis of human placenta complemented by immunoblot analysis of subcellular fractions. Brightfield immunohistochemical analysis of placental tissue indicated that the PLAC1 protein localizes to the differentiated syncytiotrophoblast in the apical region of the cell. Deconvlution immunofluorescence microscopy confirmed localization to the apical region of the syncytiotrophoblast. Its distribution included both intracellular compartments as well as loci in close association with the maternal-facing, microvillous brush border membrane (MVM). These findings were supported by immunoblot analysis of subcellular fractions. A 30 kDa band was associated with the microsomal fraction of placental lysates but not the mitochondrial, nuclear, or soluble fractions, suggesting PLAC1 is targeted to a membrane location. Plasma membranes were obtained from the fetal-facing, basal surface (BM) and the maternal-facing, MVM of the syncytiotrophoblast membrane. PLAC1 immunoreactivity was only detected in membrane fractions derived from the apical MVM consistent with immunohistochemical analyses. These data demonstrate that the PLAC1 protein is restricted primarily to the differentiated trophoblast, localizing to intracellular membranous compartment(s) in the apical region of the syncytiotrophoblast and associated with its apical, microvillous membrane surface.

Protein kinase I of Mycobacterium tuberculosis: Cellular localization and expression during infection of macrophage-like cells

Protein kinase I of Mycobacterium tuberculosis, which has an unusual amino acid composition in its catalytic loop, displayed autophosphorylation and transphosphorylation activity. Immunoblot analysis of sub-cellular fractions of M. tuberculosis, using anti-PknI antibodies raised in rabbits, showed that PknI localizes to the bacterial cytosol. In contrast, PknA was membrane-bound. Relative expression of pknI, when measured by combining molecular beacons and RT-PCR, decreased during infection of THP-1 human macrophages. Expression of pknA and pknB was upregulated during infection. Thus PknI represents a group of protein kinases that is distinct from the more extensively studied enzymes PknA and PknB.

Activation of CCAAT/enhancer-binding protein beta by 2'-amino-3'-methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2.

The induction of glutathione S-transferases by flavonoids is associated with cancer chemopreventive effects. We reported that 2'-amino-3'-methoxyflavone (PD98059), an MKK1 inhibitor, induces glutathione S-transferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer-binding protein (C/EBP) and Nrf2 in the induction of rGSTA2 by PD98059. We first assessed whether the MKK1/ERK1/2 pathway regulated rGSTA2 induction. Northern and western blot analyses showed that PD98059 at the concentrations effective for the inhibition of MKK1 increased the rGSTA2 protein and mRNA levels in H4IIE cells. PD98059 also induced rGSTA2 in cells stably transfected with dominant-negative mutant of MKK1(-), which provided evidence that the inhibition of MKK1/ERK1/2 by PD98059 was not responsible for rGSTA2 induction. Gel shift assay and immunoblot analysis of subcellular fractions revealed that PD98059 caused nuclear translocation of C/EBP beta and increased C/EBP DNA binding, which was super-shifted with anti-C/EBP beta antibody. Nrf2 was not activated by PD98059. PD98059 increased the luciferase reporter gene activity in cells transfected with the C/EBP-containing -1.65 kb flanking region of the rGSTA2 gene. Deletion of the C/EBP-binding site or over-expression of dominant-negative mutant of C/EBP abolished the reporter gene activity. Flavone, a backbone structure of PD98059, also induced nuclear translocation of C/EBP beta and C/EBP-mediated rGSTA2 gene induction. Inhibition of phosphatidylinositol 3-kinase abolished C/EBP beta-mediated rGSTA2 induction by PD98059. These results provide evidence that PD98059 and flavone induce nuclear translocation of C/EBP beta and activate the C/EBP-binding site in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of the inhibition of MKK1/ERK activity.

Localization of a mitochondrial type of NADP-dependent isocitrate dehydrogenase in kidney and heart of rat: an immunocytochemical and biochemical study.

We studied the subcellular localization of the mitochondrial type of NADP-dependent isocitrate dehydrogenase (ICD1) in rat was immunofluorescence and immunoelectron microscopy and by biochemical methods, including immunoblotting and Nycodenz gradient centrifugation. Antibodies against a 14-amino-acid peptide at the C-terminus of mouse ICD1 was prepared. Immunoblotting analysis of the Triton X-100 extract of heart and kidney showed that the antibodies developed a single band with molecular mass of 45 kD. ICD1 was highly expressed in heart, kidney, and brown fat but only a low level of ICD1 was expressed in other tissues, including liver. Immunofluorescence staining showed that ICD1 was present mainly in mitochondria and, to a much lesser extent, in nuclei. Low but significant levels of activity and antigen of ICD1 were found in nuclei isolated by equilibrium sedimentation. Immunoblotting analysis of subcellular fractions isolated by Nycodenz gradient centrifugation from rat liver revealed that ICD1 signals were exclusively distributed in mitochondrial fractions in which acyl-CoA dehydrogenase was present. Immunofluorescence staining and postembedding electron microscopy demonstrated that ICD1 was confined almost exclusively to mitochondria and nuclei of rat kidney and heart muscle. The results show that ICD1 is expressed in the nuclei in addition to the mitochondria of rat heart and kidney. In the nuclei, the enzyme is associated with heterochromatin. In kidney, ICD1 distributes differentially in the tubule segments.

The Neisseria lipooligosaccharide-specific alpha-2,3-sialyltransferase is a surface-exposed outer membrane protein.

Neisseria gonorrhoeae and Neisseria meningitidis express an approximately 43-kDa alpha-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all N. gonorrhoeae strains and some N. meningitidis serogroups) or endogenous (in other N. meningitidis serogroups) sources of 5'-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect N. gonorrhoeae and N. meningitidis from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both N. gonorrhoeae and N. meningitidis. Western immunoblot analysis of subcellular fractions of N. gonorrhoeae strain F62 and N. meningitidis strain MC58 not subset 3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an approximately 43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of N. gonorrhoeae were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic lst insertion knockout mutant of N. gonorrhoeae F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of N. meningitidis MC58 not subset 3 and wild-type N. gonorrhoeae F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole N. gonorrhoeae cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable Neisseria cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2

Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.

Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities.

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.

Pantophysin Is a Phosphoprotein Component of Adipocyte Transport Vesicles and Associates with GLUT4-containing Vesicles

Pantophysin, a protein related to the neuroendocrine-specific synaptophysin, recently has been identified in non-neuronal tissues. In the present study, Northern blots showed that pantophysin mRNA was abundant in adipose tissue and increased during adipogenesis of 3T3-L1 cells. Immunoblot analysis of subcellular fractions showed pantophysin present exclusively in membrane fractions and relatively evenly distributed in the plasma membrane and internal membrane fractions. Sucrose gradient ultracentrifugation demonstrated that pantophysin and GLUT4 exhibited overlapping distribution profiles. Furthermore, immunopurified GLUT4 vesicles contained pantophysin, and both GLUT4 and pantophysin were depleted from this vesicle population following treatment with insulin. Additionally, a subpopulation of immunopurified pantophysin vesicles contained insulin-responsive GLUT4. Consistent with the interaction of synaptophysin with vesicle-associated membrane protein 2 in neuroendocrine tissues, pantophysin associated with vesicle-associated membrane protein 2 in adipocytes. Furthermore, in [(32)P]orthophosphate-labeled cells, pantophysin was phosphorylated in the basal state. This phosphorylation was unchanged in response to insulin; however, insulin stimulated the phosphorylation of a 77-kDa protein associated with alpha-pantophysin immunoprecipitates. Although the functional role of pantophysin in vesicle trafficking is unclear, its presence on GLUT4 vesicles is consistent with the emerging role of soluble N-ethylmaleimide-sensitive protein receptor (SNARE) factor complex and related proteins in regulated vesicle transport in adipocytes. In addition, pantophysin may provide a marker for the analysis of other vesicles in adipocytes.

Molecular cloning and characterization of a Candida albicans gene coding for cytochrome c haem lyase and a cell wall-related protein.

Immunoscreening of a Candida albicans cDNA library with a monoclonal antibody (mAb 4C12) recognizing an epitope present in high-molecular-weight mannoprotein (HMWM) components specific for the mycelial cell walls (a 180 kDa component and a poly-dispersed 260 kDa species) resulted in the isolation of the gene CaCYC3 encoding for cytochrome c haem lyase (CCHL). The CaCYC3 gene was transcribed preferentially in mycelial cells in which two mRNA transcripts of 0.8 and 1 kb were found. The nucleotide and the deduced amino acid sequences of this gene displayed 45% homology and 46% identity, respectively, to the Saccharomyces cerevisiae CYC3 gene and shared common features with other reported genes encoding for CCHL. The CaCYC3 gene restored the respiratory activity when transformed in a S. cerevisiae cyc3- mutant strain. A C. albicans CYC3 null mutant was constructed after sequential disruption using the hisG::URA3::hisG ('ura-blaster') cassette. Null mutant cells were unable to use lactate as a sole carbon source and had a reduced ability to form germ tubes. Western immunoblotting analysis of subcellular fractions from wild-type and null mutant strains demonstrated the presence of two gene products, a 33kDa mitochondrial protein and a 40 kDa cell wall-associated moiety reacting with antibodies against CCHL, in both yeast cells and germ tubes. mAb 4C12 still reacted with the CaCYC3 null mutant (by immunofluorescence and immunoblotting) but showed an altered pattern of immunoreactivity against cell wall HMWM species, indicating a relationship between these moieties and the CaCYC3 gene products. The results suggest that the CaCYC3 gene encodes two proteins, one targeted to the mitochondria and the other to the cell wall.

ANG II-induced translocation of cytosolic PLA2 to the nucleus in vascular smooth muscle cells.

The accumulation of radiolabeled arachidonic acid (AA), immunoblot analysis of subcellular fractions, and immunofluorescence tagging of proteins in intact cells were used to examine the coupling of ANG II receptors with the activity and location of a cytosolic phospholipase A2 (cPLA2) in vascular smooth muscle cells (VSMC). ANG II induced the accumulation of AA, which peaked by 10 min and was downregulated by 20 min. A large proportion of the AA released in response to ANG II was due to the activation of a Ca(2+)-dependent lipasc coupled to an AT1 receptor. However, regulation of Ca2+ availability failed to completely block AA release, and a small but significant reduction in ANG II-mediated AA release was observed in the presence of an AT2 antagonist. These findings, coupled with a 25% reduction in the ANG II-induced AA release by an inhibitor specific for a Ca(2+)-independent PLA2, are consistent with the presence and activation of a Ca(2+)-independent PLA2. In contrast, immunoblot analysis and immunofluorescence detection showed that the ANG II-mediated translocation of cPLA2 to a membrane fraction was exclusively AT1 dependent and regulated by Ca2+ availability. Furthermore, the nucleus was the membrane target. We conclude that ANG II regulates the Ca(2+)-dependent activation and translocation of cPLA2 through an AT1 receptor and that this event is targeted at the nucleus in VSMC.

Noc2, a Putative Zinc Finger Protein Involved in Exocytosis in Endocrine Cells

We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca2+ and phospholipids. Noc2 is expressed predominantly in endocrine tissues and hormone-secreting cell lines and at very low levels in brain. Immunoblot analysis of subcellular fractions of the insulin-secreting cell line MIN6 and immunocytochemistry reveal that Noc2 is a 38-kDa protein present in the cytoplasm. Overexpression of Noc2 in PC12 cells cotransfected with growth hormone enhances high K+-induced growth hormone secretion. Screening a mouse embryonic cDNA library with the yeast two-hybrid system shows that Noc2 interacts with the LIM domain-containing protein zyxin, a component of the cytoskeleton, and this interaction is further confirmed by the coimmunoprecipitation experiment. Accordingly, Noc2 is probably involved in regulated exocytosis in endocrine cells by interacting with the cytoskeleton.

Intracellular Location of SNAP-25 in Human Neutrophils

Exocytosis plays an essential role in the physiological functions of human neutrophils. Although SNAP-25 is considered to play a key role in vesicle-membrane fusion, it has been detected almost exclusively in the neuronal system. Using different specific antibodies to SNAP-25, we have identified in the membrane fraction of resting human neutrophils an immunoreactive band with the same molecular mass observed in brain homogenates. Immunoblot analysis of subcellular fractions of neutrophils revealed that SNAP-25 protein was found in the granule membrane fraction, but not in the cytosolic and plasma membrane fractions. Granule localization for neutrophil SNAP-25 was further demonstrated by confocal and immunoelectron microscopy. Furthermore, SNAP-25 was mainly located in the morphologically defined neutrophil peroxidase-negative granules, which are mobilizable upon cell activation. In addition, the protein was specifically cleaved by botulinal neurotoxin A, as observed in brain homogenate. These findings reveal the presence of SNAP-25 in the granule membranes of human neutrophils.

Localization of a GSH-Dependent Dehydroascorbate Reductase in Rat Tissues and Subcellular Fractions

A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.

Association of Rab1B with GDP-dissociation Inhibitor (GDI) Is Required for Recycling but Not Initial Membrane Targeting of the Rab Protein

We have identified the Rab1B effector-domain mutant (D44N) that, when geranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell-free systems or intact cells, fails to form detectable complexes with GDP-dissociation inhibitors (GDIs). GDI-Rab complexes were collected on anti-FLAG affinity beads after incubating recombinant geranylgeranylated Rab1B with FLAG epitope-tagged GDI in vitro, or transiently coexpressing Myc-tagged Rab1B with FLAG-GDI-alpha or FLAG-GDI-2 in human embryonal kidney 293 cells. [3H]Mevalonate labeling and immunoprecipitation studies confirmed that the inability of Myc-Rab1BD44N to associate with GDI in vivo was not due to failure of the mutant to undergo geranylgeranylation. Immunofluorescence localization and immunoblot analysis of subcellular fractions indicated that expressed Myc-Rab1BD44N was efficiently delivered to intracellular membranes in 293 cells. This was confirmed when the fate of the prenylated pool of Rab1BD44N in 293 cells was traced by labeling the geranylgeranyl groups attached to the nascent protein with [3H]meval onate. However, in contrast to the prenylated Rab1BWT, which was distributed in both the membrane and soluble fractions, the prenylated Rab1BD44N was completely absent from the cytosol. Overexpression of Myc-Rab1BD44N did not impair ER --> Golgi glycoprotein trafficking in 293 cells, which was assessed by monitoring the Golgi-dependent processing of coexpressed beta-amyloid precursor protein. The current findings suggest that nascent prenylated Rab1B can be delivered to intracellular membranes in intact cells without forming a stable complex with GDI, but that recycling of prenylated Rab1B to the cytosolic compartment is absolutely dependent on GDI interaction.

Effects of cadmium on glucose transport in rat adipocytes and human erythrocytes: stimulation of GLUT1 catalytic activity

The effects of cadmium on facilitative glucose transporter function In rat adipocytes were studied. A short (30 min) incubation with cadmium resulted in stimulation of 3-O-methyl-D-glucose (3OMG ) equilibrium exchange in rat adipocytes as much as four-fold. The stimulation was a saturable function of cadmium concentation with the half-maximal effect at approximately 0.5 mM CdS04. The stimulation was due to an increase in Vmax with no significant changes in KM. No further stimulation of 30MG flux was observed once adipocytes were maximally stimulated by insulin. Semiquantitative immunoblot analysis of subcellular fractions revealed that the stimulation was accompanied by an insignificant and only a modest (less than 50%) increase in plasma membrane GLUT4 and GLUT1 levels, respectively, suggesting that the stimulation involves largely an increased catalytic activity of either or both of GLUTI and GLUT4. We next studied effect of cadmium on GLUT1 selectively using human erythrocytes and purified GLUT1 reconstituted in liposomes. With purified GLUTI, cadmium inhibited cytochalasin B binding and stimulated 30MG flux, indicating that cadmium directly interacts with GLUTI. Cadmium, however, did not affect the 30MG flux in intact human erythrocytes or their resealed ghosts. These findings strongly suggest that cadmium stimulates the catalytic activity of GLUT1 and GLUT4 in adipocytes, and this effect is suppressed by a cell-specific factor or factors in human erythrocytes.