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At least 865 records · Page 48Linked to original sources
PubMed2000

Hepatitis B virus X protein colocalizes to mitochondria with a human voltage-dependent anion channel, HVDAC3, and alters its transmembrane potential.

Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection. In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay. This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3). In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods. Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria. This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria. Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential. Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.

Animals↗
PubMed2000

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly.

The replicase gene (gene 1) of the coronavirus mouse hepatitis virus (MHV) encodes two co-amino-terminal polyproteins presumed to incorporate all the virus-encoded proteins necessary for viral RNA synthesis. The polyproteins are cotranslationally processed by viral proteinases into at least 15 mature proteins, including four predicted cleavage products of less than 25 kDa that together would comprise the final 59 kDa of protein translated from open reading frame 1a. Monospecific antibodies directed against the four distinct domains detected proteins of 10, 12, and 15 kDa (p1a-10, p1a-12, and p1a-15) in MHV-A59-infected DBT cells, in addition to a previously identified 22-kDa protein (p1a-22). When infected cells were probed by immunofluorescence laser confocal microscopy, p1a-10, -22, -12, and -15 were detected in discrete foci that were prominent in the perinuclear region but were widely distributed throughout the cytoplasm as well. Dual-labeling experiments demonstrated colocalization of the majority of p1a-22 in replication complexes with the helicase, nucleocapsid, and 3C-like proteinase, as well as with p1a-10, -12, and -15. p1a-22 was also detected in separate foci adjacent to the replication complexes. The majority of complexes containing the gene 1 proteins were distinct from sites of accumulation of the M assembly protein. However, in perinuclear regions the gene 1 proteins and nucleocapsid were intercalated with sites of M protein localization. These results demonstrate that the complexes known to be involved in RNA synthesis contain multiple gene 1 proteins and are closely associated with structural proteins at presumed sites of virion assembly.

Cell Line↗
PubMed2001

Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane.

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.

Alfalfa mosaic virus↗
PubMed2001

Actin rearrangement-inducing factor of baculoviruses is tyrosine phosphorylated and colocalizes to F-actin at the plasma membrane.

In previous studies we have identified actin rearrangement-inducing factor 1 as an early gene product of Autographa californica multicapsid nuclear polyhedrosis virus that is involved in the remodeling of the actin cytoskeleton. We have constructed viral recombinants with a mutated Arif-1 open reading frame that confirm the causal link of Arif-1 expression and the actin rearrangement observed as accumulation of F-actin at the plasma membrane at 3 to 7 h postinfection. Infection with Arif mutant viruses leads to the loss of actin accumulation at the plasma membrane in TN-368 cells, although in the course of infection, early actin cables and nuclear F-actin are observed as in wild-type-infected cells. By immunofluorescence studies, we have demonstrated the localization of Arif-1 at the plasma membrane, and confocal imaging reveals the colocalization to F-actin. Accordingly, the approximately 47-kDa Arif-1 protein is observed exclusively in membrane fractions prepared at 4 to 48 h postinfection, with a decrease at 24 h postinfection. Phosphatase treatment suggests that Arif-1 is modified by phosphorylation. Antibodies against phosphotyrosine precipitate Arif-1 from membrane fractions, indicating that Arif-1 becomes tyrosine phosphorylated during the early and late phases of infection. In summary, our results indicate that functional Arif-1 is tyrosine phosphorylated and is located at the plasma membrane as a component of the actin rearrangement-inducing complex.

Actins↗
PubMed2003

An activity associated with human chromosome 21 permits nuclear colocalization of the adenovirus E1B-55K and E4orf6 proteins and promotes viral late gene expression.

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.

Adenoviridae↗
PubMed2003

Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin.

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.

Animals↗
PubMed2003

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids.

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.

Animals↗
PubMed2004

A virulence factor of myxoma virus colocalizes with NF-kappaB in the nucleus and interferes with inflammation.

NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.

Amino Acid Sequence↗
PubMed2000

A human condensin complex containing hCAP-C-hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocalizes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation.

Structural maintenance of chromosomes (SMC) family proteins play critical roles in structural changes of chromosomes. Previously, we identified two human SMC family proteins, hCAP-C and hCAP-E, which form a heterodimeric complex (hCAP-C-hCAP-E) in the cell. Based on the sequence conservation and mitotic chromosome localization, hCAP-C-hCAP-E was determined to be the human ortholog of the Xenopus SMC complex, XCAP-C-XCAP-E. XCAP-C-XCAP-E is a component of the multiprotein complex termed condensin, required for mitotic chromosome condensation in vitro. However, presence of such a complex has not been demonstrated in mammalian cells. Coimmunoprecipitation of the endogenous hCAP-C-hCAP-E complex from HeLa extracts identified a 155-kDa protein interacting with hCAP-C-hCAP-E, termed condensation-related SMC-associated protein 1 (CNAP1). CNAP1 associates with mitotic chromosomes and is homologous to Xenopus condensin component XCAP-D2, indicating the presence of a condensin complex in human cells. Chromosome association of human condensin is mitosis specific, and the majority of condensin dissociates from chromosomes and is sequestered in the cytoplasm throughout interphase. However, a subpopulation of the complex was found to remain on chromosomes as foci in the interphase nucleus. During late G(2)/early prophase, the larger nuclear condensin foci colocalize with phosphorylated histone H3 clusters on partially condensed regions of chromosomes. These results suggest that mitosis-specific function of human condensin may be regulated by cell cycle-specific subcellular localization of the complex, and the nuclear condensin that associates with interphase chromosomes is involved in the reinitiation of mitotic chromosome condensation in conjunction with phosphorylation of histone H3.

Amino Acid Sequence↗
PubMed2002

hSnm1 colocalizes and physically associates with 53BP1 before and after DNA damage.

snm1 mutants of Saccharomyces cerevisiae have been shown to be specifically sensitive to DNA interstrand crosslinking agents but not sensitive to monofunctional alkylating agents, UV, or ionizing radiation. Five homologs of SNM1 have been identified in the mammalian genome and are termed SNM1, SNM1B, Artemis, ELAC2, and CPSF73. To explore the functional role of human Snm1 in response to DNA damage, we characterized the cellular distribution and dynamics of human Snm1 before and after exposure to DNA-damaging agents. Human Snm1 was found to localize to the cell nucleus in three distinct patterns. A particular cell showed diffuse nuclear staining, multiple nuclear foci, or one or two larger bodies confined to the nucleus. Upon exposure to ionizing radiation or an interstrand crosslinking agent, the number of cells exhibiting Snm1 bodies was reduced, while the population of cells with foci increased dramatically. Indirect immunofluorescence studies also indicated that the human Snm1 protein colocalized with 53BP1 before and after exposure to ionizing radiation, and a physical interaction was confirmed by coimmunoprecipitation assays. Furthermore, human Snm1 foci formed after ionizing radiation were largely coincident with foci formed by human Mre11 and to a lesser extent with those formed by BRCA1, but not with those formed by human Rad51. Finally, we mapped a region of human Snm1 of approximately 220 amino acids that was sufficient for focus formation when attached to a nuclear localization signal. Our results indicate a novel function for human Snm1 in the cellular response to double-strand breaks formed by ionizing radiation.

BRCA1 Protein↗
PubMed1997

Colocalization of antisense RNAs and ribozymes with their target mRNAs.

The use of complementary RNA sequences such as antisense RNAs and ribozymes to regulate the expression of specific genes in eukaryotic cells has been well-documented, particularly with their application to both human gene therapy and plant biotechnology. Despite the simplicity of this approach, this technique usually results in only partial suppression of gene expression and, in some instances, even fails to regulate the gene of interest. The variation observed with antisense RNA and ribozyme-mediated regulation is further complicated by the many factors with the potential to impact on the effectiveness of these RNAs. Recent advances in the understanding of the global architecture of the nucleus, chromatin structure, and RNA metabolism provide useful and necessary information for designing novel approaches to improving antisense RNA and ribozyme regulation. These studies predict that the position of genes within the nucleus is not random and that transcripts produced from these genes follow specific tracks in migrating to the cell cytoplasm. These observations have the potential to impact significantly on the ways in which RNA-mediated forms of gene regulation are applied. The purpose of this review is to discuss the concept of colocalizing antisense RNAs and ribozymes with their target mRNAs and to introduce a variety of approaches aimed at achieving this goal.

Cytoplasm↗
PubMed1998

Aquaporin-2 and -3: representatives of two subgroups of the aquaporin family colocalized in the kidney collecting duct.

Since the molecular identification of the first aquaporin in 1992, the number of proteins known to belong to this family has been rapidly increasing. These members may be separated into two subgroups based on gene structure, sequence homology, and function. Regulation of the water permeability of the collecting ducts of the kidney is essential for urinary concentration. Aquaporin-2 and -3, which are representative of these subgroups, are colocalized in the collecting ducts. Understanding these subgroups will elucidate the differences between aquaporin-2 and -3. Aquaporin-2 is a vasopressin-regulated water channel located in the apical membrane, and aquaporin-3 is a constitutive water channel located in the basolateral membrane. In contrast to aquaporin-3, which appears to be less well regulated, many studies have now identified multiple regulational mechanisms at the gene, protein, and cell levels for aquaporin-2, thus reflecting its physiological importance. Evidence of the participation of aquaporin-2 in the pathophysiology of water-balance disorders is accumulating.

Amino Acid Sequence↗
PubMed2006

Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex.

Results of previous studies suggested a role of mitochondria in intracellular and cell-cell lactate shuttles. Therefore, by using a rat-derived L6 skeletal muscle cell line and confocal laser-scanning microscopy (CLSM), we examined the cellular locations of mitochondria, lactate dehydrogenase (LDH), the lactate-pyruvate transporter MCT1, and CD147, a purported chaperone protein for MCT1. CLSM showed that LDH, MCT1, and CD147 are colocalized with the mitochondrial reticulum. Western blots showed that cytochrome oxidase (COX), NADH dehydrogenase, LDH, MCT1, and CD147 are abundant in mitochondrial fractions of L6 cells. Interactions among COX, MCT1, and CD147 in mitochondria were confirmed by immunoblotting after immunoprecipitation. These findings support the presence of a mitochondrial lactate oxidation complex associated with the COX end of the electron transport chain that might explain the oxidative catabolism of lactate and, hence, mechanism of the intracellular lactate shuttle.

Animals↗
PubMed2002

Colocalization of the apical Cl-/HCO3- exchanger PAT1 and gastric H-K-ATPase in stomach parietal cells.

The apical Cl-/HCO exchanger called the putative anion transporter (PAT1; SLC26A6) is expressed on apical membranes of villus cells in the duodenum, but its location in the stomach remains unknown. Here we examined the cell distribution and membrane location of PAT1 in mouse stomach. Immunofluorescence labeling studies with anti-PAT1 antibodies and Dolichos biflorus agglutinin indicated the exclusive expression of PAT1 in gastric parietal cells. Double immunocytochemical staining revealed colocalization of PAT1 with the gastric H-K-ATPase, consistent with expression in tubulovesicles and/or the secretory canaliculus. Radiolabeled 36Cl flux studies demonstrated the functional presence of Cl-/HCO exchange in purified tubulovesicles of parietal cells. The expression of PAT1 was significantly decreased in parietal cells of gastric H-K-ATPase-null mice, which exhibit a sharp reduction in tubulovesicle membranes. These data indicate that the Cl-/HCO exchanger PAT1 is localized on tubulovesicular membranes, and they are consistent with the hypothesis that it functions in the maintenance of intravesicular ion concentrations in the resting state and dehydration of vesicles derived from the secretory membranes following the transition from the stimulated to the resting state.

Animals↗
PubMed2002

Asynchronous expression and colocalization of Bsep and Mrp2 during development of rat liver.

In the liver, function of the bile salt export pump (Bsep), a major canalicular exporter of bile salts, is complemented by activity of the multidrug resistance protein 2 (Mrp2), a canalicular organic anions transporter. Mrp2 was found capable of transporting various anticancer drugs out of cells, eventually undermining their therapeutic potential and contributing to multidrug resistance. We employed a RT-PCR, immunoblotting, and immunofluorescence to examine their gene, protein expression, and distribution of antigenic sites in the rat liver during development from 16-day-old fetus to adult animal. Bsep mRNA was almost undetectable before birth. It was first clearly expressed in the liver of newborn rats. On the contrary, Mrp2 mRNA was seen before birth, although at low levels. In concert with mRNA expression, Bsep protein was undetectable before birth, while Mrp2 protein was already expressed. Both proteins were clearly detectable in the postnatal period. Confocal immunofluorescent microscopy showed asynchronous appearance of Bsep and Mrp2 proteins during development but their colocalization in the bile canaliculi once each one is expressed. During the gestational period, a weak immunofluorescence for Mrp2 was observed only in livers of 16-day-old embryos. No fluorescence for Bsep was seen. Both proteins were clearly visualizable after birth, although the pattern of immunostaining varied. These findings provide molecular evidence that expression of both Bsep and Mrp2 during development is transcriptionally regulated. They also point out the differences in relevance to the liver function of the systems responsible for canalicular transport of bile salts versus organic anions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

NADPH diaphorase and nitric oxide synthase colocalization in enteric neurons of canine proximal colon.

Considerable evidence has recently been presented that suggests that nitric oxide (NO) is a nonadrenergic noncholinergic (NANC) neurotransmitter in gastrointestinal tissues. One of the criteria that must be satisfied before this hypothesis can be accepted is that enteric neurons must be shown to contain the enzymatic apparatus necessary to synthesize NO. Specific antibodies have been developed for NO synthase (NOS) isolated from rat cerebellum, and studies have shown that NOS copurifies and colocalizes with NADPH diaphorase activity, a commonly used neural marker. We used antibodies raised against the cerebellar NOS to determine the distribution of NOS-like immunoreactivity (NOS-LI) in enteric neurons of the canine proximal colon. We also tested whether NADPH diaphorase staining would label the population of neurons containing NOS-LI in this species. A subpopulation of neurons in myenteric and submucosal ganglia displayed NOS-LI and were colabeled with NADPH diaphorase. Labeled neurons had morphological characteristics similar to the Dogiel type I morphology. Cryostat sections showed NOS-positive nerve trunks throughout the circular and longitudinal muscle layers, but a high density of NOS-LI was observed within the submucosal pacemaker region, as predicted from physiological studies. These studies provide the first morphological support for the hypothesis that NO serves as a NANC neurotransmitter in the canine colon. The study also shows that the NADPH diaphorase reaction provides a useful method to label cells with NOS-LI.

Amino Acid Oxidoreductases↗
PubMed2006

TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle.

Transient receptor potential canonical (TRPC) proteins form Ca(2+)-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na(+)-K(+)-ATPase (NKA) pump and the Na(+)/Ca(2+) exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca(2+)-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

Animals↗