Subcellular localization of cucumovirus-associated satellite double-stranded RNAs

Viral satellites are small RNAs that depend on the presence of the specific helper virus for replication and that can modulate viral disease expression. The in vivo subcellular location of the double-stranded (ds) form of different Cucumovirus-associated satellite RNAs, which accumulate in large quantities in infected tobacco plants, is reported here. Subcellular fractions were obtained by differential centrifugation and characterized by their specific nucleic acid content and by electron microscopy. Results indicate that the viral and satellite ds-RNAs copurify with a size-homogeneous vesicular fraction. A similar vesicular fraction was also isolated from healthy tobacco plants. The results suggest that the replication of satellite RNAs occurs in close association with these vesicles and are consistent with the hypothesis of the satellite dependence on the viral-coded replicase.     

Subcellular localization of casein kinase I

An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus.     

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.     

Subcellular localization of oestrogen-induced uterine peroxidase.

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.     

Subcellular localization of transglutaminase. Effect of collagen.

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.     

Protease of adenovirus type 2. Subcellular localization.

The subcellular localization of the adenovirus type 2 core polypeptide specific protease activity was investigated using an in vitro assay system. The protease activity was recovered exclusively from infected cell nuclei and was insoluble, sedimenting with the membrane fraction. Endogenous activity could be demonstrated in young virions which contain precursor PVII molecules. This protease activity only became sensitive to L-1-tosylamide-2-phenylethylchloromethyl ketone- or phenylmethylsulfonyl fluoride-mediated inhibition after disruption of the virus particles by sonication, suggesting that the enzyme was internally located. The putative precursors to virus particles, referred to as top components, which do not contain a full complement of viral DNA, did not contain protease activity. The protease released from sonicated virions converted exogenous PVII substrate molecules to polypeptide VII. The noninfectious H2ts1 virus particles synthesized at the nonpermissive temperature phenotypically resemble young virions, but unlike their wild type counterparts, were devoid of protease activity. The results show that the protease enters the precursor particles concurrently with the viral chromosome and that its presence is a prerequisite for the processing and subsequent maturation of infectious adenovirions.     

Subcellular localization of gamma-glutamyltransferase in calf thymocytes.

The subcellular localization of gamma-glutamyltransferase in calf thymocytes was investigated and compared with that of alkaline phosphodiesterase I, alkaline nitrophenyl phosphatase, succinate-tetrazolium oxidoreductase (succinate-INT reductase) and lactate dehydrogenase after two different methods of cell disruption and differential centrifugation. Most of the activity was recovered in the crude membrane fractions (43.0%), but significant amounts co-pelleted with the large-granule (mitochondria) fractions (31%). The specific activity of the gamma-glutamyltransferase in the purified plasma membrane was 30-50 times that of the enzyme in the cell homogenate and had a similar subcellular distribution to the plasma-membrane markers, alkaline phosphodiesterase I and alkaline nitrophenyl phosphatase. It was concluded that gamma-glutamyltransferase was primary a plasma-membrane-bound enzyme, and that its location in other subcellular fractions was probably due to their contamination with plasma-membrane vesicles.     

人类GABARAPL2基因的亚细胞定位

为了对GABARAPL2（GABAA受体相关蛋白相似蛋白2）基因的功能进行初步分析,首先通过同源比较的方法将序列与其同源物进行比较,发现GABARAPL2的氨基酸序列与GABARAP（GABAA受体相关蛋白）高度同源,而GABARAP已证实通过结合细胞骨架的微蛋白,使GABAA受体聚集,定位在细胞膜上,本文采用PCR法从人脑组织的cDNA文库中扩增出GABARAPL2的cDNA,克隆至T质粒载体中进行测序验证,然后以此为模板引物中引入酶切位点再次PCR,扩增出GABARAPL2的开放阅读框,并将其插入到加强型绿色荧光蛋白融合表达载体EGFP中,将绿色荧光蛋白标记的GABARAPL2和GABARAP分别转染HLF细胞株,结果两种蛋白的分布情况基本一致,在细胞质内和核内均有分布,而且核内的分布较胞质为多,结构功能域分析表明,GABARAPL2含有蛋白激酶C磷酸化位点和酪氨酸激酶磷酸化位点,可能通过磷酸化参与细胞骨架的变化,结论 GABARAPL2和GABARAP不仅在胞质中作为受体相关蛋白协助受体的聚集、定位,还参与体内许多其它重要的生理过程。     

Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.     

Subcellular localization of phosphatidylinositol synthesis

It is well-established that the endoplasmic reticulum is the major site of phosphatidylinositol (PtdIns) synthesis. The PtdIns synthetic ability of other organelles, such as plasma membrane and nucleus, remains controversial. In the present study, we re-examine this question by comparing PtdIns synthesis in isolated cytoplasts (enucleated cells) with that in corresponding karyoplasts (nuclei surrounded by plasma membrane but lacking most cytoplasmic components). We report that cytoplasts are competent to carry out both basal and stimulated PtdIns synthesis as well as polyphosphoinositide hydrolysis, while karyoplasts can neither synthesize PtdIns nor hydrolyze phosphoinositides in response to agonists. The karyoplasts are, however, capable of synthesizing phosphatidylcholine (PtdCho), as previously reported. From these data, we conclude that PtdIns synthesis is limited to cytoplasmic components, and cannot be sustained by either plasma membrane or nucleus under conditions that permit robust PtdCho synthesis.     

Subcellular localization of gamma-glutamyl carboxypeptidase and of folates.

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

Subcellular localization of presenilins during mouse preimplantation development.

The genes defective in familial Alzheimer's disease encode the proteins presenilin 1 and 2 (PS1 and 2). Expression of presenilins (PSs) and their proteolytic processing are regulated during neuronal development. Even though these proteins are detected and regulated mainly in Golgi and endoplasmic reticulum, their subcellular distribution during the development is not known. The present study aimed to investigate the localization of PSs and their role during early developmental stage using mouse embryo model. At preimplantation stage, PSs were detected not only in cytoplasm, but also in the nucleus from oocyte to 2.5 dpc (day postcoitum), then disappeared in the nucleus at blastocyst stage (3.5 dpc). Antisense against PS1 and PS2 decreased the transition to blastocyst stage, whereas each antisense alone had no effect. Treatment with lactacystin (26S proteosome inhibitor), which arrest cell cycle at M phase, redistributed PSs into centrosome-kinetochore microtubule. PS2 overexpression in HEK 293 cell arrested cell cycle at S phase. These data suggest that PSs play key roles in cell division and differentiation during early development.     

Subcellular localization of hexokinases I and II directs the metabolic fate of glucose

Background

The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK''s channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use).

Methodology/Principal Findings

To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P.

Conclusions/Significance

These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HK''s allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury.     

Subcellular localization of superoxide dismutase in rat liver.

The subcellular localization of superoxide dismutase was investigated in rat liver homogenates. Most of the superoxide dismutase activity is present in the soluble fraction (84%), the rest being associated with mitochondria. No indications for the occurrence of superoxide dismutase in other subcellular structures, particularly in peroxisomes, was found. Mitochondrial activity is not due to adsorption, since the sedimentable activity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shock and sonication shows that half of the mitochondrial superoxide dismutase activity is localized in the intermembrane space, the rest of the enzyme being a component of the matrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the inner membrane, from which it can be desorbed by low (0.04M) concentration of KCl. Superoxide dismutase activity was found in all rat organs investigated. Maximal activity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highest specific activity is associated with the medulla.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

Subcellular localization of procollagen I and prolyl 4-hydroxylase in corneal endothelial cells

To investigate the molecular mechanism of intracellular degradation of type I collagen in normal corneal endothelial cells (CEC), we studied the role of prolyl 4-hydroxylase (P4-H) and protein disulfide-isomerase (PDI; the beta subunit of P4-H) during procollagen I biosynthesis. When the subcellular localization of P4-H and PDI was determined, P4-H demonstrated a characteristic diffuse endoplasmic reticulum (ER) pattern, whereas PDI showed a slightly more restricted distribution within the ER. When colocalization of procollagen I with the enzymes was examined, procollagen I and PDI showed a large degree of colocalization. P4-H and procollagen I were predominantly colocalized at the perinuclear site. When colocalization of type IV collagen with PDI and P4-H was examined, type IV collagen was largely colocalized with PDI, which showed a wider distribution than type IV collagen. Type IV collagen is similarly colocalized with P4-H, except in some perinuclear sites. The colocalization profiles of procollagen I with both PDI and P4-H were not altered in cells treated with alpha,alpha'-dipyridyl compared to those of the untreated cells. The underhydroxylated type IV collagen demonstrated a colocalization profile with PDI similar to that observed with procollagen I, while the underhydroxylated type IV collagen was predominantly colocalized with P4-H at the perinuclear sites. Immunoblot analysis showed no real differences in the amounts of the beta subunit/PDI and the catalytic alpha subunit of P4-H in CEC compared to those of corneal stromal fibroblasts (CSF). When protein-protein association was determined, procollagen I was associated with PDI much more in CEC than it was in CSF, whereas type IV collagen showed no differential association specificity to PDI in both cells. Limited proteolysis of the newly synthesized intracellular procollagen I with pepsin showed that procollagen I in CEC was degraded by pepsin, whereas CSF contained type I collagen composed of alpha1(I) and alpha2(I). These findings suggest that procollagen I synthesized in CEC is not in triple helical conformation and that the improperly folded procollagen I may be preferentially associated with PDI before targeting to the intracellular degradation.     

Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. I. Subcellular localization and functional role.

Our investigations indicate that a variety of neutral serine proteases exist in highly purified, IL-2-activated rat NK (A-NK) cells. These enzymatic activities are not restricted to only cytolysin-containing granules and are not defined by only the assay of N-alpha-benzyloxycarbonyl-L-lysine thiobenzylesterase activity. These activities, which we term A-NKP 1, A-NKP 2, A-NKP 3, and A-NKP 4, cleave, respectively, the following fluorogenic peptide substrates: Boc-Phe-Ser-Arg-7-amino-4-methylcoumarin (AMC, trypsin-like); Suc-Ala-Ala-Phe AMC (chymotrypsin-like); Suc-Gly-Pro-Leu-Gly-Pro AMC (collagenase-like), and Z-Phe-Arg AMC (another trypsin-like enzyme). The proteases A-NKP 1, A-NKP 2, and A-NKP 3 are not cell surface-associated and appear to be cytosolic as defined by isopycnic sucrose density gradient centrifugation. In contrast, A-NKP 4 appears to be located in lysosomes. Treatment of rat A-NK cells with protease inhibitors that inhibit A-NKP 2 and A-NKP 3 also substantially inhibit A-NK cell-mediated cytotoxicity against both NK-sensitive and -resistant targets (YAC-1 and P815, respectively). These results indicate that A-NKP2 and A-NKP 3 may play a role in IL-2-activated NK cell-mediated cytotoxicity. A variety of proteolytic enzymes, in addition to granzymes, therefore exist in A-NK cells. Our studies indicate that a prerequisite to a thorough understanding of the role of proteases in killer cell function is the investigation of several classes of enzymes in addition to granzymes contained in lytic granules.