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PubMed1981

Subcellular Localization of IAA Oxidase in Peas.

Indoleacetic acid (IAA) oxidase has been reported to be involved in plant growth because of its alleged role in the control of endogenous IAA levels. This purported role was reevaluated in terms of the properties and subcellular location of the enzyme in etiolated pea (Pisum sativum L. var. Alaska) epicotyls.The enzymic properties of IAA oxidase in the floating pea epicotyl segments are similar to those previously reported in the literature. Other experiments indicate that approximately 70% of the activity is at the cut surfaces of the tissue. In addition, up to 50% of the IAA oxidase activity could be pelleted in a membranous fraction when the released enzyme was centrifuged at 10,000(g). Higher centrifugal forces reduced the proportion of the enzyme in the pellet, suggesting that vesicles containing IAA oxidase rupture at these forces.Subcellular localization of IAA oxidase was accomplished by use of sucrose density gradient centrifugation and fractionation. It was found that the enzyme is associated most closely with Golgi and also to a lesser degree with the lysosomes and endoplasmic reticulum. It is suggested that little if any IAA oxidase is freely soluble in the cytoplasm and is therefore unlikely to have a role in controlling normal growth via IAA levels. The enzyme may, however, have a role in diseased or damaged tissue.

Journal Article↗
PubMed1993

Subcellular Localization of Oxygen Defense Enzymes in Soybean (Glycine max [L.] Merr.) Root Nodules.

Soybean (Glycine max [L.] Merr.) root nodules contain the enzymes of the ascorbate-glutathione pathway to minimize oxidative damage. In the present study, fractionation and immunocytochemistry were used to determine the subcellular location of the enzymes of this pathway. All four enzymes (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase) were present in the soluble fraction from nodule plant cells and in isolated mitochondria. No activity was detected in peroxisomes. Bacteroids contained glutathione reductase but not the other enzymes of this pathway. Immunogold localization indicated that ascorbate peroxidase was present in the cytosol of infected and uninfected cells but not in the peribacteroid space. Results of immunogold and immunofluorescence studies indicated that monodehydroascorbate reductase was located primarily in the cell wall, suggesting that ascorbate regeneration in the cytoplasm may proceed primarily through the action of dehydroascorbate reductase. The possible roles of monodehydroascorbate reductase in cell wall metabolism are discussed.

Journal Article↗
PubMed2005

Subcellular localization of iron regulatory proteins to Golgi and ER membranes.

Interaction between iron regulatory proteins and iron responsive elements on certain mRNAs is at the core of regulation of intracellular iron homeostasis. Previous results suggested that in cultured cells iron regulatory proteins (IRPs) exist in cytosolic and microsomal subcellular locations and that this distribution is affected by cellular iron status. In this study, we tested the hypothesis that the membrane-associated fractions of iron regulatory proteins are specifically in the endoplasmic reticulum and Golgi membranes. Confocal microscopy revealed that IRP1 could be co-localized to the endoplasmic reticulum and the Golgi apparatus. To examine the intracellular distribution of IRPs biochemically, we used rats fed normal or iron-deficient diets. As expected, the IRPs were found predominantly in the cytosolic fraction. However, subfractionation of crude microsomal preparations revealed IRP1 in the Golgi apparatus. In animals fed an iron-deficient diet, IRP1 was found in the Golgi apparatus and the endoplasmic reticulum. To identify the mechanisms and factors involved in the localization of iron regulatory proteins in the cytosol and membrane fractions, cells were treated with a phorbol ester, a protein kinase C inhibitor (chelerythrine), hydrogen peroxide, interleukin-1beta, and 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N'N'-tetraacetic acid tetraacetoxy-methyl ester. The results indicate that iron-regulatory-protein-binding activity in the membrane fraction can be altered by cell stress or iron status and that phosphorylation plays a role in the translocation. As a result of this study we propose a novel model for intracellular distribution of IRPs and identify differences between the two iron regulatory proteins.

Alkaloids↗
PubMed1994

Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.

In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.

Amino Acid Sequence↗
PubMed1995

Reassessment of the subcellular localization of p63.

p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.

Amino Acid Sequence↗
PubMed1985

Subcellular localization of phosphatidylethanolamine N-methylation activity in rat heart.

Synthesis of phosphatidylcholine (PC) by S-adenosyl-L-methionine (AdoMet)-dependent methylation of phosphatidylethanolamine (PE) has been recently characterized in rat heart sarcolemma obtained by hypotonic shock-LiBr treatment method. The present study, employing different procedures for the isolation of purified cardiac sarcolemmal membranes in rat, confirms the existence of three catalytic sites which are specifically involved in the sequential methyl transfer reactions from PE to PC. Other subcellular organelles such as sarcoplasmic reticulum (microsomes) and mitochondria showed methyltransferase activity which was absent in myofibrils and in cytosolic fraction. Experiments with several concentrations of AdoMet revealed that the kinetic pattern of methyltransferase activity in both microsomes and mitochondria was comparable to that obtained in sarcolemma. In addition, the characteristics of three catalytic sites as identified by the synthesis of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and PC in these subcellular organelles were similar to those of sarcolemma. The results are consistent with the view that methyltransferase activity is localized in different membrane systems of the myocardium.

Animals↗
PubMed1989

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism.

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.

Acetyltransferases↗
PubMed1995

Two isoforms of Drosophila dynamin in wild-type and shibire(ts) neural tissue: different subcellular localization and association mechanisms.

The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD and 94 kD have been detected in wild-type and shi(n) adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2.000 x g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65-75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na2CO3 (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi(n) alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis arrest by GTP-gamma-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi(n) mutations and GTP-gamma-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).

Amino Acid Sequence↗
PubMed1982

The subcellular localization of enzymes of dolichol metabolism in rat liver.

Dolichyl phosphate is an intermediate in the glycosylation of N-glycosamidic linked glycoproteins in mammalian systems, and its availability may be a limiting factor in glycoprotein biosynthesis. The basic kinetics and subcellular distribution of enzymes which may influence the concentration of dolichyl phosphate in rat liver have therefore been investigated. These include dolichyl phosphate phosphatase, dolichol phosphokinase, dolichyl fatty acyl ester synthetase, GDP-mannose dolichyl phosphate mannosyl transferase, and UDP-glucose dolichyl phosphate glucosyl transferase. The specific activity of the enzymes was highest in the microsomes, except for dolichyl phosphate phosphatase and dolichyl fatty acyl ester synthetase, which were most concentrated in the plasma membrane and the cytosol fraction, respectively. The nuclei contained all of the enzyme activities while the mitochondria and cytoplasm were generally less active. The presence of both dolichol phosphokinase and dolichyl phosphate phosphatase in microsomes and nuclei, which contain the highest glycosyl transferase activities, may provide a means for direct enzymatic control of levels of dolichyl phosphate.

Acyltransferases↗