Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 ϫ g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmicphase cells. In contrast, sterol ⌬ 24 -methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.
IntroductionDiamine oxidase (DAO; EC 1.4.3.6) catalyzes the oxidative deamination of histamine and other biogenic amines in a reaction that produces the corresponding aldehyde as well as ammonia and hydrogen peroxide [1]. Hydrogen peroxide is a highly reactive compound and may cause cellular damage if not scavenged appropriately [2]. Therefore, we reasoned that the hydrogen peroxide producing and degrading enzymes might be co-localized in mammalian tissues and analyzed the cellular and subcellular distribution of DAO and of catalase (EC 1.11.1.6), the major hydrogen peroxide scavenging enzyme [3].
Materials and methodsPorcine kidney specimens were prepared immediately post mortem and fixed in 4% buffered paraformaldehyde for 24 h at 4°C. The specimens were then embedded in paraffin and sections of 5 to 25 mm were cut. The sections were dewaxed in xylene, rehydrated in ethanol, and incubated with antibodies specific for DAO and for catalase. DAO was detected with a rabbit polyclonal anti-DAO antibody (1: 2000 dilution) [4] followed by incubation with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbit immunoglobulins (1:50 dilution) (Dako, Glostrup, Denmark). Catalase was detected with a mouse monoclonal anti-catalase antibody (1:200 dilution) [5] followed by incubation with tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat antimouse immunoglobulins (1:50 dilution) (Sigma, St. Louis, MO, USA). All antibodies were diluted in 50 mM Tris hydrochloride pH 7.5/ 150 mM NaCl/1% bovine serum albumin and sections were washed with 50 mM Tris hydrochloride pH 7.5/150 mM NaCl/0.05% Tween 20. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI, 1 mg/l) to visualize cell nuclei. The sections were mounted with fluorescence mounting medium (Dako) and analyzed with a Leica TCS 4D confocal laser scanning fluorescence microscope. False-color images of individual optical sections and projections as well as stereo views with depths ranging from 5 to 20 mm were prepared by digital image processing, using the Leica ScanWare TM software.
Results and discussionLittle attention has previously been paid to the fact that diamine oxidase and related amine oxidases in addition to inactivating primary amines also produce hydrogen peroxide, a compound dangerous for cells due to its oxidative potential and as a precursor of oxygen radicals [2]. Our interest in the fate of this potentially harmful reaction product prompted us to use antibodies specific for DAO [4] and for catalase [5], the major hydrogen peroxide scavenging enzyme, to investigate the cellular and subcellular localisation of these enzymes and to ask whether they are co-localized in porcine kidney.Interestingly, we found DAO and catalase to be expressed in the same cells, namely in proximal tubular epithelial cells (Fig. 1). Both enzymes showed decreasing levels of expression from the outer cortex towards the medulla. However, the proteins were present in different subcellular compartments. In agreement with earlier work [6], we found DAO to be present in sm...
Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.
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