The human ribosomal protein L7a is a component of the major ribosomal subunit. We transiently expressed in HeLa cells L7a--galactosidase fusion proteins and studied their subcellular localization by indirect immunofluorescence staining with anti--galactosidase antibodies. We have identified three distinct domains responsible for the nuclear targeting of the protein: domain I, amino acids 23-51; domain II, amino acids 52-100; domain III, amino acids 101-220, each of which contains at least one nuclear localization signal (NLS). Through subcellular localization analysis of deletion mutants of L7a--galactosidase chimeras, we demonstrate that domain II plays a special role because it is necessary, although not sufficient, to target the chimeric -galactosidase to the nucleoli. In fact, we demonstrate that the nucleolar targeting process requires the presence of domain II plus an additional basic domain that can be represented by an NLS or a basic stretch of amino acids without NLS activity. Thus, when multiple NLS are present, each NLS exerts distinct functions. Domain II drives nucleolar accumulation of a reporter protein with the cooperative action of a short basic amino acid sequence, suggesting a mechanism requiring protein-protein or protein-nucleic acid interactions.The biogenesis of eukaryotic ribosomes is a complex process that takes place in the cell nucleus. Soon after synthesis in the cytoplasm, ribosomal proteins (r-proteins) 1 are transported to the nucleus and subsequently accumulated in the nucleoli where they are associated with the precursor-rRNAs (pre-rRNAs), which are concomitantly processed into mature rRNA molecules (1). The assembled ribosomal subunits are eventually exported to the cytoplasm to function in protein biosynthesis. Thus, the biogenesis of eukaryotic ribosomes entails an intensive traffic of molecules across the nuclear membrane and, therefore, r-proteins are a good model with which to study the mechanism of nuclear transport and nucleolar accumulation of proteins. The nuclear transport of proteins depends upon the presence of one or more nuclear localization signals (NLS). These sequences have been found throughout the polypeptide chain (2) and, in most cases, consist of either short basic amino acid sequences like the NLS of the SV40 large T-antigen ( 126 PKKKRKV 132 ) (3, 4) or longer bipartite sequences consisting of two stretches of basic amino acids separated by about 10 amino acids (5). NLS are both necessary and sufficient to target a cytoplasmic protein to the nucleus (4). Much less is known about the mechanism of nucleolar targeting of proteins.Studies on the nucleolar accumulation of viral proteins have suggested that, like nuclear transport, nucleolar transfer is mediated by short amino acid sequences, namely nucleolar localization signals (NOS) (6 -9). A NOS motif, however, is not present in the cellular nucleolar proteins that have been identified so far, e.g. NO38 (10), nucleolin (11, 12), NSR1 (13), GAR1 (14). Studies on the targeting mechanism of the nucleolar p...
The role of iron-dependent oxidative metabolism in protecting the oxidable substrates contained in mature adipocytes is still unclear. Because differentiation increases ferritin formation in several cell types, thereby leading to an accumulation of H-rich isoferritins, we investigated whether differentiation affects iron metabolism in 3T3-L1 pre-adipocytes. To this aim, we evaluated the expression of the genes coding for the H and L ferritin subunits and for cytoplasmic iron regulatory protein (IRP) during the differentiation of 3T3-L1 cells in adipocytes induced by the addition of isobutylmethylxanthine, insulin, and dexamethasone. Differentiation enhanced ferritin formation and caused overexpression of the H subunit, thus altering the H/L subunit ratio. Northern blot analysis showed increased levels of H subunit mRNA. A gel retardation assay of cytoplasmic extract from differentiated cells, using an iron-responsive element as a probe, revealed enhanced an RNA binding capacity of IRP1, which correlated with the increase of IRP1 mRNA. The observed correlation between differentiation and iron metabolism in adipocytes suggests that an accumulation of H-rich isoferritin may limit the toxicity of iron in adipose tissue, thus exerting an antioxidant function.Ferritin, the intracellular protein required for iron storage, and transferrin, which transports iron into the cells through membrane-specific receptors, are the main proteins controlling cellular iron homeostasis. Ferritin has an approximate mass of 450 kDa and is composed of 24 subunits of two types, namely H and L, in any ratio (1). Changes in iron availability regulate ferritin expression primarily at translational level through specifically regulated protein-RNA interactions between iron regulatory proteins (IRPs) 1 and iron-responsive elements (IREs) contained within the 5Ј-untranslated region of the H-and Lferritin mRNA (2). When intracellular concentrations of iron are low, IRP binding to IRE cis-elements represses ferritin translation; and conversely, when intracellular concentrations of iron are high, IRP is unable to bind IRE, and ferritin mRNA is efficiently translated (3).Two distinct IRPs have been identified: IRP1 and IRP2 (for recent reviews, see Refs. 4 -7). IRP1 has been identified as the cytosolic counterpart of aconitase, a key enzyme in the mitochondrial citric acid cycle (8, 9). We have recently shown that its RNA binding activity is inhibited by oxalomalate, a competitive inhibitor of aconitase (10). IRP2 has a different pattern of tissue specificity (11) and binds IRE-containing mRNA with an affinity similar to that of IRP1 (12).Ferritin synthesis is stimulated during development, cellular differentiation, and inflammation, as well as by some hormones and cytokines (13). With the aim of evaluating the role of iron metabolism on the protection of the highly concentrated oxidable substrates in adipocytes, we investigated the expression of the genes encoding the H-and L-ferritin subunits and IRP1 during differentiation of 3T3-L1 cells to adip...
cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits.In the brain the major regulatory isoform RII and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RII and AKAP75; conversely, granule cerebellar cells express predominantly RI␣ and RII␣. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RII synthesis by antisense oligonucleotides inhibited cAMPinduced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RII and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter.These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.cAMP formed by adenylyl cyclases after stimulation of Gprotein-coupled receptors binds the regulatory subunits (R) of the tetrameric PKA 1 holoenzyme and promotes dissociation of the catalytic subunits (C-PKA). A fraction of C-PKA translocates to the nucleus and stimulates cAMP-dependent gene expression (1-3). Multiple isoforms of PKA are determined by their specific regulatory subunits. Four regulatory subunits (RI␣, RI, RII␣, and RII) have been cloned. PKA containing RII is the predominant PKA isoform in the brain and is expressed in the cortex, whereas in the brainstem and cerebellum (except Purkinje cells) RII has not been found (4 -6). In mammalian brain, signals triggered by cAMP are targeted to specific effector sites by the tethering of cAMP-dependent protein kinases to intracellular compartments (4,7,8). PKAII is bound to membranes via specific anchor proteins (AKAPs), which bind R subunits. Bovine brain AKAP75 has been studied as prototype of kinase A anchor protein and shares high homology with human AKAP79 and rat AKAP150 (9, 10). These proteins have similar properties, related sequences, and are recognized by the same antibodies (9 -11). AKAP150/75 and RII are co-localized in the dendritic cytoskeleton and perikarya of forebrain neurons; both proteins have not been found in cerebellar granule cells (6).Although the structure and expression pattern of the PKA regulatory subunits and AKAPs are well documented, the functional role of these proteins in the transduction of cAMP signals is still poorly understood. It is not known how the different PKA isoforms in different districts of the central nervous system receive and transmit cAMP signals.We have chosen primary cortical and granule cerebellar neurons as prototype cells with different PKA composition and localization. PKA in cortical neurons is mainly of II type and is membrane-anchored by AKAP150/75. Conversely, granule cerebellar cel...
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