Improper Localization Research Articles

Oxidized LAT and Hyporesponsiveness

Correct localization to lipid rafts is essential for LAT (linker for activation of T cells)-mediated signaling through the T cell antigen receptor (TCR). Under conditions of chronic oxidative stress, T cells are hyporesponsive to stimuli. For example, in rheumatoid arthritis (RA), T cell insensitivity is thought to contribute to the disorder. Grinhuis et al . found that LAT localization in T cells is perturbed during chronic oxidative stress and results in decreased signaling outputs. LAT isolated from T cells treated with an inhibitor of glutathione (GSH) synthesis exhibited faster migration than LAT from untreated T cells under nonreducing conditions in gel electrophoresis. This suggested that LAT might form intramolecular disulfide bonds under conditions of chronic oxidative stress. Mutational analysis of Cys residues on LAT revealed that C117S or C26/29S mutants were partially insensitive to chronic oxidative stress (presumably because they could not become oxidized and form disulfide bridges), as shown by their continued presence in lipid rafts and by the ability of cells expressing these mutants to stimulate gene expression from interleukin-2 promoter-driven reporter genes. Oxidative stress also greatly reduced the TCR- and LAT-dependent activation of the transcription factors AP-1, NFAT, and NF-κB. However, expression of oxidation-insensitive LAT mutants in T cells partially reversed this inhibition. The authors suggest that the transmembrane helix of LAT might be sufficiently disrupted by chronic oxidative conditions such that LAT cannot remain localized to the lipid rafts. Other data suggested that disulfide bonds might form between Cys117 and probably Cys29. The data suggest that T cell hyporesponsiveness in chronic oxidative conditions results, in part, from improper localization of wild-type LAT, and thus ineffective signaling. For an excellent review on the function of the LAT protein, see the review by Wange . S. I. Gringhuis, E. A. M. Papendrecht-van der Voort, A. Leow, E. W. N. Levarht, F. C. Breedveld, C. L. Verweij, Effect of redox balance alterations on cellular localization of LAT and downstream T-cell receptor signaling pathways. Mol. Cell. Biol. 22 , 400-411 (2002). [Abstract] [Full Text] R. L. Wange, LAT, the linker for activation of T cells: A bridge between T cell-specific and general signaling pathways. Science's STKE (2000), http://www.stke.org/cgi/content/full/OC_sigtrans;2000/63/re1. [Abstract] [Full Text]

Considerations in the design and use of chimeric G protein-coupled receptors

This chapter describes methods that can be employed to generate hybrid G protein-coupled receptors (GPCRs) and that are useful for the proper analysis of such mutant receptors. All examples discussed in this chapter involve the mutational analysis of different members of the muscarinic acetylcholine receptor family (M 1 –M 5 ). These receptors are ideally suited for hybrid receptor studies, as the five muscarinic receptor subtypes show distinct pharmacological and functional properties, while sharing a high degree of sequence homology. A meaningful interpretation of hybrid receptor data requires that experimental conditions are carefully chosen, such that differences in receptor numbers or improper receptor folding or localization do not interfere with proper data analysis. Given the extraordinary size of the GPCR superfamily and the ease with which chimeric GPCRs can be created by the use of modern PCR technology, hybrid GPCRs will continue to serve as important tools for researches in the GPCR field for many years to come.

N-terminal RAG1 frameshift mutations in Omenn's syndrome: internal methionine usage leads to partial V(D)J recombination activity and reveals a fundamental role in vivo for the N-terminal domains.

Omenn's syndrome is an autosomal recessive primary immunodeficiency characterized by variable numbers of T lymphocytes of limited clonality, hypereosinophilia, and high IgE levels with a paradoxical absence of circulating B lymphocytes. We have previously attributed this disorder to missense mutations that render the RAG1/RAG2 recombinase only partially active. Here we report seven Omenn's patients with a novel class of genetic lesions: frameshift mutations within the 5' coding region of RAG1. Interestingly, we demonstrate in transient expression experiments that these frameshift deletion alleles remain partially functional for both deletional and inversional recombination and can hence explain the partial rearrangement phenotype observed in these patients. The rearrangement activity is mediated by truncated RAG1 proteins that are generated by alternative ATG usage 3' to the frameshift deletion and that demonstrate improper cellular localization. Taken together, our results suggest a novel mechanism for the development of immunodeficiency in a subset of Omenn's syndrome patients.

An alternatively spliced isoform of B-Myb is a transcriptional inhibitor.

B-Myb is a highly conserved member of the Myb transcription factor family. The primary transcript of the B-myb gene is spliced alternatively in two mRNAs which either contain or lack a sequence corresponding to the so-called exon 9A of c-myb. Recent studies showed that full-length B-Myb containing the exon 9A encoded amino acids is a cell cycle regulated transcription factor whose activity is stimulated by cyclin A/Cdk 2-dependent phosphorylation at the carboxyl-terminus of B-Myb. We have now investigated in more detail the transactivation potential of the shorter isoform of B-Myb lacking exon 9A. Here, we show that B-Myb lacking exon 9A has no transactivation activity even in the presence of cyclin A. This inactivity of the shorter isoform of B-Myb is not due an improper subcelluar localization. Our work suggests that B-Myb lacking exon 9A may act as an inhibitor for full-length B-Myb mediated transactivation. Furthermore, by analysing the transactivation potential of Gal4/B-Myb fusion proteins we have identified the amino-terminal part of the exon 9A as the principal transactivation domain of full-length B-Myb. The results presented here demonstrate that B-myb encodes both an activator and an inhibitor of transcription and, thus, reveal an additional level of regulation of B-Myb activity beside the known cyclin dependent mechanisms.

The Absence of Myelin P0 Protein Produces a Novel Molecular Phenotype in Schwann Cell.

In order to better understand the pathogenesis of demyelination in P0 knockout (P0-/-) mice, we analyzed the myelin gene expression and the localization of myelin proteins in P0 null mouse sciatic nerve. We have demonstrated that the severe demyelinating neuropathy of P0-knockout mouse is associated with changes in the program of myelin gene expression. Some changes in myelin gene expression occur early, others occur during adulthood. We also provide evidence that the absence of P0 is associated with changes in the localization of specific paranodal proteins in the peripheral nerve. These data suggest that P0 plays an important role, either directly or indirectly, in the program of Schwann cell gene expression and in the specific distribution of peripheral myelin proteins. Furthermore, myelin gene dysregulation and improper localization of paranodal proteins may account, in part, for the pathogenesis of demyelination in P0-knockout mice, as well as in human demyelinating peripheral neuropathy associated with mutations in the P0 gene.

Aggregation ofDictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein ?-subunit, G?2

The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol.

Septal localization of penicillin-binding protein 1 in Bacillus subtilis.

Previous studies have shown that Bacillus subtilis cells lacking penicillin-binding protein 1 (PBP1), encoded by ponA, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg2+, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of B. subtilis. In addition, we have used fluorescence and electron microscopy to show that growing ponA mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most ponA mutant cells, a significant proportion of ponA mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in B. subtilis. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.

The E1B 19K protein associates with lamins in vivo and its proper localization is required for inhibition of apoptosis.

Expression of the E1B 19K protein is required to inhibit apoptosis induced by E1A during adenovirus infection and transformation. E1B 19K is homologous to Bcl-2 in function and the two proteins also share limited amino acid sequence homology. Consequently, the E1B 19K and Bcl-2 proteins bind to and inhibit the cellular death-inducing proteins Bax, Bak and Nbk/Bik. Both E1B 19K and Bcl-2 localize to membranes of the nucleus and the endoplasmic reticulum. In addition to membrane association, and unlike Bcl-2, the E1B 19K protein is found associated with intermediate filament proteins in the cytoplasm and the nuclear lamina and copurifies with the lamins both during infection and transformation. While a membrane targeting domain at the C-terminus of Bcl-2 ensures its proper localization, the mechanism by which the E1B 19K protein localizes is unknown. Not surprisingly, lamin A fragments were cloned from a yeast two-hybrid screen for E1B 19K-interacting proteins. The interaction was demonstrated in yeast and mammalian cells in vivo and in vitro and was unique and specific to E1B 19K, with no interaction evident between Bcl-2 and lamin A. Mutants of lamin A/C which localized inappropriately in the cytoplasm or nucleus but retained E1B 19K binding, interfered with the nuclear envelope and cytoplasmic membrane targeting of the E1B 19K protein. Improper localization impaired the ability of the E1B 19K protein to inhibit apoptosis. Thus, proper localization of the E1B 19K protein is required for its function and the interaction of the E1B 19K protein with lamin A/C may represent a means for nuclear envelope localization.

Par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3.

The generation of asymmetry in the one-cell embryo of Caenorhabditis elegans is necessary to establish the anterior-posterior axis and to ensure the proper identity of early blastomeres. Maternal-effect lethal mutations with a partitioning defective phenotype (par) have identified several genes involved in this process. We have identified a new gene, par-6, which acts in conjunction with other par genes to properly localize cytoplasmic components in the early embryo. The early phenotypes of par-6 embryos include the generation of equal-sized blastomeres, improper localization of P granules and SKN-1 protein, and abnormal second division cleavage patterns. Overall, this phenotype is very similar to that caused by mutations in a previously described gene, par-3. The probable basis for this similarity is revealed by our genetic and immunolocalization results; par-6 acts through par-3 by localizing or maintaining the PAR-3 protein at the cell periphery. In addition, we find that loss-of-function par-6 mutations act as dominant bypass suppressors of loss-of-function mutations in par-2.

The hemolysin B protein, expressed in Saccharomyces cerevisiae, accumulates in binding-protein (BiP)-containing structures.

The hemolysin B (Hly B) protein of Escherichia coli, a member of the ABC-transporter family, was expressed in Saccharomyces cerevisiae and tested for its ability to complement a defect in the a-factor transporter Ste6. We found that HlyB was not able to restore mating ability to a STE6 deletion strain. The HlyB protein did not co-fractionate with Ste6 on sucrose gradients, indicating that improper localization of the HlyB protein could contribute to the lack of complementation. Immunofluorescence experiments suggest that HlyB is localized to structures derived from the endoplasmic reticulum (ER). The HlyB-expressing cells revealed a perinuclear staining typical of ER-localized proteins and intensely staining ring-like structures (HlyB-bodies). Double-label immunofluorescence experiments show that the HlyB structures also contain the ER binding protein (BiP), the product of the KAR2 gene. The HlyB protein, however, did not co-fractionate with another ER marker protein, the NADPH cytochrome c reductase. The HlyB bodies could be derivatives of a novel compartment of the early secretory pathway which contains BiP but not other resident ER proteins. In this case, HlyB could serve as a tool for the biochemical characterization of this compartment.

Improved late potential analysis in frequency domain

This work presents a technique to improve the identification of late potentials (LP) in patients affected by greater arrhythmogenic right ventricular disease (GARVD). Several authors have documented the correlation between GARVD and LP by means of time domain analysis. Moreover, the high incidence of bundle branch block in patients affected by GARVD suggests LP analysis in the frequency domain be performed. The method of spectral mapping of the ECG with Fourier transform was adopted. This consists in dividing the ST segment into 25 subsegments and estimating their frequency components by means of the fast Fourier transform. Recently, it was documented that this technique suffers from poor reproducibility of results. Low reproducibility is the consequence of an improper localization of the analysed QRS segments. An algorithm to increase the QRS end point identification reproducibility is proposed. An optimal QRS filter was adopted as well as a technique based on the Hilbert transform. This technique allowed the reliability of the normality factor estimates to be improved. The computed normality factors on the XYZ leads and on the vector magnitude were used to classify patients and healthy subjects; 28 patients affected by greater arrhythmogenic right ventricular disease and 35 healthy subjects were analysed in the study. High sensitivity was obtained with respect to GARVD and clinical sustained ventricular tachycardia by means of a cluster analysis technique. By applying the technique proposed in this paper the identification of LP in GARVD was increased from 47% to 88%, when clinical sustained ventricular tachycardia was documented, whereas in patients affected by GARVD but not prone to sustained ventricular tachycardia LP identification increases from 18% to 64%.

N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase.

In a screen designed to identify novel mutations in the mating response pathway of Saccharomyces cerevisiae, we isolated conditional alleles of NMT1, the gene encoding N-myristoyl transferase. Genetic data indicate that Nmt1 deficiency results in the activation of the pheromone response at the level of Gpa1, the alpha subunit of the pheromone-responsive G protein. We show that Gpa1 is myristoylated by Nmt1, and without this normally stable modification, Gpa1 is unable to inhibit pheromone signaling. This loss of Gpa1 function is probably not the result of improper subcellular localization. Unlike the mammalian G alpha i proteins alpha i and alpha o, nonmyristoylated Gpa1 is able to associate with membranes. In addition to Gpa1, our data indicate that Nmt1 myristoylates other proteins essential to vegetative growth.

Molecular analysis of the bicoid gene from Drosophila pseudoobscura: identification of conserved domains within coding and noncoding regions of the bicoid mRNA.

The specification of anterior positional information during Drosophila embryogenesis is largely dependent upon the function of the maternal-effect gene bicoid (bcd). Two aspects of bcd function are particularly striking. First, the bcd protein product forms a gradient during early embryogenesis, which regulates the transcription of at least one zygotic segmentation gene, hunchback, in a concentration dependent manner. Secondly, formation of the bcd protein gradient is dependent upon the specific localization of bcd mRNAs at the anterior end of the oocyte/embryo during oogenesis, a process which requires a cis-acting 625 nucleotide sequence within the 3' untranslated region of the bcd mRNA. We have cloned and sequenced the bcd gene from Drosophila pseudoobscura as a tool in identifying important functional domains within this transcription unit. DNA sequence comparisons reveal: (i) varying degrees of amino acid sequence conservation among the proposed functional domains of the bcd protein, (ii) the conservation of potential RNA secondary structures within the bcd mRNA localization element, and (iii) the maintenance of a short open reading frame within the 5' untranslated leader that may play a role in translational regulation. Finally, the D.pseudoobscura bcd gene partially rescues the phenotype of a bcd- mutation when placed into the D.melanogaster genome by germline transformation. The lack of full phenotypic rescue can be explained in part by the observed improper localization of the D.pseudoobscura bcd mRNA when expressed in D.melanogaster.

Cell‐free N‐glycosylation in Dictyostelium discoideum: Analysis of wild‐type and mutants defective in lipid‐linked oligosaccharide biosynthesis

N-glycosylation was measured in wild-type cell lysates of Dictyostelium discoideum and in two mutant strains that synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2 lacking terminal mannose and glucose residues. Endogenous lipid-linked oligosaccharide (LLO) was transferred to octanoyl-Asn-[125I]Tyr-ThrNH2 by membrane fractions. About 50% of the glycopeptide product remained associated with membranes. Taurocholate and saponin promoted and preserved glycosylation, but NP-40 and Triton X-100 did not. Using this artificial assay, the rate and extent of transfer of the truncated lipid-linked oligosaccharide in extracts of the two mutant strains, HL241 and HL243, was reduced 5-10-fold relative to that of wild-type. The low activity found in the mutant strains appears to result from either reduced affinity of the truncated LLO for the transferase or from its improper topological localization in the membrane. When protein N-glycosylation is measured in living cells it is nearly normal in HL241, but it is 3-4-fold decreased in HL243. Although the results of the in vitro and in vivo assays differ, they are not in conflict. Rather, they suggest that the static in vitro assay may be capable of revealing subtleties in the productive positioning of LLO and the oligosaccharyl transferase. The decrease in glycosylation seen in intact HL243 cells may be a consequence of the pleiotropic effects of the primary mutation rather than a direct result of the altered LLO structure. Genetic analysis showed that the mutation in HL241 is recessive, while the mutation in HL243 is dominant and prevents normal development. Thus, the two mutants share a lesion in lipid-linked oligosaccharide biosynthesis and in cell-free glycosylation, but differ in their in vivo glycosylation. Their primary defects are probably different.

Biochemical and genetic analysis of an antigenic determinant found on N‐linked oligosaccharides in Dictyostelium

Dictyostelium discoideum synthesizes many highly immunogenic carbohydrates of unknown structure and function. We have used monoclonal antibodies prepared against one of these called CA1 to investigate its structure and the consequences of its loss. CA1 is preferentially expressed on lysosomal enzymes as a specific arrangement of mannose-6-SO4 residues on N-linked oligosaccharides. Mutant strains HL241 and HL243 do not express CA1, and synthesize a truncated lipid-linked oligosaccharide (LLO) precursor that lacks the critical mannose residues needed for expression. The lesion appears to result from the loss of mannosyl transferase activity involved in LLO biosynthesis. The truncated LLO is poorly transferred to an artificial peptide acceptor in a cell-free N-glycosylation assay, and this appears to result from improper topological localization of the LLO or to a lower affinity of the LLO for the oligosaccharyl transferase. Although both mutants share these lesions, they are biochemically and genetically distinct. Only HL243 is lower in N-glycosylation in intact cells, and this is not a result of an altered structure of the LLO. There are other differences between the strains. HL241 can form fruiting bodies at a slower rate than normal while HL243 cannot aggregate. Genetic analysis of defects shows that the CA1 lesion in HL241 is recessive, while the lesion in both CA1 and in development are dominant and co-segregate in HL243 and are, therefore, likely to be in the same gene. Lysosomal enzyme targeting is normal but enzyme processing proceeds at a 2-3 fold slower rate in HL241 and HL243 compared to wild-type. Strain HL244 does not express CA1 since it completely lacks protein sulfation, but lysosomal enzyme targeting and processing proceeds at a normal rate, showing that sulfate is not essential for these processes. Alterations in oligosaccharide structure can have individualized effects on the biosynthesis of lysosomal enzymes. The results presented here illustrate how this approach can be used to study both the structure and function of carbohydrate epitopes.

Regulation of endocytic pH by the Na+,K+-ATPase in living cells.

Acidification of endocytosed ligands destined for lysosomes is biphasic, with a rapid drop to pH 6, followed by a slow decrease to pH 5. Continuous measurements of transferrin acidification have confirmed that the pH minimum in early (presorting) endosomes is approximately pH 6. On the basis of measurements of endosomal acidification in vitro, it has been proposed that the pH in the early endosome is limited by the internalization of the Na+,K+-ATPase, which generates an interior-positive membrane potential in this compartment [Fuchs, R., Schmid, S. & Mellman, I. (1989) Proc. Natl. Acad. Sci. USA 86, 539-543]. We present two lines of evidence that strongly implicate the Na+,K+-ATPase as a major regulatory element of endocytic pH in vivo. First, ouabain, a specific inhibitor of the Na+,K+-ATPase, interferes with the regulation of acidification in early endocytic compartments. Transferrin is normally rapidly acidified to pH 6.0-6.2, followed by alkalinization during recycling. In the presence of ouabain, the minimum pH of transferrin-containing endosomes decreases from 6.0-6.2 to less than 5.3. Second, ouabain eliminates the resistance to both the growth inhibitory and vacuologenic effects of chloroquine in the lysosomal acidification defective cell line CHL60-64. The phenotype of this cell line is consistent with a defect in the removal or inactivation of the early acidification regulatory elements from the late endocytic compartments. The ouabain data suggest that the defect in this cell line is due to improper localization of the Na+,K+-ATPase. A model for pH regulation and vacuolation by weak bases is discussed.

Targeting of E. coli beta-galactosidase to the nucleus in yeast.

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.