Ferric iron reduction and iron uptake in eucaryotes: studies with the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
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Biological Transport↗
Biomedical subjects
R D Klausner
Publications and source records attributed to R D Klausner.
Familial occurrence of pleomorphic adenoma.
Pleomorphic adenoma is the most common neoplasm found in the parotid gland. The familial occurrence of any salivary gland neoplasm is rare. There are 8 previous reports in the literature of familial occurrence of salivary gland tumors. Two of these report the occurrence of pleomorphic adenoma of the parotid gland in siblings; all 4 patients were adults. We report here an 11-year-old male who presented with a left parotid mass and underwent parotidectomy. The mass proved to be a pleomorphic adenoma. His sister presented 9 years later at the age of 15 years with a similar mass also in the left parotid area. She underwent superficial parotidectomy which revealed the mass to be a pleomorphic adenoma. We believe these are the youngest siblings reported to date in which familial pleomorphic adenoma has occurred.
Adenoma, Pleomorphic↗
ARF: a key regulatory switch in membrane traffic and organelle structure.
The small GTP-binding protein ADP ribosylation factor (ARF) regulates, through a GTP cycle, the reversible binding of cytosolic coat proteins to Golgi membranes. By determining the binding and release of coat proteins from membranes, ARF controls the production and lifetime of coated-membrane structures. In the past year, studies suggesting a role for ARF in phospholipid metabolism have broadened our perspective on ARF function within the cell.
ADP-Ribosylation Factors↗
Role of the CDR1 region of the TCR beta chain in the binding to purified MHC-peptide complex.
Single alanine substitutions were introduced into the CDR1 region of the beta chain of a Kd-restricted TCR. Mutants and wild-type TCR were attached to the zeta chain of the CD3 complex and expressed at the surface of a rat basophil cell line. Transfectants were tested for the binding of purified soluble Kd-peptide complexes. With this experimental system, accessory molecules are unlikely to play a major role and the contribution of each residue to the interaction can be addressed. Results show that all positions in the CDR1 region are involved in the binding to the Kd-peptide complex but at varying degrees. These effects are discussed in relation to a molecular model of the TCR. Comparison of these results with previous data obtained in a T cell hybridoma system suggests the existence of a threshold in the TCR affinity necessary for mature T cell activation.
Animals↗
Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif.
The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.
Amino Acid Sequence↗
A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.
Study of the T-cell repertoire in humans has been hampered by the lack of monoclonal antibodies (mAbs) to the T-cell receptor (TCR) variable region (V) gene products. We describe a method for producing mAbs to the human TCR beta-chain V (V beta) gene products in which mice were immunized with a rat basophil cell line (RBL-2H3) transfected with the extracellular domain of the TCR heterodimer fused to the lambda chain of CD3. These cells acted as excellent immunogens for raising anti-TCR mAb and also formed the basis of a rapid screening assay. We generated mAbs against V beta protein of the TCR, showed that these mAbs stained approximately 1% of peripheral blood T cells, and further showed that the mAbs could stimulate proliferation of these T cells. We then characterized the mAbs by amplifying TCR cDNA derived from mAb-stimulated cells and sequencing the beta chain. All clones sequenced used the V beta 7.1 chain, proving conclusively that the mAbs generated were specific for V beta 7.1 subfamily. This method generates mAbs to human TCR V beta proteins efficiently and might allow production of a complete panel of mAbs directed against human TCR V beta proteins.
Amino Acid Sequence↗
Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56lck tyrosine kinase: basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules.
Signals transduced through the T cell antigen receptor (TCR) are modulated by the src family tyrosine kinase p56lck (lck), which associates in mature T cells with the coreceptor molecules CD4 and CD8. Here we describe a novel function of lck in immature CD4+CD8+ thymocytes, that of regulating TCR expression. Activation of lck in immature CD4+CD8+ thymocytes by intrathymic engagement of CD4 maintains low TCR expression by causing most TCR components to be retained and degraded within the endoplasmic reticulum. Importantly, activation of lck in immature CD4+CD8+ thymocytes results from engagement of surface CD4 molecules, but not surface CD8 molecules, despite the nearly fourfold greater surface expression of CD8 than CD4. The competence of CD4 to activate lck in CD4+CD8+ thymocytes relates to the fact that a relatively large fraction of surface CD4 molecules (25-50%) are associated with intracellular lck molecules, whereas only 2% of surface CD8 molecules are associated with lck. The amount of lck associated with CD4 in CD4+CD8+ thymocytes is diminished by chronic CD4 engagement in the thymus, as activated lck molecules subsequently dissociate from CD4. Indeed, the amount of lck associated with CD4 in CD4+CD8+ thymocytes is markedly increased in major histocompatibility complex (MHC) class II- mice that lack the intrathymic ligand for CD4 and in which surface CD4 molecules are consequently not engaged. Thus, the present study demonstrates that (a) activation of lck in CD4+CD8+ thymocytes regulates distribution and expression of TCR components; (b) unlike CD4 molecules, CD8 molecules on CD4+CD8+ thymocytes cannot efficiently activate lck despite their significantly greater surface expression; and (c) the amount of lck associated with CD4 in the CD4+CD8+ thymocytes is inversely related to the extent of CD4 engagement by MHC class II molecules in the thymus.
Animals↗
The interaction between the iron-responsive element binding protein and its cognate RNA is highly dependent upon both RNA sequence and structure.
To assess the influence of RNA sequence/structure on the interaction RNAs with the iron-responsive element binding protein (IRE-BP), twenty eight altered RNAs were tested as competitors for an RNA corresponding to the ferritin H chain IRE. All changes in the loop of the predicted IRE hairpin and in the unpaired cytosine residue characteristically found in IRE stems significantly decreased the apparent affinity of the RNA for the IRE-BP. Similarly, alteration in the spacing and/or orientation of the loop and the unpaired cytosine of the stem by either increasing or decreasing the number of base pairs separating them significantly reduced efficacy as a competitor. It is inferred that the IRE-BP forms multiple contacts with its cognate RNA, and that these contacts, acting in concert, provide the basis for the high affinity of this interaction.
Base Sequence↗
Modification of a free Fe-S cluster cysteine residue in the active iron-responsive element-binding protein prevents RNA binding.
The iron-responsive element-binding protein (IRE-BP) binds to specific RNA stem-loop structures called iron-responsive elements (IREs), which mediate the post-transcriptional regulation of a variety of genes involved in iron metabolism. The IRE-BP is cytosolic aconitase, and a [4Fe-4S] cubane cluster is required for aconitase activity but is associated with loss of IRE binding affinity. Chemical modification of the IRE-BP can abrogate RNA binding and the 3 cysteines predicted to coordinate the Fe-S cluster in the IRE-BP could be targets for modification. We report the expression of recombinant IRE-BP in which the three putative cluster cysteines (Cys-437, Cys-503, and Cys-506) have been mutated to serine residues. Replacement of any or all of these cysteine residues results in a complete loss of aconitase activity. While all of the mutants bind RNA, substitution of Cys-437 specifically renders the IRE-BP resistant to inactivation by low concentrations of N-ethylmaleimide or diamide. These results identify Cys-437 as the target of in vitro regulation of RNA binding in the IRE-BP and suggest that, in the RNA-binding form of the protein, Cys-437 is free and therefore available for modifications that inhibit RNA binding.
Amino Acid Sequence↗
Brefeldin A reversibly inhibits secretion in Saccharomyces cerevisiae.
Brefeldin A has proven to be a useful pharmacologic tool, which, when added to mammalian cells, results in a block in secretion as well as the structural disruption of specific intracellular organelles. In spite of our understanding of some of the biochemistry underlying the action of brefeldin A, the most proximal molecular target(s) of the drug remain elusive. In attempting to address this problem, a genetic approach will undoubtedly prove useful and complementary to the biochemical identification of such a site(s). As a result of the relatively resistant nature of wild-type Saccharomyces cerevisiae to brefeldin A, an approach utilizing yeast genetics has not been possible. We report the selective sensitivity of three drug-sensitive strains of S. cerevisiae (ise-1, ISE-2, and erg6) with enhanced membrane permeability allowing uptake of brefeldin A. Upon addition of the drug, growth is dramatically inhibited and invertase secretion is rapidly, specifically, and reversibly blocked at the level of the endoplasmic reticulum. In addition, only structural analogues of brefeldin A effective in mammalian cells are active in these yeast strains.
Antifungal Agents↗
Transmembrane domain length affects charge-mediated retention and degradation of proteins within the endoplasmic reticulum.
Previous studies have shown that the presence of potentially charged amino acid residues within the transmembrane domains of type I integral membrane proteins can result in protein retention and, in some cases, degradation within the endoplasmic reticulum (ER). An apparent exception to this observation is the CD3-epsilon chain of the T-cell antigen receptor complex, which is relatively stable in spite of having a transmembrane aspartic acid residue. A chimeric protein (T epsilon T) made by replacing the transmembrane domain of the Tac antigen with that of CD3-epsilon was normally transported to the cell surface, indicating that the transmembrane domain of CD3-epsilon was essentially unable to confer the phenotype of ER retention and degradation to another protein. Progressive shortening of the T epsilon T transmembrane domain, however, resulted in increasing retention and degradation of the mutant proteins in the ER. Conversely, a mutant Tac protein containing a single aspartic acid residue in its transmembrane domain was found to be retained and degraded in the ER, but when the transmembrane domain was lengthened, ER retention and degradation of the protein were abrogated. The aspartic acid residue in the transmembrane domain of all of these mutant proteins could mediate assembly with another protein having an arginine residue in its transmembrane domain, independent of the length of the transmembrane sequence. These findings demonstrate that the length of the hydrophobic transmembrane sequence has a critical influence on the ability of potentially charged transmembrane residues to cause protein retention and degradation in the ER.
Amino Acid Sequence↗
Regulating the fate of mRNA: the control of cellular iron metabolism.
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Animals↗
A double life: cytosolic aconitase as a regulatory RNA binding protein.
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Aconitate Hydratase↗
The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.
We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.
Alleles↗
Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding.
The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.
Aconitate Hydratase↗
Tyrosine kinases and tyrosine-based activation motifs. Current research on activation via the T cell antigen receptor.
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Animals↗
Iron regulates the activity of the iron-responsive element binding protein without changing its rate of synthesis or degradation.
The iron-responsive element binding protein (IRE-BP) interacts with specific sequence/structure motifs (iron-responsive elements) within the mRNAs encoding ferritin and the transferrin receptor and thereby post-transcriptionally regulates the expression of these two proteins involved in cellular iron homeostasis. The activity of the IRE-BP is itself regulated by iron such that when cells are treated with an iron source, the RNA binding activity is decreased. The expression of recombinant human IRE-BP in murine cells has been examined as have the expressions of the endogenous IRE-BP of both human and rabbit cells. In all cases, iron down-modulated the RNA binding activity of the IRE-BP, but in no instance was this decrease in activity accompanied by a decrease in the level of the protein as judged by quantitative Western blots. Moreover, the rate of synthesis of the IRE-BP and its rate of degradation have been found to be unaltered by iron manipulation of cells in culture. Consistent with IRE-BP regulation occurring post-translationally, the iron regulation of its activity was found to be unaffected by cycloheximide. These data are discussed in terms of a model of IRE-BP regulation involving the modification of the protein's iron-sulfur center.
Animals↗
Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.
The fungal metabolite brefeldin A is a powerful tool for investigating membrane traffic in eukaryotic cells. The effects of brefeldin A on traffic are partly explained by its ability to prevent binding of cytosolic coat proteins onto membranes. The non-clathrin coatomer complex binds reversibly to Golgi membranes in a GTP-controlled cycle. The low-molecular-mass GTP-binding protein ADP-ribosylation factor (ARF), which also associates reversibly with Golgi membranes, is required for coatomer binding and probably accounts for the control by guanine nucleotide of the coatomer-membrane interaction. Brefeldin A prevents the assembly of coatomer onto the membrane by inhibiting the GTP-dependent interaction of ARF with the Golgi membrane, but the nature of this interaction has not been established. Here we demonstrate that Golgi membranes can specifically catalyse the exchange of GTP onto ARF and that brefeldin A prevents this function.
ADP-Ribosylation Factors↗