In many vertebrate and invertebrate cells, inositol 1,4,5-trisphospate production induces a biphasic Ca2+ signal. Mobilization of Ca2+ from internal stores drives the initial burst. The second phase, referred to as storeoperated Ca2+ entry (formerly capacitative Ca2+ entry), occurs when depletion of intracellular Ca2+ pools activates a non-voltage-sensitive plasma membrane Ca2+ conductance. Despite the prevalence of store-operated Ca2+ entry, no vertebrate channel responsible for store-operated Ca2+ entry has been reported. trp (transient receptor potential), a Drosophila gene required in phototransduction, encodes the only known candidate for such a channel throughout phylogeny. In this report, we describe the molecular characterization of a human homolog of trp, TRPCI. TRPC1 (transient receptor potential channel-related protein 1) was 40%o identical to Drosophila TRP over most of the protein and lacked the charged residues in the S4 transmembrane region proposed to be required for the voltage sensor in many voltage-gated ion channels. TRPCI was expressed at the highest levels in the fetal brain and in the adult heart, brain, testis, and ovaries. Evidence is also presented that TRPC1 represents the archetype of a family of related human proteins. Several signaling mechanisms for store-operated Ca2+ entry have been proposed (for review, see refs. 1, 3-5, 11). In one model, a diffusible messenger is produced or released upon depletion of the intracellular Ca2+ pool, and this messenger then activates the plasma membrane Ca2+ channels (12). A second model suggests that emptying the intracellular Ca2+ stores causes a conformational change in the storage organelle and/or its surface proteins, such as the InsP3 receptor, which is transmitted to plasma membrane Ca2+ channels either by direct coupling or via the cytoskeleton (13,14). Despite the prevalence of store-operated Ca2+ entry, no vertebrate SOC gene has been identified. Elucidation of the mechanism responsible for store-operated Ca2+ entry would be considerably aided by cloning and characterizing the channels.
In Drosophila, the store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. Here, we show that TRP forms a complex with phospholipase C-beta (NORPA), rhodopsin (RH1), calmodulin, and the PDZ domain containing protein INAD. Proteins with PDZ domains have previously been shown to cluster ion channels in vitro. We show that in InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. These results provide evidence that a PDZ domain protein is required, in vivo, for anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. We propose that the TRP channel is linked to NORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules.
Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated M r 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.It is now well established that the initiation of protein synthesis in eukaryotic cells begins with the recognition of the initiation AUG codon of mRNA by the small ribosomal subunit (40S) containing bound initiatior methionyl-tRNA (Met-tRNA f ) to form the 40S initiation complex (40S⅐mRNA⅐Met-tRNA f ). Subsequently, a 60S ribosomal subunit joins the 40S initiation complex to form the 80S initiation complex (80S⅐mRNA⅐Met-tRNA f ) that is competent to undergo peptide bond synthesis during the elongation phase of protein synthesis (for a review, see refs. 1-5). After termination of translation, ribosomes are released from the polysomal complex as 80S particles that are in equilibrium with 40S and 60S ribosomal subunits (6, 7). Because a new round of initiation requires separated subunits, a mechanism must exist for maintaining a pool of both ribosomal subunits or for generating them from 80S ribosomes. Two protein factors, eukaryotic translation initiation factor 3 (eIF3) and eIF6, have been implicated in this process (1-5). It has been reported that eIF3, a multisubunit protein complex of M r Ͼ 500,000, binds to the 40S ribosomal subunit and prevents its association with the 60S ribosomal subunit to form 80S ribosomes (8-11). In contrast to eIF3, eIF6, a monomeric protein of about 25 kDa, has been shown to bind to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit (12)(13)(14)(15). This ribosomal subunit anti-association property was used as an assay to identify and purify eIF6 from both wheat germ (12, 13) and mammalian cell extracts (14, 15). However, unlike eIF3 whose requirement for the binding of Met-tRNA f and mRNA to the 40S ribosomal subunit to form the 40S initiation complex is firmly established, the requirement of eIF6 in the initiation of protein synthesis both in vivo and in vitro has not yet been defined. This is in part because of the relatively low abundance of eIF6 in eukaryotic cells (14,15) and the inability to obtain sufficient quantities of the pure protein in stable form to carry out structural and detaile...
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