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PubMed2004

Identification of extraretinal photoreceptors in the teleost Phoxinus phoxinus.

The existence of cells capable of detecting changes of the photoperiod within the deep brain, the so-called deep brain photoreceptors, was proposed in the early years of the twentieth century. By using immunocytochemistry with antisera against phototransductory proteins on paraffin and vibratome sections, we have localized several positive areas in the brain of the teleost Phoxinus phoxinus. These areas were restricted to two encephalic regions: the epithalamus and the hypothalamus. Immunopositive (rod-opsin- and a-transducin-like) pinealocytes and parapinealocytes, as well as some sparse neurons in the habenula, were seen in the epithalamus. The immunoreaction of the hypothalamus was represented by a-transducin-like positive (magnocellular and parvicellular) neurons of the Nucleus Preopticus, as well as by a-transducin- and arrestin-like positive fibers corresponding to the hypothalamic-hypophyseal tract and a few fibers running towards the basal telencephalon. These findings corroborate the data published on other teleost fish and fully support the hypothesis of the presence of photosensitive cells in the encephalon of lower vertebrates. The labelling with antisera against different components of the phototransductory cascade also strengthens the idea that such cells employ a biochemical mechanism similar to that in the retinal visual photoreceptor cells, rods and cones. Although the function is still unclear, the detection of the photoperiod seems to be the most likely role for these extraretinal photoreceptors.

Animals↗
PubMed2006

Neural interaction of gonadotropin-regulating hormone immunoreactive neurons and the suprachiasmatic nucleus with the paraventricular organ in the Japanese grass lizard (Takydromus tachydromoides).

Our previous study demonstrated that the paraventricular organ (PVO) in the hypothalamus of the Japanese grass lizard (Takydromus tachydromoides) showed immunoreactivity against the light signal-transducing G-protein, transducin. This finding suggested that the PVO was a candidate for the deep-brain photoreceptor in this species. To understand functions of the PVO, we investigated distributions of transducin, serotonin, gonadotropin-releasing hormone (GnRH), and gonadotropin-inhibitory hormone (GnIH) in the lizard's brain. We immunohistochemically confirmed co-localization of transducin and serotonin in PVO neurons that showed structural characteristics of cerebrospinal fluid (CSF)-contacting neurons. GnRH-immunoreactive (ir) cells were localized in the posterior commissure and lateral hypothalamic area. Some of the serotonin-ir fibers extending from the PVO to the lateral hypothalamic area contacted the GnRH-ir cell bodies. GnIH-ir cells were localized in the nucleus accumbens, paraventricular nucleus, and upper medulla, and GnIH-ir fibers from the paraventricular nucleus contacted the lateral processes of serotonin-ir neurons in the PVO. In addition, we found that serotonin-ir fibers from the PVO extended to the suprachiasmatic nucleus (SCN), and the retrograde transport method confirmed the PVO projections to the SCN. These findings suggest that the PVO, by means of innervation mediated by serotonin, plays an important role in the regulation of pituitary function and the biological clock in the Japanese grass lizard.

Animals↗
PubMed1987

Immunoelectron microscopic localization of photoreceptor-specific markers in the monkey retina.

Antibodies for several molecules that function in the visual process were used to localize these molecules in primate rod and cone cells. These antibodies (monoclonal or polyclonal) were prepared against Interphotoreceptor Retinoid-binding Protein (IRBP), S-antigen (S-Ag), opsin, alpha-transducin and also against cyclic GMP (cGMP). Lowicryl-embedded tissues were labeled with secondary antibodies linked to colloidal gold. Although IRBP is predominantly an extracellular protein, the relatively small amount found intracellularly was localized mainly in rods, with little in cones. Opsin, S-Ag and cGMP were found mainly in rod cell outer segments. A polyclonal antiserum raised against transducin-alpha purified from rod outer segments predominantly labeled rod cells, but an antiserum against the carboxyterminal decapeptide of transducin-alpha labeled both rod and cone cells. Thus, most of these specialized molecules are present predominantly in rod cells, confirming major differences in components of the visual cycle in rods and cones.

Animals↗
PubMed1990

Ca2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin.

Arrestin (also called S-antigen or 48-kDa protein) binds to photoexcited and phosphorylated rhodopsin and, thereby, blocks competitively the activation of transducin. Using Ca2+ titration in the presence of the indicator arsenazo III and 45Ca2+ autoradiography, we show that arrestin is a Ca2(+)-binding protein. The Ca2+ binding capacity of arresting-containing protein extracts from bovine rod outer segments is about twice as high as that of arrestin-depleted extracts. The difference in the Ca2+ binding of arrestin-containing and arrestin-depleted protein extracts was attributed to arrestin. Both, these difference-measurements of protein extracts and the measurements of purified arrestin yield dissociation constants for the Ca2+ binding of arrestin between 2 and 4 microM. The titration curves are consistent with a molar ratio of one Ca2+ binding site per arrestin. No Ca2+ binding in the micromolar range was found in extracts containing mainly transducin and cGMP-phosphodiesterase. Since arrestin is one of the most abundant proteins in rod photoreceptors occurring presumably up to millimolar concentrations in rod outer segments, we suggest that aside from its function to prevent the activation of transducin, arrestin acts probably as an intracellular Ca2+ buffer.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Identification of proteins in retinas and IPM from eyes with retinitis pigmentosa.

Opsin, the alpha-subunit of transducin, S-antigen, interphotoreceptor retinoid-binding protein (IRBP) and cathepsin D were assessed in autopsy eyes from patients with retinitis pigmentosa (RP) and normal autopsy eyes. Immunochemical methods were used to determine the presence of these proteins on Western blots of retinal homogenates from five RP donors and on blots of interphotoreceptor matrix (IPM) preparations from six other RP eyes. The amounts of immunoreactive opsin, S-antigen, alpha-transducin, and IRBP appeared below normal in retinas from RP eyes. All six IPM samples from patients with advanced RP had reduced amounts of S-antigen and no detectable IRBP or transducin. Cathepsin D (an RPE protein) was present in IPM or RP eyes in amounts comparable to that in IPMs from normal eyes. Small amounts of cathepsin D were also detected in retinas from both normal and RP eyes. These studies show that proteins specific to the photoreceptor-pigment epithelium complex in normal eyes can be detected in autopsy eyes from patients with RP and suggest that the observed reductions in photoreceptor-specific proteins occur as a consequence of photoreceptor loss.

Adult↗
PubMed1997

Mutation of a conserved cysteine in the X-linked cone opsins causes color vision deficiencies by disrupting protein folding and stability.

PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.

Amino Acid Sequence↗
PubMed1997

Mutation of a conserved proline disrupts the retinal-binding pocket of the X-linked cone opsins.

PURPOSE: To test the effects of disruption of a conserved proline in the green cone opsin molecule on light-activated isomerization, transducin activation, protein accumulation, glycosylation, and transport. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a plasmid containing wild-type or mutant (P307L) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of CdCl2 and analyzed by spectra, transducin activation, Western blotting, and immunocytochemistry. RESULTS: The P307L mutation diminished ability of the visual pigment to absorb light at the appropriate wavelength and to activate transducin. Protein glycosylation and transport to the cell membrane were unaffected. Although there was some diminution in the accumulation of the opsin, this was insufficient to account for the observed effect. CONCLUSIONS: Like rhodopsin, the formation of the cone opsins visual pigments is dependent on the binding of retinal into a hydrophobic pocket that is formed by the second and fourth transmembranous loops. Disruption of a conserved proline near the retinal binding site represents a cause of color vision deficiency that is unrelated to spectral shifts of the photopigment.

Biological Transport↗
PubMed1991

Identification of a retinal protein in Drosophila with antibody to the alpha subunit of bovine brain G(o) protein.

An antibody directed against the alpha(o) subunit of bovine brain G(o) (R4) was used to identify a Drosophila retinal protein which may be the analogue of vertebrate transducin. The immunoreactivity appears predominantly in the retinal and occellar rhabdomeres. On a Western blot, the antibody recognizes a 41 kDa protein that is present in the heads of yellow white flies, but not in the heads of eyeless mutant flies, eyes absent. This protein is not recognized by an antibody raised against Drosophila alpha(o). Antibody R4 intensely stains rhabdomeres and, to a lesser extent, the neuropil of the central nervous system in tissue sections of adult flies. Antibody to Drosophila alpha(o) stains the neuropil of the central nervous system, but does not stain rhabdomeres. In developing flies, faint immunoreactivity appears in the retinal rhabdomeres at about 70% of the time through pupal development and increases to its apparent adult maximal level about 1 day after eclosion. Tissue sections from a phototransduction mutant, norp A, have retinal immunoreactivity at normal levels up to about 1 week after eclosion, but by 2 weeks, immunoreactivity has largely disappeared. This disappearance parallels the degeneration of the retina in norp A mutants. In Drosophila and other invertebrates, light activates a phospholipase C in the retina. The identification of a protein in Drosophila rhabdomeres with an antibody raised against a mammalian G protein alpha subunit thought to be involved in phospholipase C activation suggests that there may be common structural features between the putative Drosophila transducin and alpha(o). The identification of regions common to mammalian alpha(o) and Drosophila transducin may then provide clues to the structural requirements for PLC activation.

Animals↗
PubMed1987

GTP binding proteins: a key role in cellular communication.

One of the major steps in the understanding of the hormonal and sensory transduction mechanisms in eukaryotic cells has been the discovery of a family of GTP binding proteins which couple receptors to specific cellular effectors. The absolute requirement of GTP for hormonal stimulation of adenylate cyclase was the initial observation which led to the purification of the protein involved: Gs. Gs couples stimulatory receptors to adenylate cyclase. It is a heterotrimer composed of an alpha chain (45 or 52 kDa), a beta chain (35-36 kDa) and a gamma chain (8 kDa). Several other G proteins of known functions have been purified: Gi, which couples inhibitory receptors to adenylate cyclase, and transducin which couples photoexcited rhodopsin to cyclic GMP phosphodiesterase. Some G proteins of uncertain function have also been purified: Go, a G protein mainly localized in nervous tissues and Gp, a G protein isolated from placenta and platelets. All these G proteins have a common design. Like Gs they all consist of 3 chains: alpha, beta and gamma. The beta chains are nearly identical, whereas the gamma chains are more variable. The alpha chains are different, but share common domains (especially at the level of the GTP binding site). These domains of homologies are also similar to those of other GTP binding proteins, such as the product of the ras gene (p21) and the initiation or elongation factors. alpha Chains are also ADP ribosylated by bacterial toxins. Gs and transducin are targets for cholera toxin, whereas Gi, Go and transducin are targets for pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

GTP-binding proteins as possible targets for protein kinase C action.

The alpha-subunits of two guanine nucleotide binding proteins Gi and transducin, as well as the beta-subunit of transducin, serve as substrates for phosphorylation by the Ca2+- and phospholipid-dependent protein kinase C (PKC). Phosphorylation of the alpha-subunit of transducin is strictly dependent on its conformation and it is only the inactive form that is subjected to phosphorylation by PKC. This review will focus on the proposition that G proteins may serve as cellular targets for modulatory actions of PKC.

GTP-Binding Proteins↗
PubMed2000

Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin.

BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.

Amino Acid Sequence↗
PubMed1990

Activation mechanism of retinal rod cyclic GMP phosphodiesterase probed by fluorescein-labeled inhibitory subunit.

The cyclic GMP phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is kept inactive in the dark by its gamma subunits and is activated following illumination by the GTP form of the alpha subunit of transducin (T alpha-GTP). Recent studies have shown that the stoichiometry of the inhibited holoenzyme is alpha beta gamma 2. T alpha-GTP and gamma act reciprocally. We have investigated the activation mechanism using fluorescein-labeled gamma subunit (gamma F) as a probe. gamma F containing a single covalently attached fluorescein was prepared by reaction of PDE with 5-(iodoacetamido)fluorescein and purification by reversed-phase high-pressure liquid chromatography (HPLC). gamma F, like native gamma, inhibits the catalytic activity of trypsin-activated PDE and transducin-activated PDE. Inhibition by gamma F was overcome by further addition of T alpha-GTP. gamma F binds very weakly to ROS membranes stripped of PDE and other peripheral membrane proteins. gamma F added to ROS membranes became incorporated into a component that could be extracted with a low ionic strength buffer. HPLC gel filtration showed that gamma F became part of the PDE holoenzyme. Incorporation occurred in less than 1 min in the presence of light and GTP, but much more slowly (t1/2 approximately 500 s) in the absence of GTP. This result indicates that transducin activates PDE by binding to the holoenzyme and accelerating the dissociation of gamma from the inhibitory sites. The binding of gamma F to trypsin-activated PDE alpha beta was monitored by steady-state emission anisotropy measurements and compared with PDE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Tertiary interactions between transmembrane segments 3 and 5 near the cytoplasmic side of rhodopsin.

Previous studies [Yu, H., Kono, M., and Oprian, D. D. (1999) Biochemistry 38, xxxx-xxxx] using split receptors and disulfide cross-linking have shown that native cysteines 140 and 222 on the cytoplasmic side of transmembrane segments (TM) 3 and 5 of rhodopsin, respectively, can cross-link to each other upon treatment with the oxidant Cu(phen)3(2+). In this paper we show that although the 140-222 cross-link does not affect the spectral properties of rhodopsin, it completely and reversibly inactivates the ability of the receptor to activate transducin. Following on this lead we further investigate the cytoplasmic region of TM3 and TM5 and identify three additional pairs of residues that when changed to Cys are capable of forming disulfide cross-links in the protein: 140/225, 136/222, and 136/225. These disulfides are able to form without addition of the Cu(phen)3(2+) oxidant. Similar to the 140-222 cross-link, none of the additional disulfides affect the spectral properties of rhodopsin. Also like the 140-222 bond, the 136-222 disulfide completely and reversibly inactivates the light-dependent activation of transducin by the receptor. In contrast, the 140-225 and 136-225 disulfides have no effect on the ability of rhodopsin to activate transducin. The pattern of cross-linking observed in Cys and disulfide scans of the protein is consistent with helical secondary structure in TM3 from 130 to 142 and in TM5 from 218 to 225.

Amino Acid Sequence↗
PubMed1986

Inhibitory and stimulatory G proteins of adenylate cyclase: cDNA and amino acid sequences of the alpha chains.

The G protein family of signal transducers includes five heterotrimers, which are most clearly distinguished by their different alpha chains. The family includes Gs and Gi, the stimulatory and inhibitory GTP-binding regulators of adenylate cyclase; Go, a protein of unknown function abundant in brain; and transducin 1 and transducin 2, proteins involved in retinal phototransduction. Using a bovine alpha t1 cDNA as a hybridization probe, we have isolated mouse cDNAs that encode alpha chains of two G proteins. One encodes a polypeptide of 377 amino acids (Mr 43,856), identified as alpha s because it specifically fails to hybridize with any transcript in an alpha s-deficient S49 mouse lymphoma mutant, cyc-; the other encodes a polypeptide of 355 amino acids (Mr 40,482), presumed to be alpha i. These alpha chains and those of the retinal transducins exhibit impressive sequence homology. Of the four, alpha t1 and alpha t2 are most alike (81% identical amino acid residues), whereas the presumptive alpha i is more similar than alpha s to alpha t1 (63% vs. 38% identical residues). Sequence homologies with p21ras and elongation factor Tu identify regions of the alpha chains that form the site for GTP binding and hydrolysis. Further comparison of the alpha-chain sequences suggests additional regions that may contribute to interactions with beta gamma subunits and the receptor and effector components of different signal transduction systems.

Adenosine Diphosphate Ribose↗
PubMed1989

Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase.

The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory gamma subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine gamma subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of lambda phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the lambda cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue gamma subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of gamma in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic gamma with the same amino acid sequence as that of native gamma. Both fusion protein and synthetic gamma inhibited trypsin-activated phosphodiesterase with high affinity (Kd less than 100 pM). Likewise, both were as effective as native gamma in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of gamma is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of gamma stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of gamma is critical for inhibition of the phosphodiesterase.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2003

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse.

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.

Animals↗
PubMed1999

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence.

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).

Animals↗
PubMed1994

Mutational analysis of the PRP4 protein of Saccharomyces cerevisiae suggests domain structure and snRNP interactions.

The PRP4 protein of Saccharomyces cerevisiae is an essential part of the U4/U6 snRNP, a component of the mRNA splicing apparatus. As an approach to the determination of structure-function relationships in the PRP4 protein, we have isolated more than fifty new alleles of the PRP4 gene through random and site-directed mutagenesis, and have analyzed the phenotypes of many of them. Twelve of the fourteen single-point mutations that give rise to temperature-sensitive (ts) or null phenotypes are located in the portion of the PRP4 gene that corresponds to the beta-transducin-like region of the protein; the remaining two are located in the central portion of the gene, one of them in an arginine-lysine-rich region. Nine additional deletion or deletion/insertion mutations were isolated at both the amino- and carboxy-termini. These data show that the amino-terminal region (108 amino acids) of PRP4 is non-essential, while the carboxy-terminal region is essential up to the penultimate amino acid. A deletion of one entire beta-transducin-like repeat (the third of five) resulted in a null phenotype. All ts mutants show a first-step defect in the splicing of U3 snRNA primary transcript in vivo at the non-permissive temperature. The effects on prp4 mutant growth of increased copy-number of mutant prp4 genes themselves, and of genes for other components of the U4/U6 snRNP (PRP3 and U6 snRNA) have also been studied. We suggest that the PRP4 protein has at least three domains: a non-essential amino-terminal segment of at least 108 amino acids, a central basic region of about 140 residues that is relatively refractile to mutation and might be involved in RNA interaction, and an essential carboxy-terminal region of about 210 residues with the five repeat-regions that are similar to beta-transducins, which might be involved in protein-protein interaction. A model of interactions of snRNP components suggested by these results is presented.

Amino Acid Sequence↗