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W L Pak

Publications and source records attributed to W L Pak.

At least 19 recordsLinked to original sources
PubMed1996

Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin.

Cyclophilins are ubiquitous and abundant proteins that exhibit peptidyl prolyl cis-trans isomerization (PPlase) activity in vitro. Their functions in vivo, however, are not well understood. Two new retinal cyclophilin isoforms, types I and II, are highly expressed in cone photoreceptors of the vertebrate retina. Type-II cyclophilin is identical to RanBP2, a large protein that binds the GTPase Ran. Here we report that two contiguous domains in RanBP2, Ran-binding domain 4 (RBD4) and cyclophilin, act in concert as a chaperone for the opsin molecule of the red/green-sensitive visual pigment of a dichromatic vertebrate. In Drosophila, the cyclophilin NinaA is expressed in all photoreceptors and is required for the expression of only a subset of opsins. The molecular basis of these photoreceptor class-specific effects and the functions of NinaA and other cyclophilins in vivo remain unclear. Unlike NinaA, which forms a stable complex with opsin from retinular cells R1-6, we find that the cyclophilin domain of RanBP2 does not bind opsin directly; rather, it augments and stabilizes the interaction between red/green (R/G) opsin and the RBD4 domain. This involves a cyclophilin-mediated modification of R/G opsin, possibly involving proline isomerization. The RBD4-cyclophilin supradomain of RanBP2, therefore, is a form of vertebrate chaperone of defined substrate specificity, which may be involved in the processing and/or transport of long-wavelength opsin in cone photoreceptor cells.

Amino Acid Isomerases↗
PubMed1996

Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

Alleles↗
PubMed1996

Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior.

The role of histamine as a fast neurotransmitter of imaginal insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdcJK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdcP211, hdcP217, and hdcP218, variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.

Animals↗
PubMed1995

Retina-specifically expressed novel subtypes of bovine cyclophilin.

The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retina cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well s other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina-specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins.

Alternative Splicing↗
PubMed1995

Molecular characterization of two Drosophila guanylate cyclases expressed in the nervous system.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Amino Acid Sequence↗
PubMed1995

Drosophila in vision research. The Friedenwald Lecture.

A genetic approach involving the use of ERG-defective Drosophila mutants, described in this article, was developed with the explicit goal of elucidating phototransduction and related photoreceptor events at the molecular level. Advances in the past 30 years have made this goal a reality. Although the phototransduction process in Drosophila is not yet fully understood, enormous strides are being made by many investigators now working in this field. In addition to elucidating phototransduction vents in invertebrate photoreceptors, works of these investigators are providing insights into the functional machinery of signaling systems in general. For example, mobilization of Ca2+ and replenishment of intracellular Ca2+ stores after their depletion is a current topic of intense interest (see, for example, references 93, 136 and 137). Study of trp mutants is providing insights into this process. Similarly, the mechanisms of inactivation of G-protein coupled receptors appear to be highly conserved in diverse transduction systems, and inactivation of Drosophila metarhodopsin is proving a valuable model system for this study. The identification of the NinaA protein and its role as chaperon and/or foldase for opsin would not have been possible without the genetic approach described in this article. In fact, study of the NinaA gene is providing fresh insights not only into the rhodopsin maturation process but also into the role of cyclophilins in general. Moreover, recent results suggest that related retina-specific proteins are also present in mammals. As valuable as the NinaA protein has been, it may be only the first of a number of such novel proteins or novel isozymes of known proteins required in the retina to be discovered through this approach. For two of the Drosophila proteins discussed in this article, NorpA and NinaA, related mammalian proteins expressed in photoreceptors have been identified, even though it seemed unlikely that such proteins would have any role in mammalian photoreceptors. A recent report suggests that the Trp protein has human homologs expressed most heavily in the brain (C. Montell, cited in reference 93). It may well be that almost any protein identified in the Drosophila retina has its counterpart(s) in mammals. Discovery of these mammalian homologs of Drosophila proteins will probably spur new lines of investigation in mammalian photoreceptor function. For example, because no role had been assigned to PLC in cone phototransduction, the discovery that NorpA-homologous PLCs are present in cone outer segments raises questions about what role(s) these PLCs might play in signal transduction in cone outer segments.

Amino Acid Isomerases↗
PubMed1994

Bovine phospholipase C highly homologous to the norpA protein of Drosophila is expressed specifically in cones.

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.

Amino Acid Sequence↗
PubMed1994

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Amino Acid Sequence↗
PubMed1993

Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

Amino Acid Sequence↗
PubMed1993

Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element-mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Amino Acid Sequence↗
PubMed1993

Histamine is a major mechanosensory neurotransmitter candidate in Drosophila melanogaster.

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.

Animals↗
PubMed1992

Degeneration of photoreceptors in rhodopsin mutants of Drosophila.

Five different, well-characterized mutants of the R1-6 rhodopsin gene (ninaE), which corresponds to the rod opsin gene of vertebrates, have been examined morphologically as a function of age (up to 9 weeks) to determine whether or not the photoreceptors degenerate and to assess the pattern of degeneration. Structural deterioration of R1-6 photoreceptors with age has been found in all five mutants. The structural pattern of degeneration is similar in the five mutants, but the time course of degeneration is allele dependent and varies greatly among the five, with the strongest alleles causing the fastest degeneration. The degeneration appears to be independent of either the illumination cycle to which the animals are exposed or the presence of screening pigments in the eye. Although the degeneration first appears in R1-6 photoreceptors, eventually R7/8 photoreceptors, which correspond to cones of vertebrates, are also affected. In many of these mutants, striking proliferations of membrane processes have been observed in the subrhabdomeric region of R1-6 photoreceptors. It is hypothesized that (1) this accumulation of membranes may be caused by the failure of newly synthesized membranes that are inserted into the base of microvilli to be assembled into R1-6 rhabdomeres and (2) this failure may be caused by the extremely low concentration of normal R1-6 rhodopsin in the ninaE mutants.

Aging↗
PubMed1991

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.

Animals↗
PubMed1991

A Drosophila phospholipase C gene that is expressed in the central nervous system.

A Drosophila phospholipase C (PLC) gene, designated as plc-21, was isolated by screening a genomic DNA library using a cDNA for a previously isolated Drosophila PLC gene, norpA, as probe under reduced stringency hybridization conditions. The gene maps to 21C on the left arm of the second chromosome. Two proteins of 1305 and 1312 amino acids, respectively, were deduced from two classes of cDNA which were isolated. The two putative plc-21 proteins are similar in sequence and overall structure to the beta-class of PLCs found in mammals and differ from each other only by 7 amino acid residues that are present near the C terminus of one of the proteins but not the other. Hybridization of plc-21 cDNA probes to blots of poly(A)+ RNA revealed that the gene encodes a 7.0-kilobase transcript that could be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that could be detected throughout development and in both heads and bodies of adults. In situ hybridization of cDNA sequences to tissue sections showed that the gene is expressed in the neuronal cell bodies of the optic lobe, central brain, and thoracic ganglia of adults and the brain of larvae. This tissue distribution of plc-21 transcripts is identical to the distribution of transcripts from a Drosophila Go alpha-subunit gene that we reported previously.

Amino Acid Sequence↗
PubMed1990

Genetic and molecular characterization of the optomotor-blind gene locus in Drosophila melanogaster.

The Drosophila gene optomotor-blind (omb) is involved in the development of a set of giant neurons in the optic lobes and possibly other structures in the imaginal brain. Adult flies have discrete defects in optomotor behavior. The gene has previously been mapped in chromomeres 4C5-6, together with three other genes, bifid, Quadroon and lacqueredgls. We have localized the gene in a genomic walk of 340 kb of DNA. By mapping seven chromosome breakpoints with omb phenotype we determined its minimum size to about 80 kb. From this region more than 20 RNAs of different size and temporal expression pattern are transcribed. Three of them (T3, T7 and T7') stem from primary transcripts of 40-80 kb in length. In its distal part the omb gene overlaps in at least 19 kb with four other complementation units, bifid, l(1)bifid, Quadroon and lacqueredgls. The three nonlethals affect the external appearance of the fly and seem to be unrelated to brain development.

Animals↗
PubMed1989

Molecular characterization of Drosophila gene encoding G0 alpha subunit homolog.

A Drosophila melanogaster gene (dgo) encoding a G protein alpha subunit has been isolated by screening genomic and adult head cDNA libraries using bovine transducin alpha subunit cDNA as probe. The gene, which maps to 47A on the second chromosome, encodes two proteins which are both 354 amino acids long but differ in seven amino acids in the amino-terminal region. The deduced amino acid sequences of the two proteins are 81% identical to that of a rat Go alpha subunit. Analysis of genomic clones revealed that there are eight coding exons and that the putative transcripts for the two proteins differ in the 5'-noncoding regions and the first coding exons but share the remaining six coding exons. The arrangement of two different 5'-noncoding regions on the gene suggests that two different promoters regulate the expression of the transcripts encoding the two proteins. RNA blot analysis detected three transcripts: a 3.9-kilobase (kb) transcript found at all stages of development; a 5.4-kb transcript present predominantly in adult heads; and a 3.4-kb transcript present only in adult bodies. In situ hybridizations of a cDNA probe to adult tissue sections showed that the gene is expressed abundantly in neuronal cell bodies in the brain, optic lobe, and thoracic ganglia.

Amino Acid Sequence↗
PubMed1989

Drosophila ninaA gene encodes an eye-specific cyclophilin (cyclosporine A binding protein).

Mutations in the ninaA gene of Drosophila severely reduce the amount of rhodopsin specifically in R1-6 photoreceptors. Isolation of the ninaA gene by chromosomal walking revealed that it is expressed only in the eye and encodes a 237-amino acid polypeptide that shows strong sequence similarity to cyclophilin, a putative molecular target for cyclosporine A, a potent immunosuppressant used in human organ transplantations. Unlike most cyclophilins characterized to date, the ninaA-encoded protein has a putative signal sequence and a transmembrane domain. Each of the three ehtyl methanesulfonate-induced ninaA mutant alleles analyzed shows a single nucleotide change in the mRNA coding region leading to either a nonsense or a missense mutation. We find no evidence that the ninaA-encoded protein is directly involved in phototransduction. The only detectable mutant phenotype that correlates with the severity of molecular defects in the three mutants is the amount of depletion of R1-6 rhodopsin. The above results and the recent findings that cyclophilin is a peptidylprolyl cis-trans-isomerase suggest that the ninaA-encoded protein may be required for proper folding and stability of R1-6 rhodopsin.

Amino Acid Sequence↗