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Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.

An in situ 35S-labeled guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding procedure was developed to assay light-stimulated G protein activity in Drosophila compound eyes. We found that Drosophila with mutations in G beta e, an abundant photoreceptor-specific G protein beta subunit essential for photoexcitation, are defective in light-stimulated [35S]GTP gamma S binding. We confirmed that G beta e interacts with a GTP-binding protein by demonstrating that immunoprecipitation of G beta e is sensitive to GTP gamma S. These results suggest that G beta e functions as the beta subunit of a heterotrimeric G protein that couples photoactivation of rhodopsin to downstream components in the Drosophila phototransduction cascade.

Heterogeneous N-terminal glycine acylation recently has been reported for two proteins involved in visual signal transduction. Similar N-acylations have typically involved only myristate; however, none of the previously examined proteins were isolated from retinas. To determine whether heterogeneous N-acylation is tissue-specific or protein sequence-specific, the N-terminal modifications of the catalytic subunit of cAMP-dependent protein kinase, partially purified from bovine retinas, heart, and brain tissues, were characterized. Using tandem mass spectrometry and liquid chromatography coupled directly to an electrospray mass spectrometer, we found only myristate at the N termini of catalytic subunits from brain and heart tissue, whereas the N termini of the retina-derived subunits were heterogeneously acylated in a manner similar to recoverin and transducin. Thus it appears that the nature of N-terminal glycine acylation is determined by the cell or tissue type in which it is located, and not by the sequence of the modified protein. We also examined the N-acylation of recoverin purified from human retinas, as well as transducin purified from frog retinas, to determine if heterogeneous acylation of retinal proteins is a uniquely bovine phenomenon. Interestingly, human recoverin was modified by the same family of fatty acids found on the bovine retinal proteins, while frog transducin was modified homogeneously not with myristate, but with a doubly unsaturated (C14:2) fatty acyl group.

Recoverin is a recently identified Ca(2+)-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.

A cDNA clone corresponding to a novel cGMP phosphodiesterase inhibitory subunit (PDE gamma) was isolated from a bovine retinal cDNA library using a rod PDE gamma cDNA as a probe under conditions of reduced stringency. This new clone encodes an 83-amino acid protein approximately 80% identical to rod PDE gamma. The transcript for this protein is retina specific and 940 base pairs in length. Southern blots of bovine genomic DNA show that transcripts encoding rod gamma and this new PDE gamma are derived from different genes. Polyclonal antibodies raised against peptides corresponding to the unique amino termini of the rod PDE gamma and this new clone label rod outer segments and a subset of cone outer segments, respectively, on bovine retinal sections. This subset of cones is also recognized by antibodies that label human blue cones. On Western blots, antibodies against this new cone protein specifically recognize a 13-kDa cone PDE inhibitor.

The elective isolation of a soil microorganism, tentatively assigned to the genus Arthrobacter, which produced an extracellular neuraminidase is described. The secretion of neuraminidase from washed cells in minimal medium required the presence of sialo-containing glycoproteins, whereas free N-acetyl-neuraminic asid of N-acetylmannosamine were poor inducers. No enzyme could be dected in the induction fitrated of cells, in the absence of inducer or in the culture filtrate of cells grown in a complete medium. The routine enzyme inducer was a hot-water extract of "edible bird's nest." Mild acid treatment (0.05 N H2SO4) of this extract increased enzyme activity two--to threefold and the specific activity about eightfold. Neuraminidase induction with acid-treated bird's nest was manifested at a linear rate for 6 h without increase in cell number. No other anticipated glycohydrolase or protease activities were foud. The amount of enzyme located within the cells was barely detectable as compared to that found in the induction filtrate. Experiments with chloramphenicol or chlortetracycline indicate that de novo protein synthesis was required for neuraminidase production and that this exoenzyme was not released from a preformed pool. Neuraminidase from this source has an apparent molecular weight of 87,000, a pH optimum of 5 to 6, and an apparent Km of 2.08 mg/ml for collocalia mucoid and 3.3 X 10(-3) M for N-acetylneuraminlactose and is insensitive both to Ca2+ ions and ethylenediaminetetraacetic acid. Preliminary studies indicate that the enzyme can hydrolyze alpha-2,3-, alpha-2,6-, or alph-2-8-N-acetylneuraminylglycosidic linkages. From total activity data and purification criteria, it would appear that this isolate can produce about 5 mg of enzyme per liter of induction medium.

Since 1951, the alpine habitat of the Black Rosy Finch (Leucosticte tephrocotis atrata) has been visited in many mountain ranges for the purpose of determining the breeding range of this species. The distribution of this species has long been uncertain. Available information is scattered and some of it remains unpublished in the form of museum specimens. This report adds new findings and summarizes existing data on distribution.

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.

A genomic clone spanning the mouse rod transducin alpha subunit (Tr alpha) gene has been isolated by screening a mouse genomic library with a bovine Tr alpha cDNA clone. The coding region of the mouse Tr alpha gene reveals an 88.7% nucleotide identify and 99.7% amino acid identity with bovine Tr alpha. The mouse Tr alpha gene is composed of 8 exons and 7 introns within its coding region. These introns are in the same locations as introns in human Gi alpha genes, that encode G proteins closely related to transducin. Primer extension, RNA sequencing, and S1 nuclease protection analyses indicate that the mouse Tr alpha gene transcription start site is 84 bases upstream of the initiation codon. Northern blot analysis shows that the mouse Tr alpha is expressed in the retina, but not in brain, kidney, liver, or heart.

A G protein alpha subunit gene has been isolated from a Drosophila melanogaster genomic library using a combination of bovine rod and cone transducin alpha subunit cDNAs as a probe under reduced stringency conditions. The gene, DG alpha 1, encodes a protein with an amino acid sequence 78% identical to bovine Gi alpha 1. However, unlike all reported Gi alpha subunit the DG alpha 1-encoded protein is not expected to be a pertussis toxin substrate, because it lacks a cysteine at the appropriate site. The protein coding region of the gene is split by four introns. The sequence of a head tissue cDNA clone, as well as amino acid similarities to mammalian G proteins, confirms this exon/intron structure. Northern blots of total cellular RNA reveal a major 2.3-kilobase transcript and a less abundant 1.7-kilobase transcript. These transcripts are most abundant in RNA from embryos and pupae. The DG alpha 1 gene is located on band 65C on the left arm of the third chromosome, on the basis of in situ hybridizations to Drosophila salivary gland polytene chromosomes.

Light activates rod outer segment (ROS) phosphodiesterase (PDEase), as shown by previous biochemical and physiological studies. We have further investigated the role of PDEase in this system by injecting trypsin-activated PDEase, purified from bovine ROS, into single ROS of the isolated retina of the toad Bufo marinus. Injection of about 300 molecules of activated PDEase in darkness is without immediate detectable effect, as measured by intracellular membrane-voltage recording. The effect of the activated PDEase injections only becomes evident after illumination. The light response is augmented; kinetics of repolarization are slowed. We conclude that this augmentation of the light-dependent hyperpolarization results from the hydrolysis of endogenous cyclic GMP caused by injected PDEase. These results provide evidence that PDEase affects light-dependent channels of the vertebrate scotopic photoreceptors but do not specify whether the effects are exercised for the initial hyperpolarizing phase of the receptor potential and for the recovery phase or only for the recovery phase.

Drosophila melanogaster cDNA clones coding for the alpha subunit of a Go-like G protein have been isolated. The sequence of two cDNA clones shows there is alternative splicing in the 5'-coding region which, on conceptual translation, would give rise to two proteins with slightly different amino termini. A partial genomic clone indicates there are four introns in the carboxyl-terminal half of the clone. Two transcripts, 3.8 and 5.3 kilobases long, are expressed at a high level in the heads of adult flies and also in larvae, pupae, and embryos. Hybridization of the cDNA probes to sections of adult flies indicates the RNA is present predominantly in nervous tissue (in the cortex of the brain and the thoracic ganglion); it is also expressed in the ovaries. Transcripts corresponding to both cDNAs are present in the central nervous system, but only one of them is found in detectable levels in the ovaries. The gene maps to 47A on the Drosophila second chromosome.

Retinal rod outer segments contain a phosphodiesterase specific for cyclic GMP. This enzyme is virtually inactive in the dark. Photoexcitation of rhodopsin results in the formation of hundreds of molecules of GTP-transducin, which in turn activate many molecules of phosphodiesterase. The phosphodiesterase is also known to be activated by the proteolytic action of trypsin. We have investigated the nature of the inhibitory constraint on the catalytic activity of the phosphodiesterase in the dark state. Phosphodiesterase purified by hexylagarose chromatography followed by gel filtration high pressure liquid chromatography consists of three kinds of subunits: alpha (88 kilodaltons), beta (84 kilodaltons), and gamma (11 kilodaltons). Three lines of evidence show that the phosphodiesterase in the dark state is inhibited by its gamma subunit. First, inhibitor activity copurifies with the catalytic activity of this enzyme. Second, trypsin degrades the gamma subunit, resulting in a concomitant increase in catalytic activity. The high pressure liquid chromatography elution position of trypsin-activated phosphodiesterase suggests that it is an alpha beta complex. Third, nearly all of the catalytic activity of trypsin-activated phosphodiesterase can be inhibited by the addition of gamma subunit purified either by heat treatment or by gel filtration at pH 2.1. The addition of gamma subunit to trypsin-activated phosphodiesterase decreases its Vmax from 1.2 mmol of cyclic GMP hydrolyzed/min/mg to less than 1% of this value with relatively little change in the value of Km. The gamma subunit has high affinity for trypsin-activated phosphodiesterase. The dissociation constant of this complex is 0.13 nM. These experiments show that the phosphodiesterase in the dark state has very little catalytic activity because of the inhibitory constraint imposed by its gamma subunit.

Both the light-stimulated cGMP phosphodiesterase of retinal rod outer segments (ROS) and hormone-stimulated adenylate cyclase are regulated by guanine nucleotide-binding regulatory proteins (N). Transducin serves as the signal-carrying regulatory protein in ROS, and the N protein (also called G or G/F) performs this role in the adenylate cyclase system. The GTP form of these regulatory proteins activates the corresponding enzyme, whereas the GDP form does not. Both transducin and the N protein possess a GTPase activity that restores the regulatory protein to the unstimulated state. Cholera enterotoxin catalyzes the transfer of ADP-ribose from NAD+ to the N protein, which inhibits its GTPase activity and activates adenylate cyclase. We report here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes. This modification of the guanine nucleotide-binding subunit of transducin is markedly enhanced by the bleaching of rhodopsin and by the addition of guanosine-5'-(beta, gamma-imino)triphosphate. In contrast, GDP, GTP, and guanosine-5'-(3-O)thiotriphosphate inhibit the reaction, while GMP and ATP have no effect. Under optimal conditions, toxin catalyzes labeling of 0.7 mol of the alpha-subunit of transducin/mol of bound [3H]guanosine-5'-(beta, gamma-imido)triphosphate and causes 70% inhibition of the light-dependent GTPase activity of transducin in ROS. These results indicate close functional homology between transducin of ROS and the N protein of adenylate cyclase.