Most short DNA molecules isolated from 3T3 cells are not nascent.

The population of short DNA molecules (less than 10(3) nucleotides) in 3T3 cells has been studied using in vivo and in vitro pulse labeling techniques and in vitro end-labeling. There is a large number of molecules of less than 100 nucleotides present in equal numbers in both Go and S phase cells. In S phase cells, most of these molecules are not replicating intermediates because they do not become density-labeled after a moderate period of substitution of BrdUMP, although they are detected by end-labeling in vitro. This population includes the nascent Okazaki pieces that can be labeled in a short pulse with [3H]dThd or [3H]dTTP, however, these represent less than 10% of the total population. Alkaline hydrolysis of the molecules that had been end-labeled with 32P using [gamma32P]ATP and polynucleotide kinase did not reveal significant release of [32P] 2'(3'), 5' ribonucleoside diphosphates.     

Phosphorylation of protamines by protein kinase C: involvement of sites which are phosphorylated in vivo and are not affected by cAMP-dependent protein kinase

Most fish protamines contain two phosphorylatable sites both of which incorporate phosphate in vivo. Here we show that in two protamines (salmine A1 and clupeine Y1) the site more distant from the N-terminus (residues 20-21) is unaffected by cAMP-dependent protein kinase while it represents the main target for protein kinase C. Such a phosphorylation is typically independent of Ca2+ and phospholipids: responsiveness to these effectors however is conferred by previous fragmentation of protamine with thermolysin. These results suggest that Ca2+, phospholipid-independent phosphorylation of protamine by protein kinase C might have physiological relevance and shed light on the structural basis for the specificity of such an unique process.     

Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh*), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh* to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh*. Furthermore, the catalyzing ability of the phosphorylated Rh* was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh* and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh*.     

Isoelectric focusing of phosphorylated cattle rhodopsin

³²P-rhodopsin was partially separated by isoelectric focusing into several fractions of different phosphorylation extent. It was found that the incorporated phosphate is not uniformly distributed in a population of rhodopsin molecules. In a preparation with an average phosphorylation extent of 2.4 moles of phosphate per mole of rhodopsin, most of the ³²P-phosphate was found in fractions where 4–5 phosphates are bound per rhodopsin, whereas a large fraction of the total rhodopsin was not phosphorylated at all. The maximum number of phosphate binding sites in rhodopsin appears to be at least five.Abbreviations used P/Rh moles of phosphate per mole of rhodopsin - ROS rod outer segments Presented in part at the EMBO workshop on Transduction Mechanism of Photoreceptors, held in Jülich, Germany, on 4–8 October, 1976     

NMR spectroscopy of phosphorylated wild-type rhodopsin: mobility of the phosphorylated C-terminus of rhodopsin in the dark and upon light activation

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The (31)P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 degrees C. Below this temperature, the (31)P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 degrees C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, (19)F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the (19)F and the (31)P-labels. The REDOR technique allows (19)F...(31)P distances to be measured out to approximately 12 A with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the (19)F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 A in phosphorylated rhodopsin.     

Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.     

Isolation and identification of the phosphorylated species of rhodopsin

Rhodopsin is phosphorylated in a light-dependent manner by a kinase intrinsic to the rod outer segment. We have used chromatofocusing to separate six phosphorylated species of rhodopsin and have recovered in the pH gradient fractions 60-80% of the initial phosphorylated sample loaded on the column. The isolated species of rhodopsin coincide with the species that are observed in isoelectric focusing gels in the pH range 6.1-4.7. Unphosphorylated rhodopsin focuses at a pI of 6.0. Two species having two phosphates per rhodopsin with isoelectric points of 5.45 and 5.40 have been isolated. The phosphate to rhodopsin ratios for the remaining species are 3.8, 5.0, 6.1, and 8.2 with isoelectric points of 5.16, 4.99, 4.85, and 4.73, respectively. The chromatofocusing profile suggests that there may be multiple forms of rhodopsin with the same number of phosphates among some of the other phosphorylated forms of rhodopsin.     

Phosphorylation of rhodopsin as a possible mechanism of adaptation

Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the more physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.Abbreviations used ATP adenosine 5-triphosphate - GTP guanosine 5-triphosphate - ROS rod outer segments - IBMX isobutylmethylxanthine - cyclic GMP guanosine 3, 5-monophosphate Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Phosphorylation of rhodopsin by protein kinase C in vitro

Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.     

Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

The effects of activating mutations associated with night blindness on the stoichiometry of rhodopsin interactions with G protein-coupled receptor kinase 1 (GRK1) and arrestin-1 have not been reported. Here we show that the monomeric form of WT rhodopsin and its constitutively active mutants M257Y, G90D, and T94I, reconstituted into HDL particles are effectively phosphorylated by GRK1, as well as two more ubiquitously expressed subtypes, GRK2 and GRK5. All versions of arrestin-1 tested (WT, pre-activated, and constitutively monomeric mutants) bind to monomeric rhodopsin and show the same selectivity for different functional forms of rhodopsin as in native disc membranes. Rhodopsin phosphorylation by GRK1 and GRK2 promotes arrestin-1 binding to a comparable extent, whereas similar phosphorylation by GRK5 is less effective, suggesting that not all phosphorylation sites on rhodopsin are equivalent in promoting arrestin-1 binding. The binding of WT arrestin-1 to phospho-opsin is comparable to the binding to its preferred target, P-Rh*, suggesting that in photoreceptors arrestin-1 only dissociates after opsin regeneration with 11-cis-retinal, which converts phospho-opsin into inactive phospho-rhodopsin that has lower affinity for arrestin-1. Reduced binding of arrestin-1 to the phospho-opsin form of G90D mutant likely contributes to night blindness caused by this mutation in humans.     

Phosphorylation of rhodopsin as a possible mechanism of adaptation.

Light-induced phosphorylation of rhodopsin has been extensively studied by a number of investigators from a biochemical point of view. However, little is known about the physiological function of this reaction. The slow rates measured for phosphorylation and dephosphorylation suggest that it may be involved in visual adaptation rather than in excitation. This paper presents biochemical data obtained from phosphorylation experiments in isolated photoreceptor membranes as well as in the physiological system of whole retinas and living animals. An attempt is made to compare the phosphorylation reaction with visual adaptation hypotheses taken from the electrophysiological literature. Finally, effects of cyclic nucleotide metabolism on the sensitivity of photoreceptors are presented and discussed.     

The arrestin-bound conformation and dynamics of the phosphorylated carboxy-terminal region of rhodopsin

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Most of the yeast genomic sequences are not essential for cell growth and division

To determine the fraction of the yeast Saccharomyces cerevisiae genome that is required for normal cell growth and division, we constructed diploid strains that were heterozygous for random single disruptions. We monitored the effects of approximately 200 independent disruptions by sporulating the diploids and examining the phenotype of the resulting haploid strains. We found that only 12% of the disruptions were haploid-lethal, 14% resulted in slow growth, and an additional 4% were associated with some other new phenotype (such as an auxotrophic requirement). No obvious new phenotype was detected for 70% of the disruptions.     

Role of the carboxyl-terminal region of arrestin in binding to phosphorylated rhodopsin.

The structural and functional properties of arrestin were studied by subjecting the protein to limited proteolysis. Limited proteolysis by trypsin cleaves arrestin (48 kDa), producing 20-25-kDa fragments. Prior to this stage of proteolysis, trypsin produced 46.6-, 45.4-, and 42-kDa fragments. Structural analysis of the proteolytic fragments demonstrated major cleavage at the carboxyl terminus, indicating that the carboxyl terminus is highly exposed. We found that forms of arrestin truncated at their carboxyl terminus maintained their functional properties and bound to phosphorylated rhodopsin. Native arrestin binds only to photoexcited phosphorylated rhodopsin, whereas the truncated arrestin binds to phosphorylated rhodopsin independent of its exposure to light. The truncated forms of arrestin were separated from native arrestin by a chromatographic procedure and subsequently characterized in functional studies. The binding of the truncated forms of arrestin to phosphorylated photoexcited rhodopsin is more tight than the binding of native arrestin as determined by a direct binding assay and the phosphodiesterase assay. We suggest that the acidic carboxyl-terminal region of arrestin may act as a regulator for light-dependent binding to phosphorylated rhodopsin.     

Phosphorylation of heavy sarcoplasmic reticulum vesicles: Identification and characterization of three phosphorylated proteins

Summary Heavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl₂, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 m -³²P-ATP.³²P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl₂ (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 m sodium dantrolene, an inhibitor of Ca²⁺ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.