Horseradish Peroxidase Uptake by Rod Photoreceptor Inner Segments Accompanies Outer Segment Disc Assembly

1981 ◽  
Vol 39 ◽  
pp. 486-487
Author(s):  
Joseph C. Besharse ◽  
Donna M. Forestner
Keyword(s):  
Amino Acids ◽  
Horseradish Peroxidase ◽  
Xenopus Laevis ◽  
Outer Segment ◽  
Membrane Vesicles ◽  
Ringer Solution ◽  
Rod Photoreceptor ◽  
Label Protein ◽  
Tritiated Amino Acids ◽  
Peroxidase Uptake

We have suggested that large, membrane vesicles found in the periciliary region of rod photoreceptor inner segments (RIS) contain precursors destined for incorporation into discs of the outer segment (ROS). It has also been shown, however, that endocytosis of extracellular horseradish peroxidase (HRP) occurs in inner segments, suggesting an endocytic origin for some vesicles. As part of an evaluation of the vesicle hypothesis, we examined the extent to which endocytic mechanisms contribute to vesicle populations found in the ellipsoid region of the RIS. HRP uptake was examined in isolated retinas from Xenopus laevis which had been maintained in a Ringer solution in light or darkness for 5 to 120 minutes. To verify that ROS disc assembly actually occurred during HRP incubations, we preincubated retinas for 40 minutes in a mixture of tritiated amino acids (leucine, valine and phenylalanine) to label protein precursors of ROS discs. Labeled retinas were then incubated in cold medium with or without HRP for 2 hours.

scholarly journals Identification of an Outer Segment Targeting Signal in the Cooh Terminus of Rhodopsin Using Transgenic Xenopus laevis

2000 ◽  
Vol 151 (7) ◽  
pp. 1369-1380 ◽  
Author(s):  
Beatrice M. Tam ◽  
Orson L. Moritz ◽  
Lawrence B. Hurd ◽  
David S. Papermaster
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Fusion Proteins ◽  
Outer Segment ◽  
Fluorescent Protein ◽  
Localization Signal ◽  
Targeting Signal ◽  
Retinal Degenerative Diseases ◽  
Membrane Spanning ◽  
Green Fluorescent

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and α adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.

scholarly journals Expression of the poly(A)-binding protein during development of Xenopus laevis.

1989 ◽  
Vol 9 (6) ◽  
pp. 2756-2760 ◽  
Author(s):  
B D Zelus ◽  
D H Giebelhaus ◽  
D W Eib ◽  
K A Kenner ◽  
R T Moon
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Fusion Protein ◽  
Outer Segment ◽  
Binding Protein ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
Cdna Clones ◽  
Protein Encoding

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

Expression of the poly(A)-binding protein during development of Xenopus laevis

1989 ◽  
Vol 9 (6) ◽  
pp. 2756-2760
Author(s):  
B D Zelus ◽  
D H Giebelhaus ◽  
D W Eib ◽  
K A Kenner ◽  
R T Moon
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Xenopus Laevis ◽  
Fusion Protein ◽  
Outer Segment ◽  
Binding Protein ◽  
Polyclonal Antiserum ◽  
Xenopus Laevis Oocytes ◽  
Cdna Clones ◽  
Protein Encoding

We have isolated and sequenced cDNA clones encoding the poly(A)-binding protein of Xenopus laevis oocytes. Polyclonal antiserum was raised against a fusion protein encoding 185 amino acids of the Xenopus poly(A)-binding protein. This antiserum localizes the poly(A)-binding protein to subcellular sites associated with protein synthesis; in the retina, immunoreactive protein is detected in the synthetically active inner segment of the photoreceptor but not in the transductive outer segment. Transcripts encoding the poly(A)-binding protein are present in oocytes, although no protein is detected on protein blots. In contrast, the levels of both transcripts and protein increase in development, which correlates with the observed increase in total poly(A) during Xenopus embryogenesis (N. Sagata, K. Shiokawa, and K. Yamana, Dev. Biol. 77:431-448, 1980).

scholarly journals SYNTHESIS AND STORAGE OF MICROTUBULE PROTEINS BY SEA URCHIN EMBRYOS

1971 ◽  
Vol 50 (2) ◽  
pp. 516-528 ◽  
Author(s):  
Rudolf A. Raff ◽  
Gerald Greenhouse ◽  
Kenneth W. Gross ◽  
Paul R. Gross
Keyword(s):  
Amino Acids ◽  
Sea Urchin ◽  
Messenger Rna ◽  
Binding Activity ◽  
Total Soluble Protein ◽  
Cell Stage ◽  
Lytechinus Pictus ◽  
Sea Urchin Embryos ◽  
Tritiated Amino Acids ◽  
Vinblastine Sulfate

Studies employing colchicine binding, precipitation with vinblastine sulfate, and acrylamide gel electrophoresis confirm earlier proposals that Arbacia punctulata and Lytechinus pictus eggs and embryos contain a store of microtubule proteins. Treatment of 150,000 g supernatants from sea urchin homogenates with vinblastine sulfate precipitates about 5% of the total soluble protein, and 75% of the colchicine-binding activity. Electrophoretic examination of the precipitate reveals two very prominent bands. These have migration rates identical to those of the A and B microtubule proteins of cilia. These proteins can be made radioactive at the 16 cell stage and at hatching by pulse labeling with tritiated amino acids. By labeling for 1 hr with leucine-3H in early cleavage, then culturing embryos in the presence of unlabeled leucine, removal of newly synthesized microtubule proteins from the soluble pool can be demonstrated. Incorporation of labeled amino acids into microtubule proteins is not affected by culturing embryos continuously in 20 µg/ml of actinomycin D. Microtubule proteins appear, therefore, to be synthesized on "maternal" messenger RNA. This provides the first protein encoded by stored or "masked" mRNA in sea urchin embryos to be identified.

scholarly journals Horseradish peroxidase uptake in vivo by neuronal and glial lysosomes

1973 ◽  
Vol 38 (3) ◽  
pp. 370-385 ◽  
Author(s):  
O.Z. Sellinger ◽  
Patricia D. Petiet
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Uptake
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