The effects of monensin on transport of membrane components in the frog retinal photoreceptor. I. Light microscopic autoradiography and biochemical analysis

Matheke, ML; Fliesler, Steven J.; Basinger, SF; Holtzman, E

doi:10.1523/jneurosci.04-04-01086.1984

Cited by 16 publications

(7 citation statements)

References 2 publications

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“…Total retinal proteins of individual halves were precipitated in ice-chilled 10% trichloroacetic acid (TCA), and the TCA-precipitable material was solubilized in 1 N NaOH. The specific radioactivity (disintegrations per minute per microgram protein) was determined by liquid scintillation counting and by fluorometric assay of protein content with fluorescamine (62), as described previously (35). The remaining half-retinas were pooled, solubilized in Laemmli sample buffer (31), and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE), by use of a discontinuous 7.5:15% gradient slab gel system as described in detail elsewhere ( 15,35).…”

Section: Microscopy and Autoradiographymentioning

confidence: 99%

“…The specific radioactivity (disintegrations per minute per microgram protein) was determined by liquid scintillation counting and by fluorometric assay of protein content with fluorescamine (62), as described previously (35). The remaining half-retinas were pooled, solubilized in Laemmli sample buffer (31), and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS PAGE), by use of a discontinuous 7.5:15% gradient slab gel system as described in detail elsewhere ( 15,35). Coomassie Blue staining and fluorography of gels was performed as previously described (15,35).…”

Section: Microscopy and Autoradiographymentioning

confidence: 99%

“…After rapid rinsing through four changes of icechilled chase medium, retinas were individually precipitated with TCA, and the incorporation of 3H and ~4C into total retinal TCA-soluble material (normalized to the TCA-precipitable protein content) was determined ( 15,35).…”

Section: Measurement Of 5ubstrate Uptakementioning

confidence: 99%

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Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Fliesler¹,

Rayborn²,

Hollyfield³

1985

The Journal of Cell Biology

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Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphatedependent protein glycosylation. At a TM concentration of 20 /~g/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by ~66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [31-1]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.The rod photoreceptor in the vertebrate retina is a structurally and functionally polarized and segmented postmitotic cell. The rod outer segment (ROS) ~ is composed of a highly ordered stack of individual, flattened double-membrane saccules (the disc membranes) enveloped by the plasma membrane of the cell. The photosensitive nature of the ROS is due to the presence of rhodopsin, the rod visual pigment, in the disc membranes. The ROS is attached to the adjacent rod inner segment (RIS) by the connecting cilium, a narrow cytoplasmic bridge through which intracellular exchange and flow of cytoplasmic constituents takes place. The RIS, which contains the organelles of anabolic and catabolic metabolism, is compartmentalized into two regions: the ellipsoid and the myoid. The ellipsoid (proximal to the connecting cilium) is filled with Abbreviations used in this paper. ITV, intracellular transport vesicle; TCA, trichloroacetic acid; RIS, rod inner segment; ROS, rod outer segment. a dense cluster of mitochondria, whereas the myoid (distal to the nucleus) houses an extensive network of endoplasmic reticulum and the Golgi apparatus.The rod cell renews the ROS membranes at a prodigious rate in an ongoing process throughout its lifetime (47,64,65). For instance, in the African clawed toad Xeno...

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Section: Microscopy and Autoradiographymentioning

confidence: 99%

Section: Microscopy and Autoradiographymentioning

confidence: 99%

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Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Fliesler¹,

Rayborn²,

Hollyfield³

1985

The Journal of Cell Biology

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Section: Introductionmentioning

confidence: 99%

“…Opsin is initially glycosylated cotranslationally with mannose and N-acetylglucosamine in the endoplasmic reticulum, with subsequent posttranslational addition of N-acetylglucosamine in the Golgi apparatus (2,12,13). Recent studies (14,15) have shown that passage of opsin through the Golgi apparatus is obligatory for its incorporation into ROS disc membranes.Biochemical studies have demonstrated both the formation of various intermediates and the presence of endogenous acceptors for those intermediates implicated in the lipid intermediate pathway of protein glycosylation in the retina (16). The biogenic activity of the lipid intermediate pathway is highest in the photoreceptor cells of the retina, relative to other retinal cell types (17).…”

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Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.

Fliesler¹,

Basinger²

1985

Proc. Natl. Acad. Sci. U.S.A.

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Isolated frog retinas were incubated with radiolabeled glycoprotein precursors in the presence or absence of tunicamycin (TM), a selective inhibitor of protein N-glycosylation. In dual-label incubations, TM inhibited the incorporation of [3H~mannose into total retina Cl3CCOOH-precipitable material by 85% relative to controls, whereas incorporation of [ The vertebrate retinal rod photoreceptor is a polarized cell that exhibits an extraordinary degree of morphological and metabolic compartmentation (see Fig. 1). The rod outer segmernt (ROS) is composed of a highly ordered stack of discrete, disc-shaped double membranes which are surrounded by the plasma membrane of the cell. The first several disc membranes at the base of the ROS are actually continuous infoldings of the plasma membrane, from which the discrete disc membranes of the ROS are derived. The ROS disc membranes are composed almost entirely of proteins and lipids, in approximately equal proportions by weight (1). Rhodopsin, the rod visual pigment, accounts for >95% of the total ROS membrane protein, making it the major structural as well as functional ROS membrane component (2, 3). It is composed of a glycoprotein (opsin) to which an 11-cis-retinaldehyde chromophore is covalently linked. Opsin consists of a single polypeptide to which two asparagine-linked oligosaccharides are attached near the amino terminus (2, 4-6).The oligosaccharides are unusually short hybrids of the complex type of N-linked oligosaccharides (7), primarily having the structure GlcNAc(Man)3(GlcNAc)2, with lesser amounts of GlcNAc(Man)4(GLcNAc)2 and GlcNAc(Man)5(GlcNAc)2 (5, 6). drates have been suggested (8-11), the biological significance of the oligosaccharide moieties of opsin remains to be elucidated.The ROS is joined to its adjaceM l ompartment, the rod inner segment (RIS), by a connecting cilium which serves as the sole link by which intercompartmental exchange between the ROS and RIS occurs. The RIS is subdivided into two relatively distinct compartments: the myoid (containing much of the endoplasmic reticulum and the Golgi apparatus), and the ellipsoid (characterized by a dense cluster of mitochondria). A substantial proportion of the total proteins synthesized in the rough endoplastnic reticulum of the rod cell are transported sequentially from the myoid through the ellipsoid to the periciliary plasma membrane of the RIS before being incorporated into the basal disc membranes of the ROS (for a comprehensive review, see refs. 2, 12, 13). Opsin is initially glycosylated cotranslationally with mannose and N-acetylglucosamine in the endoplasmic reticulum, with subsequent posttranslational addition of N-acetylglucosamine in the Golgi apparatus (2,12,13). Recent studies (14,15) have shown that passage of opsin through the Golgi apparatus is obligatory for its incorporation into ROS disc membranes.Biochemical studies have demonstrated both the formation of various intermediates and the presence of endogenous acceptors for those intermediates implicated in the lipid interme...

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The Photoreceptor Connecting Cilium A Model for the Transition Zone

Besharse

Horst

1990

Ciliary and Flagellar Membranes

105

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The effects of monensin on transport of membrane components in the frog retinal photoreceptor. I. Light microscopic autoradiography and biochemical analysis

Cited by 16 publications

References 2 publications

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin.

Tunicamycin blocks the incorporation of opsin into retinal rod outer segment membranes.

The Photoreceptor Connecting Cilium A Model for the Transition Zone

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