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The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

Milligan SC, Alb JG, Elagina RB, Bankaitis VA, Hyde DR - J. Cell Biol. (1997)

Bottom Line: Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo.Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism.This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

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The dominant RdgB-T59E ERG exhibits reduced photosensitivity. (A–D) ERGs were recorded to a 2-s light stimulus  from 1-d-old wild-type (A), rdgB+; P[rdgB-T59E] (B), rdgB+;  P[pitpα-rdgB] (C), and rdgB+; P[rdgB-pitp] (D) flies. The light  response amplitude of the rdgB+; P[rdgB-T59E] flies is ∼60% of  either the wild-type or rdgB+; P[pitpα-rdgB] flies. A 10-mV scale  is shown at the bottom. All the flies contained the white+ gene,  which conferred the wild-type eye color to remove potential differences due to the expression of w+ from the P element construct. (E) The ERG amplitudes of wild-type flies (diamonds),  rdgB+; P[rdgB-T59E] (squares), rdgB+; P[pitpα-rdgB] (circles),  and rdgB+; P[rdgB-pitp] (triangles) were recorded over a range of  light intensities. Neutral density units, corresponding to the filters  used to modulate the light intensity, are plotted against the light  response amplitude. Each point represents a minimum of four independent recordings and the standard deviation is shown as vertical lines.
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Figure 7: The dominant RdgB-T59E ERG exhibits reduced photosensitivity. (A–D) ERGs were recorded to a 2-s light stimulus from 1-d-old wild-type (A), rdgB+; P[rdgB-T59E] (B), rdgB+; P[pitpα-rdgB] (C), and rdgB+; P[rdgB-pitp] (D) flies. The light response amplitude of the rdgB+; P[rdgB-T59E] flies is ∼60% of either the wild-type or rdgB+; P[pitpα-rdgB] flies. A 10-mV scale is shown at the bottom. All the flies contained the white+ gene, which conferred the wild-type eye color to remove potential differences due to the expression of w+ from the P element construct. (E) The ERG amplitudes of wild-type flies (diamonds), rdgB+; P[rdgB-T59E] (squares), rdgB+; P[pitpα-rdgB] (circles), and rdgB+; P[rdgB-pitp] (triangles) were recorded over a range of light intensities. Neutral density units, corresponding to the filters used to modulate the light intensity, are plotted against the light response amplitude. Each point represents a minimum of four independent recordings and the standard deviation is shown as vertical lines.

Mentions: We examined whether the rdgB+; P[rdgB-T59E] and/or the rdgB+; P[pitpα-rdgB] flies exhibited an electrophysiological defect. All the flies that were tested were newly eclosed and subjected to ERG analysis after a 1-h dark adaptation period. Whereas the light-response amplitudes of rdgB+; P[rdgB-T59E] flies were reduced by ∼40% of wild type (Fig. 7, B and A, respectively), the rdgB+; P[pitpα- rdgB] flies were essentially wild type (Fig. 7 C). Wild-type flies expressing multiple copies of either P[rdgB-pitp] (Fig. 7 D) or P[rdgB+] (data not shown), failed to mimic the reduced amplitude observed for rdgB+; P[rdgB-T59E] flies, demonstrating the specificity of the T59E mutation. Moreover, extension of these ERG analyses to different light intensities failed to produce the wild-type light-response amplitudes in rdgB+; P[rdgB-T59E] flies (Fig. 7 E). These data demonstrated that expression of RdgB-T59E in a rdgB+ background resulted in a reduced photosensitivity, while PITPα-RdgB failed to effect the light-response amplitude.


The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation.

Milligan SC, Alb JG, Elagina RB, Bankaitis VA, Hyde DR - J. Cell Biol. (1997)

The dominant RdgB-T59E ERG exhibits reduced photosensitivity. (A–D) ERGs were recorded to a 2-s light stimulus  from 1-d-old wild-type (A), rdgB+; P[rdgB-T59E] (B), rdgB+;  P[pitpα-rdgB] (C), and rdgB+; P[rdgB-pitp] (D) flies. The light  response amplitude of the rdgB+; P[rdgB-T59E] flies is ∼60% of  either the wild-type or rdgB+; P[pitpα-rdgB] flies. A 10-mV scale  is shown at the bottom. All the flies contained the white+ gene,  which conferred the wild-type eye color to remove potential differences due to the expression of w+ from the P element construct. (E) The ERG amplitudes of wild-type flies (diamonds),  rdgB+; P[rdgB-T59E] (squares), rdgB+; P[pitpα-rdgB] (circles),  and rdgB+; P[rdgB-pitp] (triangles) were recorded over a range of  light intensities. Neutral density units, corresponding to the filters  used to modulate the light intensity, are plotted against the light  response amplitude. Each point represents a minimum of four independent recordings and the standard deviation is shown as vertical lines.
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Figure 7: The dominant RdgB-T59E ERG exhibits reduced photosensitivity. (A–D) ERGs were recorded to a 2-s light stimulus from 1-d-old wild-type (A), rdgB+; P[rdgB-T59E] (B), rdgB+; P[pitpα-rdgB] (C), and rdgB+; P[rdgB-pitp] (D) flies. The light response amplitude of the rdgB+; P[rdgB-T59E] flies is ∼60% of either the wild-type or rdgB+; P[pitpα-rdgB] flies. A 10-mV scale is shown at the bottom. All the flies contained the white+ gene, which conferred the wild-type eye color to remove potential differences due to the expression of w+ from the P element construct. (E) The ERG amplitudes of wild-type flies (diamonds), rdgB+; P[rdgB-T59E] (squares), rdgB+; P[pitpα-rdgB] (circles), and rdgB+; P[rdgB-pitp] (triangles) were recorded over a range of light intensities. Neutral density units, corresponding to the filters used to modulate the light intensity, are plotted against the light response amplitude. Each point represents a minimum of four independent recordings and the standard deviation is shown as vertical lines.
Mentions: We examined whether the rdgB+; P[rdgB-T59E] and/or the rdgB+; P[pitpα-rdgB] flies exhibited an electrophysiological defect. All the flies that were tested were newly eclosed and subjected to ERG analysis after a 1-h dark adaptation period. Whereas the light-response amplitudes of rdgB+; P[rdgB-T59E] flies were reduced by ∼40% of wild type (Fig. 7, B and A, respectively), the rdgB+; P[pitpα- rdgB] flies were essentially wild type (Fig. 7 C). Wild-type flies expressing multiple copies of either P[rdgB-pitp] (Fig. 7 D) or P[rdgB+] (data not shown), failed to mimic the reduced amplitude observed for rdgB+; P[rdgB-T59E] flies, demonstrating the specificity of the T59E mutation. Moreover, extension of these ERG analyses to different light intensities failed to produce the wild-type light-response amplitudes in rdgB+; P[rdgB-T59E] flies (Fig. 7 E). These data demonstrated that expression of RdgB-T59E in a rdgB+ background resulted in a reduced photosensitivity, while PITPα-RdgB failed to effect the light-response amplitude.

Bottom Line: Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo.Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism.This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPalpha, a classical PI transfer protein, for RdgB's PITP domain (PITPalpha or PITPalpha-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPalpha-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPalpha-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

Show MeSH
Related in: MedlinePlus