Limits...
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.

Show MeSH

Related in: MedlinePlus

Time course of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. The time course of retinal degeneration was determined based upon the loss of the deep pseudopupil.  The fraction of flies that retained a wild-type deep pseudopupil  was determined daily for 30 d after eclosion. Flies raised in constant light include rdgB2 (filled triangles), rdgB+ flies containing  two copies of the P[rdgB-T59E] transgene (open squares), rdgB+  flies containing one copy of the P[rdgB-T59E] transgene (filled  squares), rdgB+ flies containing two copies of the P[pitpα-rdgB]  transgene (open circles), and rdgB+ flies containing one copy of  the P[pitpα-rdgB] transgene (filled circles). rdgB+ flies containing  one copy of the P[rdgB-T59E] transgene were also examined  while being raised in a 12-h light/dark cycle (half-filled squares).  Each point represents the average of four trials (n > 25 flies per  trial). The standard deviation is shown as vertical lines.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139788&req=5

Figure 5: Time course of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. The time course of retinal degeneration was determined based upon the loss of the deep pseudopupil. The fraction of flies that retained a wild-type deep pseudopupil was determined daily for 30 d after eclosion. Flies raised in constant light include rdgB2 (filled triangles), rdgB+ flies containing two copies of the P[rdgB-T59E] transgene (open squares), rdgB+ flies containing one copy of the P[rdgB-T59E] transgene (filled squares), rdgB+ flies containing two copies of the P[pitpα-rdgB] transgene (open circles), and rdgB+ flies containing one copy of the P[pitpα-rdgB] transgene (filled circles). rdgB+ flies containing one copy of the P[rdgB-T59E] transgene were also examined while being raised in a 12-h light/dark cycle (half-filled squares). Each point represents the average of four trials (n > 25 flies per trial). The standard deviation is shown as vertical lines.

Mentions: Surprisingly, the rdgB-T59E transgene, which partially restored the ERG light response in rdgB2 flies, produced a dominant retinal degeneration. Expression of RdgB-T59E resulted in a light-enhanced and dose-dependent loss of the deep pseudopupil in rdgB+ flies. Degeneration of wild-type flies expressing one copy of P[rdgB-T59E] was first observed 4 d after eclosion under constant light conditions and at 13 d in a 12-h light/dark cycle (Fig. 5). The deep pseudopupil loss became increasingly prevalent so that essentially all flies in the population had experienced degeneration by 17 d after eclosion in constant light and by 26 d in a 12-h light/dark cycle. Retinal degeneration was never observed, under any circumstances, in wild-type fly controls (not shown). The severity of this dominant retinal degeneration phenotype was proportional to the ratio of P[rdgB-T59E]/rdgB+. Both the onset and rate of degeneration were accelerated in transgenic flies raised in constant light when the P[rdgB-T59E]/rdgB+ ratio was 2:1 rather than 1:1 (Fig. 5). Indeed, rdgB+ flies that possessed two copies of P[rdgB-T59E] lost their deep pseudopupil at essentially the same rate as did rdgB2 mutant flies raised in identical conditions.


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)

Time course of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. The time course of retinal degeneration was determined based upon the loss of the deep pseudopupil.  The fraction of flies that retained a wild-type deep pseudopupil  was determined daily for 30 d after eclosion. Flies raised in constant light include rdgB2 (filled triangles), rdgB+ flies containing  two copies of the P[rdgB-T59E] transgene (open squares), rdgB+  flies containing one copy of the P[rdgB-T59E] transgene (filled  squares), rdgB+ flies containing two copies of the P[pitpα-rdgB]  transgene (open circles), and rdgB+ flies containing one copy of  the P[pitpα-rdgB] transgene (filled circles). rdgB+ flies containing  one copy of the P[rdgB-T59E] transgene were also examined  while being raised in a 12-h light/dark cycle (half-filled squares).  Each point represents the average of four trials (n > 25 flies per  trial). The standard deviation is shown as vertical lines.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139788&req=5

Figure 5: Time course of the dominant RdgB-T59E and PITPα-RdgB retinal degenerations. The time course of retinal degeneration was determined based upon the loss of the deep pseudopupil. The fraction of flies that retained a wild-type deep pseudopupil was determined daily for 30 d after eclosion. Flies raised in constant light include rdgB2 (filled triangles), rdgB+ flies containing two copies of the P[rdgB-T59E] transgene (open squares), rdgB+ flies containing one copy of the P[rdgB-T59E] transgene (filled squares), rdgB+ flies containing two copies of the P[pitpα-rdgB] transgene (open circles), and rdgB+ flies containing one copy of the P[pitpα-rdgB] transgene (filled circles). rdgB+ flies containing one copy of the P[rdgB-T59E] transgene were also examined while being raised in a 12-h light/dark cycle (half-filled squares). Each point represents the average of four trials (n > 25 flies per trial). The standard deviation is shown as vertical lines.
Mentions: Surprisingly, the rdgB-T59E transgene, which partially restored the ERG light response in rdgB2 flies, produced a dominant retinal degeneration. Expression of RdgB-T59E resulted in a light-enhanced and dose-dependent loss of the deep pseudopupil in rdgB+ flies. Degeneration of wild-type flies expressing one copy of P[rdgB-T59E] was first observed 4 d after eclosion under constant light conditions and at 13 d in a 12-h light/dark cycle (Fig. 5). The deep pseudopupil loss became increasingly prevalent so that essentially all flies in the population had experienced degeneration by 17 d after eclosion in constant light and by 26 d in a 12-h light/dark cycle. Retinal degeneration was never observed, under any circumstances, in wild-type fly controls (not shown). The severity of this dominant retinal degeneration phenotype was proportional to the ratio of P[rdgB-T59E]/rdgB+. Both the onset and rate of degeneration were accelerated in transgenic flies raised in constant light when the P[rdgB-T59E]/rdgB+ ratio was 2:1 rather than 1:1 (Fig. 5). Indeed, rdgB+ flies that possessed two copies of P[rdgB-T59E] lost their deep pseudopupil at essentially the same rate as did rdgB2 mutant flies raised in identical conditions.

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