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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 protein generates a nina-like  ERG. White-eyed (cn bw) versions of wild-type (A), ninaEI17  (which fails to express any of the R1-6 opsin), (B), rdgB+;  P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] flies (D) were tested  for the ERG light response using 5 s of either orange (o) or blue  light (b) stimulation. Wild-type flies exhibit both a PDA and inactivation of the R1-6 light response by blue light. The ninaEI17  flies possess neither the PDA nor the blue light inactivation. The  rdgB+; P[rdgB-T59E] flies also fail to exhibit a PDA and R1-6 inactivation by blue light. The rdgB+; P[pitpα-rdgB] flies appear to  possess a PDA, but they failed to rapidly return to baseline after  the subsequent orange light stimulus. A 5-mV scale is shown at  the bottom.
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Figure 8: The dominant RdgB-T59E protein generates a nina-like ERG. White-eyed (cn bw) versions of wild-type (A), ninaEI17 (which fails to express any of the R1-6 opsin), (B), rdgB+; P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] flies (D) were tested for the ERG light response using 5 s of either orange (o) or blue light (b) stimulation. Wild-type flies exhibit both a PDA and inactivation of the R1-6 light response by blue light. The ninaEI17 flies possess neither the PDA nor the blue light inactivation. The rdgB+; P[rdgB-T59E] flies also fail to exhibit a PDA and R1-6 inactivation by blue light. The rdgB+; P[pitpα-rdgB] flies appear to possess a PDA, but they failed to rapidly return to baseline after the subsequent orange light stimulus. A 5-mV scale is shown at the bottom.

Mentions: Because the reduced photosensitivity (like the small rhabdomeres) was consistent with reduced functional rhodopsin, we compared the ERG light responses between white-eyed versions (cn bw) of R1-6 rhodopsin-deficient (ninaE) flies, rdgB+; P[rdgB-T59E] flies, and rdgB+; P[pitpα-rdgB] flies. Mutations that dramatically reduce functional R1-6 rhodopsin levels exhibit aberrant ERG light responses that lack both the inactivation and the PDA (Fig. 8 B), relative to wild-type flies (Fig. 8 A; Stephenson et al., 1983). The ERGs of white-eyed rdgB+; P[rdgB-T59E] flies closely resembled the ninaE mutant phenotype (Fig. 8 C), which suggested that these flies had reduced levels of functional rhodopsin.


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 protein generates a nina-like  ERG. White-eyed (cn bw) versions of wild-type (A), ninaEI17  (which fails to express any of the R1-6 opsin), (B), rdgB+;  P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] flies (D) were tested  for the ERG light response using 5 s of either orange (o) or blue  light (b) stimulation. Wild-type flies exhibit both a PDA and inactivation of the R1-6 light response by blue light. The ninaEI17  flies possess neither the PDA nor the blue light inactivation. The  rdgB+; P[rdgB-T59E] flies also fail to exhibit a PDA and R1-6 inactivation by blue light. The rdgB+; P[pitpα-rdgB] flies appear to  possess a PDA, but they failed to rapidly return to baseline after  the subsequent orange light stimulus. A 5-mV scale is shown at  the bottom.
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Related In: Results  -  Collection

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Figure 8: The dominant RdgB-T59E protein generates a nina-like ERG. White-eyed (cn bw) versions of wild-type (A), ninaEI17 (which fails to express any of the R1-6 opsin), (B), rdgB+; P[rdgB-T59E] (C), and rdgB+; P[pitpα-rdgB] flies (D) were tested for the ERG light response using 5 s of either orange (o) or blue light (b) stimulation. Wild-type flies exhibit both a PDA and inactivation of the R1-6 light response by blue light. The ninaEI17 flies possess neither the PDA nor the blue light inactivation. The rdgB+; P[rdgB-T59E] flies also fail to exhibit a PDA and R1-6 inactivation by blue light. The rdgB+; P[pitpα-rdgB] flies appear to possess a PDA, but they failed to rapidly return to baseline after the subsequent orange light stimulus. A 5-mV scale is shown at the bottom.
Mentions: Because the reduced photosensitivity (like the small rhabdomeres) was consistent with reduced functional rhodopsin, we compared the ERG light responses between white-eyed versions (cn bw) of R1-6 rhodopsin-deficient (ninaE) flies, rdgB+; P[rdgB-T59E] flies, and rdgB+; P[pitpα-rdgB] flies. Mutations that dramatically reduce functional R1-6 rhodopsin levels exhibit aberrant ERG light responses that lack both the inactivation and the PDA (Fig. 8 B), relative to wild-type flies (Fig. 8 A; Stephenson et al., 1983). The ERGs of white-eyed rdgB+; P[rdgB-T59E] flies closely resembled the ninaE mutant phenotype (Fig. 8 C), which suggested that these flies had reduced levels of functional rhodopsin.

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