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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 PITPα-RdgB protein prevents the  rapid recovery of the light-response amplitude after prolonged  saturating-light treatment. A 2-s ERG light response was recorded  from wild-type, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and  rdgB+; P[rdgB+] flies, followed by 20 min of saturating light, 30 s  of dark recovery, and another 2-s ERG light-response recording.  The difference was determined between the first and second  light-response recordings. Five flies of each genotype were recorded with the average difference in the light-response amplitude and standard deviation shown. The average initial light- response amplitudes were: wild-type (28.2), rdgB+; P[rdgB-T59E]  (19.8), rdgB+; P[pitpα-rdgB] (26.8), and rdgB+; P[rdgB+] (22.6  mV). The increased light-response amplitude of rdgB+; P[rdgB-T59E] flies in this figure, relative to Fig. 7, is due to the use of  white-eyed flies in this data (cn bw background) and wild-type  eye colored flies in Fig. 7. The other three genotypes contained  some screening pigment.
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Figure 10: The dominant PITPα-RdgB protein prevents the rapid recovery of the light-response amplitude after prolonged saturating-light treatment. A 2-s ERG light response was recorded from wild-type, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+] flies, followed by 20 min of saturating light, 30 s of dark recovery, and another 2-s ERG light-response recording. The difference was determined between the first and second light-response recordings. Five flies of each genotype were recorded with the average difference in the light-response amplitude and standard deviation shown. The average initial light- response amplitudes were: wild-type (28.2), rdgB+; P[rdgB-T59E] (19.8), rdgB+; P[pitpα-rdgB] (26.8), and rdgB+; P[rdgB+] (22.6 mV). The increased light-response amplitude of rdgB+; P[rdgB-T59E] flies in this figure, relative to Fig. 7, is due to the use of white-eyed flies in this data (cn bw background) and wild-type eye colored flies in Fig. 7. The other three genotypes contained some screening pigment.

Mentions: The rdgB+; P[pitpα-rdgB] flies appeared to have a PDA (Fig. 8 D), which was consistent with those flies expressing wild-type levels of rhodopsin (Fig. 9). However, the light response in rdgB+; P[pitpα-rdgB] flies remained inactivated after the conversion of metarhodopsin to rhodopsin by the orange light stimulus (Fig. 8 D). To further examine the effect of PITPα-RdgB on the dark recovery, ERGs to a 2-s light stimulus were recorded from flies, before and 30 s after a 20-min saturating light stimulus. The rdgB+; P[pitpα-rdgB] flies exhibited a significant difference between the initial and final ERG amplitudes (13.0 mV, Fig. 10), which was 49% of the initial amplitude. Only minor amplitude differences were observed for wild-type (2.6 mV, 9% of initial amplitude), rdgB+; P[rdgB-T59E] (3.8 mV, 19% of initial amplitude), and rdgB+; P[rdgB+] (3.1 mV, 14% of initial amplitude) flies (Fig. 10). The rdgB+; P[pitpα] flies, which lacked the dominant degeneration phenotype, were similar to the wild-type controls showing a difference of only 3.1 mV (14% of the initial amplitude) under the same regimen. These data indicated that PITPα-RdgB expression negatively affected the recovery phase of the light response in an otherwise wild-type photoreceptor cell. It is unclear if there is a direct relationship between this electrophysiological defect and the retinal degeneration. However, both of these PITPα-RdgB dominant phenotypes are similar to very mild rdgB mutant phenotypes, which suggests that PITPα-RdgB could be interacting directly with RdgB or competing for a molecule to reduce the wild-type RdgB activity.


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 PITPα-RdgB protein prevents the  rapid recovery of the light-response amplitude after prolonged  saturating-light treatment. A 2-s ERG light response was recorded  from wild-type, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and  rdgB+; P[rdgB+] flies, followed by 20 min of saturating light, 30 s  of dark recovery, and another 2-s ERG light-response recording.  The difference was determined between the first and second  light-response recordings. Five flies of each genotype were recorded with the average difference in the light-response amplitude and standard deviation shown. The average initial light- response amplitudes were: wild-type (28.2), rdgB+; P[rdgB-T59E]  (19.8), rdgB+; P[pitpα-rdgB] (26.8), and rdgB+; P[rdgB+] (22.6  mV). The increased light-response amplitude of rdgB+; P[rdgB-T59E] flies in this figure, relative to Fig. 7, is due to the use of  white-eyed flies in this data (cn bw background) and wild-type  eye colored flies in Fig. 7. The other three genotypes contained  some screening pigment.
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Related In: Results  -  Collection

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Figure 10: The dominant PITPα-RdgB protein prevents the rapid recovery of the light-response amplitude after prolonged saturating-light treatment. A 2-s ERG light response was recorded from wild-type, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+] flies, followed by 20 min of saturating light, 30 s of dark recovery, and another 2-s ERG light-response recording. The difference was determined between the first and second light-response recordings. Five flies of each genotype were recorded with the average difference in the light-response amplitude and standard deviation shown. The average initial light- response amplitudes were: wild-type (28.2), rdgB+; P[rdgB-T59E] (19.8), rdgB+; P[pitpα-rdgB] (26.8), and rdgB+; P[rdgB+] (22.6 mV). The increased light-response amplitude of rdgB+; P[rdgB-T59E] flies in this figure, relative to Fig. 7, is due to the use of white-eyed flies in this data (cn bw background) and wild-type eye colored flies in Fig. 7. The other three genotypes contained some screening pigment.
Mentions: The rdgB+; P[pitpα-rdgB] flies appeared to have a PDA (Fig. 8 D), which was consistent with those flies expressing wild-type levels of rhodopsin (Fig. 9). However, the light response in rdgB+; P[pitpα-rdgB] flies remained inactivated after the conversion of metarhodopsin to rhodopsin by the orange light stimulus (Fig. 8 D). To further examine the effect of PITPα-RdgB on the dark recovery, ERGs to a 2-s light stimulus were recorded from flies, before and 30 s after a 20-min saturating light stimulus. The rdgB+; P[pitpα-rdgB] flies exhibited a significant difference between the initial and final ERG amplitudes (13.0 mV, Fig. 10), which was 49% of the initial amplitude. Only minor amplitude differences were observed for wild-type (2.6 mV, 9% of initial amplitude), rdgB+; P[rdgB-T59E] (3.8 mV, 19% of initial amplitude), and rdgB+; P[rdgB+] (3.1 mV, 14% of initial amplitude) flies (Fig. 10). The rdgB+; P[pitpα] flies, which lacked the dominant degeneration phenotype, were similar to the wild-type controls showing a difference of only 3.1 mV (14% of the initial amplitude) under the same regimen. These data indicated that PITPα-RdgB expression negatively affected the recovery phase of the light response in an otherwise wild-type photoreceptor cell. It is unclear if there is a direct relationship between this electrophysiological defect and the retinal degeneration. However, both of these PITPα-RdgB dominant phenotypes are similar to very mild rdgB mutant phenotypes, which suggests that PITPα-RdgB could be interacting directly with RdgB or competing for a molecule to reduce the wild-type RdgB activity.

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