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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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ERG light responses of rdgB2  mutants expressing  various transgenes. Dark-reared (<12-h old) wild-type (Oregon-R), rdgB2, rdgB2; P[rdgB-pitp], rdgB2; P[rdgB-pitp-T59E], rdgB2;  P[rdgB-T59E], rdgB2; P[pitpα], and rdgB2; P[pitpα-rdgB] flies  were mounted in dim red light for ERGs. After an additional 1-h  dark adaptation, an ERG recording was made from a 2-s light  stimulus. The flies were then saturated with light for 5 min and  given 30 s to dark adapt, followed by another ERG recording to a  2-s light stimulus. An additional 5-min dark adaptation was given  before the third ERG recording, again to a 2-s light stimulus. A  10-mV scale is shown below the initial wild-type ERG.
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Figure 3: ERG light responses of rdgB2 mutants expressing various transgenes. Dark-reared (<12-h old) wild-type (Oregon-R), rdgB2, rdgB2; P[rdgB-pitp], rdgB2; P[rdgB-pitp-T59E], rdgB2; P[rdgB-T59E], rdgB2; P[pitpα], and rdgB2; P[pitpα-rdgB] flies were mounted in dim red light for ERGs. After an additional 1-h dark adaptation, an ERG recording was made from a 2-s light stimulus. The flies were then saturated with light for 5 min and given 30 s to dark adapt, followed by another ERG recording to a 2-s light stimulus. An additional 5-min dark adaptation was given before the third ERG recording, again to a 2-s light stimulus. A 10-mV scale is shown below the initial wild-type ERG.

Mentions: Incorporation of the T59E mutation, however, did not affect the stable expression of either the full-length RdgB (RdgB-T59E) or RdgB-PITP (RdgB-PITP-T59E) (Table I). We compared the ERG light responses between newly eclosed rdgB2 flies that expressed or lacked T59E-containing proteins with wild-type flies. Wild-type flies maintained an ERG light-response amplitude of ∼25 mV that returned to baseline within 3 s after termination of the light stimulus (Fig. 3). Whereas newly eclosed dark-raised rdgB2 flies exhibited a wild-type light-response amplitude, the ERGs required on average 1 min to return to baseline after termination of the light stimulus (Fig. 3). Thus, rdgB2 flies are defective in terminating the ERG light response. The rdgB2 flies expressing either RdgB-PITP-T59E or RdgB-T59E also exhibited a wild-type light-response amplitude. While the ERG light-response termination in rdgB2 flies expressing T59E-containing proteins was significantly faster than rdgB2 flies, it was still two to three times slower than wild type (Fig. 3).


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)

ERG light responses of rdgB2  mutants expressing  various transgenes. Dark-reared (<12-h old) wild-type (Oregon-R), rdgB2, rdgB2; P[rdgB-pitp], rdgB2; P[rdgB-pitp-T59E], rdgB2;  P[rdgB-T59E], rdgB2; P[pitpα], and rdgB2; P[pitpα-rdgB] flies  were mounted in dim red light for ERGs. After an additional 1-h  dark adaptation, an ERG recording was made from a 2-s light  stimulus. The flies were then saturated with light for 5 min and  given 30 s to dark adapt, followed by another ERG recording to a  2-s light stimulus. An additional 5-min dark adaptation was given  before the third ERG recording, again to a 2-s light stimulus. A  10-mV scale is shown below the initial wild-type ERG.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: ERG light responses of rdgB2 mutants expressing various transgenes. Dark-reared (<12-h old) wild-type (Oregon-R), rdgB2, rdgB2; P[rdgB-pitp], rdgB2; P[rdgB-pitp-T59E], rdgB2; P[rdgB-T59E], rdgB2; P[pitpα], and rdgB2; P[pitpα-rdgB] flies were mounted in dim red light for ERGs. After an additional 1-h dark adaptation, an ERG recording was made from a 2-s light stimulus. The flies were then saturated with light for 5 min and given 30 s to dark adapt, followed by another ERG recording to a 2-s light stimulus. An additional 5-min dark adaptation was given before the third ERG recording, again to a 2-s light stimulus. A 10-mV scale is shown below the initial wild-type ERG.
Mentions: Incorporation of the T59E mutation, however, did not affect the stable expression of either the full-length RdgB (RdgB-T59E) or RdgB-PITP (RdgB-PITP-T59E) (Table I). We compared the ERG light responses between newly eclosed rdgB2 flies that expressed or lacked T59E-containing proteins with wild-type flies. Wild-type flies maintained an ERG light-response amplitude of ∼25 mV that returned to baseline within 3 s after termination of the light stimulus (Fig. 3). Whereas newly eclosed dark-raised rdgB2 flies exhibited a wild-type light-response amplitude, the ERGs required on average 1 min to return to baseline after termination of the light stimulus (Fig. 3). Thus, rdgB2 flies are defective in terminating the ERG light response. The rdgB2 flies expressing either RdgB-PITP-T59E or RdgB-T59E also exhibited a wild-type light-response amplitude. While the ERG light-response termination in rdgB2 flies expressing T59E-containing proteins was significantly faster than rdgB2 flies, it was still two to three times slower than wild type (Fig. 3).

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