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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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RdgB-PITP is functionally related to PITPα. (A) Various RdgB-PITPs (wild-type RdgB-PITP and the missense mutants T59A and T59E) were expressed in E. coli, partially purified  and shown to be stably expressed by gel electrophoresis and immunoblot. (B) RdgB-PITP, RdgB-PITP-T59E, and RdgB-PITP-T59A were analyzed for PI and PC transfer activity in vitro. The  solid bars represent the percentage of radiolabeled PI transferred  relative to wild-type RdgB-PITP (5.3 ± 0.1%). The shaded bars  represent the percentage of radiolabeled PC transferred relative  to wild-type RdgB-PITP (6.4 ± 1.2%). SM transfer activity was  assessed for mammalian PITPβ, which served as positive control,  and for wild-type RdgB-PITP (striped bars). PITPβ transferred  2.3 ± 0.1% of total input radiolabeled SM and this value was set  at 100%. Lysates from E. coli that lack a plasmid expressing PITP  failed to exhibit any detectable PI and PC transfer activity (Alb  et al., 1995). Each sample represents three trials and the standard  deviation is shown as vertical bars.
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Figure 2: RdgB-PITP is functionally related to PITPα. (A) Various RdgB-PITPs (wild-type RdgB-PITP and the missense mutants T59A and T59E) were expressed in E. coli, partially purified and shown to be stably expressed by gel electrophoresis and immunoblot. (B) RdgB-PITP, RdgB-PITP-T59E, and RdgB-PITP-T59A were analyzed for PI and PC transfer activity in vitro. The solid bars represent the percentage of radiolabeled PI transferred relative to wild-type RdgB-PITP (5.3 ± 0.1%). The shaded bars represent the percentage of radiolabeled PC transferred relative to wild-type RdgB-PITP (6.4 ± 1.2%). SM transfer activity was assessed for mammalian PITPβ, which served as positive control, and for wild-type RdgB-PITP (striped bars). PITPβ transferred 2.3 ± 0.1% of total input radiolabeled SM and this value was set at 100%. Lysates from E. coli that lack a plasmid expressing PITP failed to exhibit any detectable PI and PC transfer activity (Alb et al., 1995). Each sample represents three trials and the standard deviation is shown as vertical bars.

Mentions: We previously demonstrated that RdgB-PITP exhibits PI-transfer activity (Vihtelic et al., 1993). To assess the range of phospholipid transfer capability, and to determine whether RdgB-PITP exhibited phospholipid transfer properties more similar to mammalian PITPα (which transfers PI and PC) or PITPβ (which transfers PI, PC, and SM), we expressed RdgB-PITP as a soluble polypeptide in E. coli (Fig. 2 A, lane 1) and assayed its ability to mobilize PI, PC, and SM between membrane bilayers in vitro. RdgB-PITP catalyzed robust transfer of both PI and PC in vitro (Fig. 2 B), with 5.3 ± 0.1% and 6.4 ± 1.2% of total radiolabeled PI and PC substrate transferred, respectively. However, we did not detect SM transfer activity with RdgB-PITP (Fig. 2 B). Thus, RdgB-PITP exhibited biochemical properties more closely resembling those of PITPα than PITPβ.


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)

RdgB-PITP is functionally related to PITPα. (A) Various RdgB-PITPs (wild-type RdgB-PITP and the missense mutants T59A and T59E) were expressed in E. coli, partially purified  and shown to be stably expressed by gel electrophoresis and immunoblot. (B) RdgB-PITP, RdgB-PITP-T59E, and RdgB-PITP-T59A were analyzed for PI and PC transfer activity in vitro. The  solid bars represent the percentage of radiolabeled PI transferred  relative to wild-type RdgB-PITP (5.3 ± 0.1%). The shaded bars  represent the percentage of radiolabeled PC transferred relative  to wild-type RdgB-PITP (6.4 ± 1.2%). SM transfer activity was  assessed for mammalian PITPβ, which served as positive control,  and for wild-type RdgB-PITP (striped bars). PITPβ transferred  2.3 ± 0.1% of total input radiolabeled SM and this value was set  at 100%. Lysates from E. coli that lack a plasmid expressing PITP  failed to exhibit any detectable PI and PC transfer activity (Alb  et al., 1995). Each sample represents three trials and the standard  deviation is shown as vertical bars.
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Related In: Results  -  Collection

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Figure 2: RdgB-PITP is functionally related to PITPα. (A) Various RdgB-PITPs (wild-type RdgB-PITP and the missense mutants T59A and T59E) were expressed in E. coli, partially purified and shown to be stably expressed by gel electrophoresis and immunoblot. (B) RdgB-PITP, RdgB-PITP-T59E, and RdgB-PITP-T59A were analyzed for PI and PC transfer activity in vitro. The solid bars represent the percentage of radiolabeled PI transferred relative to wild-type RdgB-PITP (5.3 ± 0.1%). The shaded bars represent the percentage of radiolabeled PC transferred relative to wild-type RdgB-PITP (6.4 ± 1.2%). SM transfer activity was assessed for mammalian PITPβ, which served as positive control, and for wild-type RdgB-PITP (striped bars). PITPβ transferred 2.3 ± 0.1% of total input radiolabeled SM and this value was set at 100%. Lysates from E. coli that lack a plasmid expressing PITP failed to exhibit any detectable PI and PC transfer activity (Alb et al., 1995). Each sample represents three trials and the standard deviation is shown as vertical bars.
Mentions: We previously demonstrated that RdgB-PITP exhibits PI-transfer activity (Vihtelic et al., 1993). To assess the range of phospholipid transfer capability, and to determine whether RdgB-PITP exhibited phospholipid transfer properties more similar to mammalian PITPα (which transfers PI and PC) or PITPβ (which transfers PI, PC, and SM), we expressed RdgB-PITP as a soluble polypeptide in E. coli (Fig. 2 A, lane 1) and assayed its ability to mobilize PI, PC, and SM between membrane bilayers in vitro. RdgB-PITP catalyzed robust transfer of both PI and PC in vitro (Fig. 2 B), with 5.3 ± 0.1% and 6.4 ± 1.2% of total radiolabeled PI and PC substrate transferred, respectively. However, we did not detect SM transfer activity with RdgB-PITP (Fig. 2 B). Thus, RdgB-PITP exhibited biochemical properties more closely resembling those of PITPα than PITPβ.

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