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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 preferentially affects  rhodopsin protein levels. Immunoblot analyses were performed  on head extracts from <1-d-old dark-raised flies of the following  genotypes: rdgB+ (wild-type), ninaEI17, rdgB2, rdgB+; P[rdgB-T59E],  rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+]. Two head equivalents per sample were electrophoresed, transferred to nitrocellulose, and incubated with either anti–NinaE polyclonal or anti–Trp  polyclonal antisera. Triplicate blots were used to generate the average percent of wild-type protein and standard deviation.
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Figure 9: The dominant RdgB-T59E protein preferentially affects rhodopsin protein levels. Immunoblot analyses were performed on head extracts from <1-d-old dark-raised flies of the following genotypes: rdgB+ (wild-type), ninaEI17, rdgB2, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+]. Two head equivalents per sample were electrophoresed, transferred to nitrocellulose, and incubated with either anti–NinaE polyclonal or anti–Trp polyclonal antisera. Triplicate blots were used to generate the average percent of wild-type protein and standard deviation.

Mentions: We confirmed that RdgB-T59E expression reduced steady-state R1-6 rhodopsin levels to ∼1% of wild-type rhodopsin levels (Fig. 9). The reduced rhodopsin levels were not a result of RdgB overexpression, because overexpressing wild-type RdgB (rdgB+; P[rdgB+] flies) did not adversely affect rhodopsin levels (117 ± 34% of the wild-type levels, Fig. 9). Also, rdgB2 mutants failed to show reduced steady-state levels of rhodopsin (Fig. 9), which demonstrates that functional RdgB is not required for producing the wild-type rhodopsin levels. We also found that rdgB+; P[rdgB-T59E] flies elicited near wild-type levels of the trp-encoded Ca2+ channel (Fig. 9), the dgq-encoded Gα subunit, the gbe-encoded Gβe subunit, the rdgC-encoded serine/threonine phosphatase, and the ninaC-encoded unconventional myosins, suggesting that rhodopsin is selectively sensitive to expression of RdgB-T59E (data not shown). Therefore, the dominant retinal degeneration phenotype, the reduced photoreceptor sensitivity, loss of the PDA, and the gross reduction in mature rhodopsin levels in rdgB+; P[rdgB-T59E] flies all resulted from expression of RdgB-T59E and its interaction with some other protein and/or organelle.


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 preferentially affects  rhodopsin protein levels. Immunoblot analyses were performed  on head extracts from <1-d-old dark-raised flies of the following  genotypes: rdgB+ (wild-type), ninaEI17, rdgB2, rdgB+; P[rdgB-T59E],  rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+]. Two head equivalents per sample were electrophoresed, transferred to nitrocellulose, and incubated with either anti–NinaE polyclonal or anti–Trp  polyclonal antisera. Triplicate blots were used to generate the average percent of wild-type protein and standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: The dominant RdgB-T59E protein preferentially affects rhodopsin protein levels. Immunoblot analyses were performed on head extracts from <1-d-old dark-raised flies of the following genotypes: rdgB+ (wild-type), ninaEI17, rdgB2, rdgB+; P[rdgB-T59E], rdgB+; P[pitpα-rdgB], and rdgB+; P[rdgB+]. Two head equivalents per sample were electrophoresed, transferred to nitrocellulose, and incubated with either anti–NinaE polyclonal or anti–Trp polyclonal antisera. Triplicate blots were used to generate the average percent of wild-type protein and standard deviation.
Mentions: We confirmed that RdgB-T59E expression reduced steady-state R1-6 rhodopsin levels to ∼1% of wild-type rhodopsin levels (Fig. 9). The reduced rhodopsin levels were not a result of RdgB overexpression, because overexpressing wild-type RdgB (rdgB+; P[rdgB+] flies) did not adversely affect rhodopsin levels (117 ± 34% of the wild-type levels, Fig. 9). Also, rdgB2 mutants failed to show reduced steady-state levels of rhodopsin (Fig. 9), which demonstrates that functional RdgB is not required for producing the wild-type rhodopsin levels. We also found that rdgB+; P[rdgB-T59E] flies elicited near wild-type levels of the trp-encoded Ca2+ channel (Fig. 9), the dgq-encoded Gα subunit, the gbe-encoded Gβe subunit, the rdgC-encoded serine/threonine phosphatase, and the ninaC-encoded unconventional myosins, suggesting that rhodopsin is selectively sensitive to expression of RdgB-T59E (data not shown). Therefore, the dominant retinal degeneration phenotype, the reduced photoreceptor sensitivity, loss of the PDA, and the gross reduction in mature rhodopsin levels in rdgB+; P[rdgB-T59E] flies all resulted from expression of RdgB-T59E and its interaction with some other protein and/or organelle.

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