Ceramidase expression facilitates membrane turnover and endocytosis of rhodopsin in photoreceptors

Abstract

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. Here, we show that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaE I17 mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration.S phingolipids are integral components of eukaryotic cell membranes. They are essential for survival of yeast, Drosophila, and mammals. Sphingolipids have a hydrophobic backbone moiety, ceramide, linked to a variable polar head group, e.g., phosphorylcholine in sphingomyelin and a saccharide in glycosphingolipids. Ceramide itself consists of a long chain base linked to a fatty acid. Ceramide, sphingosine, and sphingosine-1-P are lipid second messengers modulating a variety of cellular functions (1-3). Ceramide is at the branch point of the sphingolipid biosynthetic pathway and serves as a substrate for several enzymes, e.g., sphingomyelin synthase, ceramidase, ceramide kinase, and glucosylceramide synthase. In Drosophila, transgenic expression of a neutral ceramidase is accompanied by a decrease in the levels of ceramide (4). We have shown that modulation of enzymes of the sphingolipid biosynthetic pathway suppressed retinal degeneration in certain phototransduction mutants (4). Transgenic expression of ceramidase or a loss of one copy of the rate-limiting enzyme in the sphingolipid biosynthetic pathway, namely, Serine Palmitoyl CoA transferase, suppressed retinal degeneration in arrestin and phospholipase C (norpA) mutants. These backgrounds also suppressed degeneration in a dynamin mutant, suggesting that ceramidase was modulating the endocytic pathway. To gain further insight into the mechanism of ceramidase action, we have investigated the effects of ceramidase expression in the rhodopsin null mutant, ninaE I17 . Rhodopsin, the G protein-coupled receptor, transduces light signal in Drosophila photoreceptors R1-R6 (5, 6). Rhodopsin is also involved in the formation of a rhabdomere terminal web (RTW), an actin-based cytoskeletal scaffold implicated in rhabdomere biogenesis (7,8). Rhabdomeres are densely organized, specialized areas of plasma membrane that house the signal transduction machinery of photoreceptors. In ninaE I17 mutants, the RTW is not organized during morphogenesis and thus photoreceptors have improperly formed rhabdomeres (8). In addition, in 3-dayold flies, rhabdomeric e...

Phototransduction and retinal degeneration in Drosophila

“…Thus, a defect in degradation of internalized rhodopsin may be toxic. The cell death resulting from loss of arr2 is countered by overexpressing ceramidase [224,225], which cleaves ceramide to produce sphingosine. The ceramidase may decrease the toxicity resulting from absence of Arr2 by increasing endocytosis [224,225].…”

“…The cell death resulting from loss of arr2 is countered by overexpressing ceramidase [224,225], which cleaves ceramide to produce sphingosine. The ceramidase may decrease the toxicity resulting from absence of Arr2 by increasing endocytosis [224,225]. Unlike the light-dependent cell death in sun [31] and arr2 flies [95], the degeneration in arr1 is light-independent [99].…”

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

“…The sphingolipid biosynthetic pathway is an evolutionarily conserved route that generates and interconverts various sphingolipids such as ceramide, sphingosine, ceramide 1-phosphate and sphingosine 1-phosphate (9). We showed earlier that modulating this biosynthetic pathway by targeted overexpression of Drosophila neutral ceramidase (CDase), an enzyme that converts ceramide to sphingosine, rescues retinal degeneration in an arrestin mutant, and facilitates membrane turnover in a rhodopsin null mutant by modulating the endocytic machinery (10)(11)(12). Although these studies established that ceramide metabolism is important for survival of photoreceptors, they did not evaluate its role in signaling events during phototransduction.…”

Sphingolipids and membrane biology as determined from genetic models

“…Newly eclosed flies show inappropriately formed rhabdomeric components that involute into the cytosol [62]. Ceramidase overexpression facilitates the clearing of these inappropriately formed rhabdomeric components by internalization [63]. These results suggest a potential role for sphingolipid metabolites in endocytosis and membrane turnover.…”