We have developed an assay using permeabilized cells to monitor fragmentation of the Golgi complex that occurs during mitosis. Golgi stacks, in permeabilized interphase normal rat kidney (NRK) cells, upon incubation with mitotic extracts undergo extensive fragmentation, and the fragmented Golgi membranes are dispersed throughout the cytoplasm. We find that the continued presence of p34cdc2, the mitosis initiation kinase, is not necessary for Golgi fragmentation. Instead, fragmentation depends on cytosolic mitogen-activated protein kinase kinase 1 (MEK1 or MAPKK1). However, the known cytoplasmic substrates for MEK1, ERK1, and ERK2 are not required for this process. Interestingly, we find a Golgi-associated ERK, which we propose as the likely target for MEK1 in Golgi fragmentation.

We have reconstituted the fusion and assembly of vesiculated Golgi membranes (VGMs) into functionally active stacks of cisternae. A kinetic analysis of this assembly process revealed that highly dispersed VGMs of 60-90 nm diameter first fuse to form larger vesicles of 200-300 nm diameter that are clustered together. These vesicles then fuse to form tubular elements and short cisternae, which finally assemble into stacks of cisternae. We now provide evidence that the sequential stack formation from VGMs reflects two distinct fusion processes: the first event is N-ethyl-maleimide (NEM)-sensitive factor (NSF) dependent, and the second fusion event requires an NSF-like NEM-sensitive ATPase called p97. Interestingly, while the earliest steps in stack formation share some similarities with events catalyzing fusion of transport vesicles to its target membrane, neither GTP gamma S nor Rab-GDI, inhibitors of vesicular protein traffic, inhibit stack formation.

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. Here, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescued retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppressed degeneration in these mutants. Both genetic backgrounds modulated the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlated with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein.We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.aging ͉ Drosophila ͉ membrane fluidity ͉ sphingolipids ͉ sphingomyelin