Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins.

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin alpha/beta-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.

Distinct AAA-ATPase p97 complexes function in discrete steps of nuclear assembly.

Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. Here we show that p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation.

GTP hydrolysis by Ran is required for nuclear envelope assembly.

Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTP gamma S, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTP gamma S and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTP gamma S. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.

Linear mitochondrial plasmids of F. oxysporum are novel, telomere-like retroelements.

Diverse types of linear RNA and DNA autonomously replicating genetic elements exist in prokaryotic and eukaryotic hosts, yet linear elements that replicate by reverse transcription have not been identified. Here, we report the sequence and organization of two linear mitochondrial plasmids of the fungal plant pathogen F. oxysporum and the characterization of a plasmid-associated reverse transcriptase activity. Plasmids pFOXC2 and pFOXC3 are 1.9 kb in length and have a "clothespin" genomic structure, which includes a terminal hairpin and a telomere-like iteration of a 5 bp sequence at the other terminus. The retroplasmid replication cycle involves novel strategies for copying terminal sequences, which may provide clues concerning the origin of telomerase as well as the evolution of linear DNAs.