Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner and sphingolipids in the outer leaflet. This asymmetry is maintained by ATP-driven lipid transporters whose identities are unknown. The yeast plasma membrane contains two P-type ATPases, Dnf1p and Dnf2p, with structural similarity to ATPase II, a candidate aminophospholipid translocase from bovine chromaffin granules. Loss of Dnf1p and Dnf2p virtually abolished ATP-dependent transport of NBD-labeled phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine from the outer to the inner plasma membrane leaflet, leaving transport of sphingolipid analogs unaffected. Labeling with trinitrobenzene sulfonic acid revealed that the amount of phosphatidylethanolamine exposed on the surface of ⌬dnf1⌬dnf2 cells increased twofold relative to wild-type cells. Phosphatidylethanolamine exposure by ⌬dnf1⌬dnf2 cells further increased upon removal of Drs2p, an ATPase II homolog in the yeast Golgi. These changes in lipid topology were accompanied by a cold-sensitive defect in the uptake of markers for bulk-phase and receptor-mediated endocytosis. Our findings demonstrate a requirement for Dnf1p and Dnf2p in lipid translocation across the yeast plasma membrane. Moreover, it appears that Dnf1p, Dnf2p and Drs2p each help regulate the transbilayer lipid arrangement in the plasma membrane, and that this regulation is critical for budding endocytic vesicles.

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Mutations in RVS161 and RVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis. Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant. Here we demonstrate that the two proteins function together in vivo. We find that the steady state level of Rvs167p is strongly reduced in an rvs161⌬ strain. Similarly, the level of Rvs161p is strongly reduced in an rvs167⌬ strain. We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein. Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in an abp1⌬ strain suggesting that Abp1p is not essential for this interaction.

Long chain sphingoid bases (LCBs) and their phosphates (LCBPs) are not only important intermediates in ceramide biosynthesis but also signaling molecules in the yeast, Saccharomyces cerevisiae. Their cellular levels, which control multiple cellular events in response to external and intrinsic signals, are tightly regulated by coordinated action of metabolic enzymes such as LCB kinase and LCBP phosphatase. However, little is known about the mechanisms by which the two enzymes generate biosynthetic or signaling outputs. It has been shown that the LCBP phosphatase, Lcb3p, is required for efficient ceramide synthesis from exogenous LCB. Here we present direct evidence that the major LCB kinase, Lcb4p, but not the minor kinase, Lcb5p, regulates synthesis of ceramide from exogenously added LCB. Surprisingly, our biochemical evidence suggests that the LCBP used for ceramide synthesis must be generated on the membrane. Our data show that Lcb4p is tightly associated with membranes and is localized to the endoplasmic reticulum where it can work in concert with Lcb3p. These results raise the conceptually attractive possibility that membrane-associated and cytosolic Lcb4p play distinct roles to differentially generate biosynthetic and signaling pools of LCBP.Sphingolipid metabolites, including ceramide, sphingosine, and sphingosine 1-phosphate (S1P) 1 function as important second messengers in mammalian cells, regulating diverse biological processes such as cell growth, differentiation, apoptosis, stress responses, calcium homeostasis, and cell migration (1-4). Several lines of evidence strongly suggest that the dynamic balance between intracellular ceramide/sphingosine and S1P is an important factor that determines their cellular processes (3, 5). However, the mechanisms by which cells regulate intracellular levels of these lipids as well as their localization and mechanisms of action are largely unknown.The level of S1P is regulated by the metabolic enzymes responsible for its formation, which is catalyzed by sphingosine kinase (6, 7), and its degradation, which is catalyzed by an endoplasmic reticulum (ER)-bound S1P lyase (8, 9) and a specific phosphatase (10, 11). In mammalian cells, two sphingosine kinase isoforms have been cloned and characterized (6, 7). Although sphingosine kinase type 1 (SPHK1) and type 2 (SPHK2) have a high degree of homology, they have differential tissue expression, temporal developmental expression, and properties, suggesting that they have distinct cellular functions and may regulate levels of S1P differently. Furthermore, SPHK1 is a cytoplasmic enzyme, whereas SPHK2 has several predicted transmembrane regions, suggesting that it is a membrane protein (3). However, both kinase activities are present in the cytosol and in membranes (6, 7). Another study suggested the presence of additional sphingosine kinases in mammalian tissues: one cytosolic and two membrane-bound activities that are associated with the ER and with plasma membrane (12).In the yeast, Saccharomyces cerevisiae, two genes that e...

No abstract