Isolation and Characterization of the Saccharomyces cerevisiae DPP1 Gene Encoding Diacylglycerol Pyrophosphate Phosphatase

Diacylglycerol pyrophosphate (DGPP) is involved in a putative novel lipid signaling pathway. DGPP phosphatase (DGPP phosphohydrolase) is a membrane-associated 34-kDa enzyme fromSaccharomyces cerevisiae which catalyzes the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyzes the dephosphorylation of PA to yield diacylglycerol. Amino acid sequence information derived from DGPP phosphatase was used to identify and isolate the DPP1(diacylglycerol pyrophosphatephosphatase) gene encoding the enzyme. Multicopy plasmids containing the DPP1 gene directed a 10-fold overexpression of DGPP phosphatase activity in S. cerevisiae. The heterologous expression of the S. cerevisiae DPP1 gene in Sf-9 insect cells resulted in a 500-fold overexpression of DGPP phosphatase activity over that expressed in wild-type S. cerevisiae. DGPP phosphatase possesses a Mg2+-independent PA phosphatase activity, and its expression correlated with the overexpression of DGPP phosphatase activity in S. cerevisiae and in insect cells. DGPP phosphatase was predicted to be an integral membrane protein with six transmembrane-spanning domains. The enzyme contains a novel phosphatase sequence motif found in a superfamily of phosphatases. Adpp1Δ mutant was constructed by deletion of the chromosomal copy of the DPP1 gene. The dpp1Δ mutant was viable and did not exhibit any obvious growth defects. The mutant was devoid of DGPP phosphatase activity and accumulated (4-fold) DGPP. Analysis of the mutant showed that the DPP1 gene was not responsible for all of the Mg2+-independent PA phosphatase activity in S. cerevisiae

CD95 Rapidly Clusters in Cells of Diverse Origins

We have shown that CD95-mediated cell death requires a clustering of the receptor in distinct sphingolipid-rich domains of the cell membrane (Grassmé et al., 2000, Cremesti et al., 2000). These domains form in response to acid sphingomyelinase (ASM)-induced ceramide generation. However, recent studies challenged the finding of early CD95 clustering (Algeciras-Schimnich et al., 2002). Here, six independent groups tested clustering of CD95 in diverse cell type including primary cells ex vivo and established cell lines. The studies show clustering of CD95 within seconds to minutes in all cell types tested by the different groups. In addition, clustering of CD95 was detected after stimulation of cells using three agonistic anti-CD95 antibodies (CH11, APO-1-3 and JO2), CD95 ligand and stimuli that induce an upregulation and activation of the endogenous CD95/CD95 ligand system. The data confirm our previous studies and suggest rapid, i.e., within seconds to minutes, CD95 clustering as a general phenomenon occurring in many cell types

A BAR Domain in the N Terminus of the Arf GAP ASAP1 Affects Membrane Structure and Trafficking of Epidermal Growth Factor Receptor

SummaryBackgroundArf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1.ResultsASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1•GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect.ConclusionsThe N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector