Fertilization induces a transient exposure of phosphatidylserine in mouse eggs

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca2+ concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca2+ spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca2+ release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca2+ ionophore, suggesting that the Ca2+ source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca2+ rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.Fil: Curia, Claudio Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ernesto, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Stein, Paula. University of Pennsylvania; Estados UnidosFil: Busso, Dolores. Pontificia Universidad Católica de Chile; ChileFil: Schultz, Richard. University of Pennsylvania; Estados UnidosFil: Cuasnicu, Patricia Sara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Cohen, Debora Juana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

ABC Transporter Pdr10 Regulates the Membrane Microenvironment of Pdr12 in Saccharomyces cerevisiae

The eukaryotic plasma membrane exhibits both asymmetric distribution of lipids between the inner and the outer leaflet and lateral segregation of membrane components within the plane of the bilayer. In budding yeast (Saccharomyces cerevisiae), maintenance of leaflet asymmetry requires P-type ATPases, which are proposed to act as inward-directed lipid translocases (Dnf1, Dnf2, and the associated protein Lem3), and ATP-binding cassette (ABC) transporters, which are proposed to act as outward-directed lipid translocases (Pdr5 and Yor1). The S. cerevisiae genome encodes two other Pdr5-related ABC transporters: Pdr10 (67% identity) and Pdr15 (75% identity). We report the first analysis of Pdr10 localization and function. A Pdr10-GFP chimera was located in discrete puncta in the plasma membrane and was found in the detergent-resistant membrane fraction. Compared to control cells, a pdr10∆ mutant was resistant to sorbate but hypersensitive to the chitin-binding agent Calcofluor White. Calcofluor sensitivity was attributable to a partial defect in endocytosis of the chitin synthase Chs3, while sorbate resistance was attributable to accumulation of a higher than normal level of the sorbate exporter Pdr12. Epistasis analysis indicated that Pdr10 function requires Pdr5, Pdr12, Lem3, and mature sphingolipids. Strikingly, Pdr12 was shifted to the detergent-resistant membrane fraction in pdr10∆ cells. Pdr10 therefore acts as a negative regulator for incorporation of Pdr12 into detergent-resistant membranes, a novel role for members of the ABC transporter superfamily