No abstract

CD47, a ‘self’ recognition marker expressed on tissue cells, interacts with immunoreceptor SIRPα expressed on the surface of macrophages to initiate inhibitory signaling that prevents macrophage phagocytosis of healthy host cells. Previous studies have suggested that cells may lose the surface CD47 during aging or apoptosis to enable phagocytic clearance. In the present study, we demonstrate that the level of cell surface CD47 is not decreased but the distribution pattern of CD47 is altered during apoptosis. On non-apoptotic cells, CD47 molecules are clustered in lipid rafts forming ‘punctates’ on the surface, whereas on apoptotic cells, CD47 molecules are diffused on the cell surface following the disassembly of lipid rafts. We show that clustering of CD47 in lipid rafts provides a high binding avidity for cell surface CD47 to ligate macrophage SIRPα, which also presents as clusters, and elicit SIRPα-mediated inhibitory signaling that prevents phagocytosis. In contrast, dispersed CD47 on the apoptotic cell surface is associated a significant reduction of the binding avidity to SIRPα and failure to trigger SIRPα signal transduction. Disruption of lipid rafts with methyl-β-cyclodextrin (MβCD) disrupted CD47 cluster formation on the cell surfaces, leading to decrease of the binding avidity to SIRPα and a concomitant increase of cells being engulfed by macrophages. Taken together, our study reveals that CD47 normally is clustered in lipid rafts on non-apoptotic cells but is diffused in the plasma membrane when apoptosis occurs, and this transformation of CD47 greatly reduces the strength of CD47-SIRPα engagement, resulting in the phagocytosis of apoptotic cells.

5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil  ), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1 ), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues.

The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.Killer toxin production is a frequently realized intra-and interspecies strategy among yeasts to restrict the growth of competitors. While target cell killing is the common purpose, the structures of toxins, their mechanisms of action, and the organizations of encoding genes are rather diverse (25,32). The Kluyveromyces lactis and Pichia acaciae toxin systems depend on double-stranded DNA elements (8,16,40). The two species each harbor a pair of extranuclear linear plasmids, i.e., pGKL1 (8.9 kb) and pGKL2 (13.5 kb) (K. lactis) and pPac1-1 (13.6 kb) and pPac1-2 (6.8 kb) (P. acaciae) (1,8,37). The larger plasmids are autonomous elements displaying almost identical gene contents that include loci essential for cytoplasmic replication, transcription, and transcript modification (14,15). In contrast, the smaller plasmids carry structurally distinct toxin genes (32); these elements are nonautonomous and rely on the respective larger autonomous plasmid for extranuclear replication and transcription (29).The K. lactis toxin, termed zymocin, consists of three subunits encoded by the pGKL1-borne ORF2 (the ␣ and ␤ subunits) and ORF4 (the ␥ subunit) (36). Docking to the primary cell wall receptor chitin is facilitated by the ␣ subunit (12), and the remarkably hydrophobic ␤ subunit presumably assists in the uptake of the ␥ subunit, which is a tRNase (24,27,37).Like the K. lactis toxin zymocin, the P. acaciae toxin (PaT) comprises a heteromeric complex (26). The polypeptide encoded by pPac1-2 ORF1, possessing both chitin-binding and hydrophobic domains, is akin to the K. lactis counterparts; however, the intracellularly acting toxic subunit (encoded by pPac1-2 ORF2) is obviously unrelated to the K. lactis ␥ subunit (22).Zymocin action depends on the protein complex Elongator (4). Recently published data indicate that Elongator is instrumental in tRNA modification, i.e., in placing 5-methoxycarbonylmethyl (mcm 5 ) and 5-carbamoylmethyl (ncm 5 ) moieties on uridines at the wobble position (11, 24). Loss of Elongatordependent wobble nucleoside modifications in tRNA Glu , tRNA Lys , and tRNA Gln prevents recognition and cleavage by the zymocin ␥ subunit and confers exotoxin resistance (13, 24).For PaT function, in contrast, Elongator is not required, indicating the functional diversity of the toxins. Moreover, terminal toxin responses to PaT and zymocin differ: while the latter toxin arrests target cells in G 1 , PaT has been shown to induce S-phase arrest and DNA damage checkpoint induction followed by apoptotic cell death (20,21). It has been shown that self-protection from zymocin is mediated by pGKL1 ORF3; however, in the pPac killer system...