Coligation of the Fc receptor on B cells, Fc gamma RIIB1, with the B cell antigen receptor (BCR) leads to abortive BCR signaling. Here it was shown that the Fc gamma RIIB1 recruits the phosphotyrosine phosphatase PTP1C after BCR coligation. This association is mediated by the binding of a 13-amino acid tyrosine-phosphorylated sequence to the carboxyl-terminal Src homology 2 domain of PTP1C and activates PTP1C. Inhibitory signaling and PTP1C recruitment are dependent on the presence of the tyrosine within the 13-amino acid sequence. Inhibitory signaling mediated by Fc gamma RIIB1 is deficient in motheaten mice which do not express functional PTP1C. Thus, PTP1C is an effector of BCR-Fc gamma RIIB1 negative signal cooperativity.

Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid precursor inositol (M. L. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8:47734779, 1988). In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function. PGPS derepressed two-to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol) Cardiolipin (CL) is found only in the mitochondrial inner membrane (7,9,20) and is necessary for several aspects of mitochondrial function. In the yeast Saccharomyces cerevisiae, CL is required for cytochrome oxidase (CO) activity (41, 42) and may be involved in import of proteins into the mitochondrion (10, 11). In higher eucaryotes, CL is an effector of the cytochrome P-450-dependent cholesterol sidechain cleavage enzyme (31) and is required for activities of CO (34) and the mitochondrial phosphate carrier protein (21). An understanding of the regulation of CL biosynthesis would therefore provide insight into mitochondrial membrane biogenesis as well as the role played by this phospholipid in mitochondrial function.The synthesis of CL involves three sequential reactions (7, 28, 37). The enzyme phosphatidylglycerolphosphate synthase (PGPS) catalyzes the committed step in CL synthesis, involving the conversion of the liponucleotide CDP-diglyceride (CDP-DG) and glycerol 3-phosphate to phosphatidylglycerolphosphate (PGP). PGP phosphatase (PGPase) dephosphorylates PGP to phosphatidylglycerol, which subsequently is converted to CL by CL synthase (CLS). In procaryotes, CL is synthesized from two molecules of phosphatidylglycerol, while in higher eucaryotes, the CLS reaction involves the condensation of phosphatidylglycerol and CDP-DG. Tamai and Greenberg (39) have recently shown that S. cerevisiae, * Corresponding author. like higher eucaryotes, utilizes CDP-DG as a substrate in the synthesis of CL.We initially postulated that mitochondrial phospholipid synthesis may be affected by at least two sets of factors: (i) those that affect general phospholipid synthesis and (ii) those that affect mitochondrial development. In a previous study (14), our laboratory showed that expression of PGPS in S. cerevisiae is indeed regulated by the water-soluble phospholipid precursors inositol and choline. These precursors also repress the enzymes of the phosphatidylinositol (PI) and phosphatidylcholine (PC) branches of phospholipid synthesis (6). However, inositol repression of PGPS is not mediated by the same genetic regulatory circuit that controls the PI and PC branche...

Enrichment procedures, such as those utilizing inositol-less death, have proven to be extremely powerful for increasing the efficiency of identification of spontaneous mutants in a variety of procaryotic and eucaryotic organisms. We characterized inositol-less death in several widely used strains of the inositol-requiring yeast Schizosaccharomycespombe and determined conditions under which this phenomenon can be used to enrich for mutants. Conflicting reports in the literature on the effects of inositol starvation upon viability of S. pombe had cast doubt on the suitability of using inositol-less death in a mutant enrichment procedure for this organism. We determined that inositol-less death was strain dependent, with differences in viability of up to 5 orders of magnitude observed between the most-sensitive strain, 972, and the least-sensitive strain, SP837. Inositol-less death was also dependent upon the cell concentration at the time of initiation of starvation. While inositol-less death occurred at all four temperatures tested, the kinetics of death was slower at 16°C than at 23, 30, or 37°C. Inositol-less death was observed during growth in fermentable and nonfermentable carbon sources, although loss of viability in glycerol-ethanol was significantly slower than that in glucose, sucrose, or raffinose. The feasibility of exploiting inositol-less death to enrich for spontaneous mutants was demonstrated by the identification of amino acid auxotrophs, nucleotide auxotrophs, carbon source utilization mutants, and temperature-sensitive mutants. By varying starvation conditions, some mutants were recovered at frequencies as high as 5.7 x 10-2, orders of magnitude higher than the spontaneous mutation rate.

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Fcγ receptors (FcγRII) on B lymphocytes negatively regulate B cell receptor (BCR)-dependent activation upon cross-linking of the two receptors. The mechanism reflects the ability of the FcγRII cytoplasmic tail to recruit specific phosphatases that inactivate elements of the BCR-signaling cascade. We now show that cross-linking also blocks the processing and presentation of BCR-bound Ag. This occurs because the FcγRII isoform typically expressed by B cells (FcγRII-B1) is incompetent for endocytosis. When cross-linked, FcγRII-B1 acts as a dominant negative inhibitor of BCR endocytosis. In contrast, cross-linking of endocytosis-competent FcγRII isoforms did not inhibit endocytosis or processing of BCR-bound Ag. Thus, FcγRII-B1 acts not only to prevent B cell activation under conditions of Ab excess, but also to prevent clonotypic T cell activation by inhibiting the ability of B cells to generate specific MHC class II-bound TCR ligands.