Although astrocytes constitute nearly half of the cells in our brain, their function is a long-standing neurobiological mystery. Here we show by quantal analyses, FM1-43 imaging, immunostaining, and electron microscopy that few synapses form in the absence of glial cells and that the few synapses that do form are functionally immature. Astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold and are required for synaptic maintenance in vitro. We also show that most synapses are generated concurrently with the development of glia in vivo. These data demonstrate a previously unknown function for glia in inducing and stabilizing CNS synapses, show that CNS synapse number can be profoundly regulated by nonneuronal signals, and raise the possibility that glia may actively participate in synaptic plasticity.

Abstract. Usolp, a Saccharomyces cerevisiae protein required for ER to Golgi transport, is homologous to the mammalian intra-Golgi transport factor pl15. We have used genetic and biochemical approaches to examine the function of Usolp. The temperature-sensitive phenotype of the usol-1 mutant can be suppressed by overexpression of each of the known ER to Golgi v-SNAREs (Betlp, Boslp, Sec22p, and Ykt6p). Overexpression of two of them, Betlp and Sec22p, can also suppress the lethality of Ausol, indicating that the SNAREs function downstream of Usolp. In addition, overexpression of the small GTP-binding protein Yptlp, or of a gain of function mutant (SLY1-20) of the t-SNARE associated protein Slylp, also confers temperature resistance. Usolp and Yptlp appear to function in the same process because they have a similar set of genetic interactions with the v-SNARE genes, they exhibit a synthetic lethal interaction, and they are able to suppress temperature sensitive mutants of one another when overexpressed. Usolp acts upstream of, or in conjunction with, Yptlp because overexpression of Yptlp allows a Ausol strain to grow, whereas overexpression of Usolp does not suppress a Ayptl strain. Finally, biochemical analysis indicates that Usolp, like Yptlp, is required for assembly of the v-SNARE/ t-SNARE complex. The implications of these findings, with respect to the mechanism of vesicle docking, are discussed.

Post‐translational processing of a distinct group of proteins and polypeptides, including the a‐factor mating pheromone and RAS proteins of Saccharomyces cerevisiae, results in the formation of a modified C‐terminal cysteine that is S‐isoprenylated and alpha‐methyl esterified. We have shown previously that a membrane‐associated enzymatic activity in yeast can mediate in vitro methylation of an isoprenylated peptide substrate and that this methyltransferase activity is absent in ste14 mutants. We demonstrate here that STE14 is the structural gene for this enzyme by expression of its product as a fusion protein in Escherichia coli, an organism in which this activity is lacking. We also show that a‐factor, RAS1 and RAS2 are physiological methyl‐accepting substrates for this enzyme by demonstrating that these proteins are not methylated in a ste14 null mutant. It is notable that cells lacking STE14 methyltransferase activity exhibit no detectable impairment of RAS function or cell viability. However, we did observe a kinetic delay in the rate of RAS2 maturation and a slight decrease in the amount of membrane localized RAS2. Thus, methylation does not appear to be essential for RAS2 maturation or localization, but the lack of methylation can have subtle effects on the efficiency of these processes.

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH2-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDSPAGE. We determined the biochemical composition of each by defining their NH2-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH2-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH2-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH2-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.