Control of synapse number by glia.

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.

Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p.

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

Biogenesis of the Saccharomyces cerevisiae mating pheromone a-factor.

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 SDS-PAGE. 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.

Assembly of the ER to Golgi SNARE complex requires Uso1p.

Uso1p, a Saccharomyces cerevisiae protein required for ER to Golgi transport, is homologous to the mammalian intra-Golgi transport factor p115. We have used genetic and biochemical approaches to examine the function of Uso1p. The temperature-sensitive phenotype of the uso1-1 mutant can be suppressed by overexpression of each of the known ER to Golgi v-SNAREs (Bet1p, Bos1p, Sec22p, and Ykt6p). Overexpression of two of them, BET1p and Sec22p, can also suppress the lethality of delta uso1, indicating that the SNAREs function downstream of Uso1p. In addition, overexpression of the small GTP-binding protein Ypt1p, or of a gain if function mutant (SLY1-20) of the t-SNARE associated protein Sly1p, also confers temperature resistance. Uso1p and Ypt1p 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. Uso1p acts upstream of, or in conjunction with, Ypt1p because overexpression of Ypt1p allows a delta uso1 strain to grow, whereas overexpression of Uso1p does not suppress a delta ypt1 strain. Finally, biochemical analysis indicates that Uso1p, like Ypt1p, 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.

p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p.

A recently discovered vesicular transport factor, termed p115, is required along with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins for in vitro Golgi transport. p115 is a peripheral membrane protein found predominantly on the Golgi. Biochemical and electron microscopic analyses indicate that p115 is an elongated homodimer with two globular "heads" and an extended "tail" reminiscent of myosin II. We have cloned and sequenced cDNAs for bovine and rat p115. The predicted translation products are 90% identical, and each can be divided into three domains. The predicted 108-kDa bovine protein consists of an N-terminal 73-kDa globular domain followed by a 29-kDa coiled-coil dimerization domain, a linker segment of 4 kDa, and a highly acidic domain of 3 kDa. p115 is related to Uso1p, a protein required for endoplasmic reticulum to Golgi vesicular transport in Saccharomyces cerevisiae, which has a similar "head-coil-acid" domain structure. The p115 and Uso1p heads are similar in size, have approximately 25% sequence identity, and possess two highly homologous regions (62% and 60% identity over 34 and 53 residues, respectively). There is a third region of homology (50% identity over 28 residues) between the coiled-coil and acidic domains. Although the acidic nature of the p115 and Uso1p C termini is conserved, the primary sequence is not. We discuss these results in light of the proposed function of p115 in membrane targeting and/or fusion.

The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins.

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.