Protein components of a rat brain synaptic junctional protein preparation.

Antisera against a rat brain synaptic protein preparation, the postsynaptic density (PSD) fraction, were used to isolate cDNA clones by expression screening of a rat brain cDNA library. About one fifth of more than 200 analyzed cDNAs encoding potential synapse-associated proteins were previously unknown. Identifiable proteins include, among others, components of the pre- and postsynaptic cytoskeleton, synaptic vesicle proteins and several protein kinases and kinase substrates. This demonstrates that both pre- and postsynaptic elements purify with the PSD fraction.

Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix.

In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.

SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A.

A novel synapse-associated protein, SAP90, accumulates around the axon hillock of Purkinje cells in rat cerebellum. By immuno-electron microscopy, SAP90 has been localized to the presynaptic termini of basket cells forming inhibitory, gamma-aminobutyric acid (GABA)ergic synapses onto Purkinje cell axon hillocks. The amino acid sequence for SAP90 has been deduced from the nucleotide sequence of a series of overlapping cDNA clones. SAP90 is related to the gene product encoded by the Drosophila tumor suppressor gene dlg-A. SAP90 and the dlg-A product share an overall sequence identity of 54%. Three distinct domains can be identified: (i) a potential cytoskeletal region consisting of three repeats of 90 amino acids in length, (ii) a domain with similarity to SH3, a putative regulatory motif found in the src family of non-receptor protein tyrosine kinases and several proteins associated with the cortical cytoskeleton, and (iii) a carboxyl-terminal domain homologous to yeast guanylate kinase. These features suggest a possible role for SAP90 in a guanine nucleotide-mediated signal transduction pathway at a subset of GABAergic synapses in the rat cerebellum.

Hormone-induced expression of the CHS1 gene from Saccharomyces cerevisiae.

When MATa cells of Saccharomyces cerevisiae have been treated with the mating hormone alpha-factor an increase in chitin synthase zymogen, as well as chitin content in the cell-wall fraction, have been reported. With a DNA probe derived from the cloned CHS1 gene that codes for chitin synthase I [Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L. and Robbins, P. (1986) Cell 46, 213-225] a Northern analysis was conducted of CHS1-specific transcripts. alpha-Factor-treated MATa cells revealed more than sixfold elevated steady-state levels of CHS1 mRNA as compared to control cells. MAT alpha cells responded the same way when treated with a-factor although induction rate was somewhat smaller. After hormone application a rapid increase in CHS1 mRNA levels could be observed that occurred also in the absence of ongoing protein synthesis. In order to minimize possible side effects of CHS1-coding sequences on expression and mRNA stability a CHS1::SUC2 chimaeric gene was constructed where 730 bp of the CHS1 promoter region (+20 bp of the coding region) were fused in frame to a fragment of the SUC2 coding region. The fusion protein exhibits invertase activity that has been used to monitor CHS1 promoter activity. By analysis of shortened versions of the CHS1 promoter a 94-bp DNA fragment has been identified that confers hormone inducibility to the CHS1 promoter. According to the published sequence of the CHS1 gene, this fragment contains four repeats of a TGAAACA consensus sequence previously identified in the alpha-factor-inducible BAR1 promoter [Kronstad, J. W., Holly, J. A. and MacKay, V. L. (1987) Cell 50, 369-377]. This heptamer may represent the cis-acting element involved in mating-hormone-mediated gene expression in yeast.

Saccharomyces cerevisiae mating pheromones specifically inhibit the synthesis of proteins destined to be N-glycosylated.

alpha Factor specifically inhibits the synthesis of N-glycosylated proteins in Saccharomyces cerevisiae mating type a cells but not in alpha cells or in a/alpha diploids. a Factor has the same effect of alpha cells. The synthesis of O-glycosylated proteins is not inhibited. Although the mating pheromones act like a 'physiological tunicamycin', the mechanism of inhibition is different: not the glycosylation of proteins as such but rather the synthesis of those proteins destined to be N-glycosylated is inhibited. Thus none of a number of glycosylating enzymes tested in vitro is reduced in activity in alpha-factor-treated cells. The synthesis of the glycoprotein carboxypeptidase Y, on the other hand, is strongly inhibited by tunicamycin as well as by alpha factor; but only in the former case did carbohydrate-free protein accumulate in the cells. alpha Factor causes maximal inhibition of glycoprotein formation after as little as 30 min, long before all cells in the population are arrested in G1; moreover, release from this inhibition precedes the increase in budding index (resumption of cell division). It is postulated, therefore, that N-glycosylated proteins are required for the G1/S-phase transition in the yeast cell cycle. This is supported by previous reports that first cycle arrest in G1 occurs when (a) tunicamycin is added to growing cultures, and (b) a temperature-sensitive N-glycosylation mutant is shifted to its restrictive temperature.