Enzyme II of the Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System: Protein-Protein and Protein-Phospholipid Interactions

The mannitol-specific enzyme II (EII), purified free of phospholipid, exhibits a concentration dependence in its specific activity with P-HPr and mannitol as the donor and acceptor substrates, respectively. This concentration dependence, previously observed only in the case of the mannitol ↔ mannitol phosphate exchange reaction, indicates that an oligomeric form of the enzyme is responsible for catalyzing the phosphorylation reaction (P-HPr + mannitol → mannitol-P + HPr) as well as the exchange reaction. Kinetic analysis revealed that the monomeric enzyme has a much lower specific activity than the associated species. The specific activity can be increased by raising the steady-state level of phosphorylation of EII and also by adding phospholipid, demonstrating that phosphorylation and the binding of phospholipid facilitate the association process. Kinetic measurements and fluorescence energy transfer measurements demonstrate a strong preference of EII for phospholipids with specific head group and fatty acid composition.

Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTAs*). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTAs*. Tetracycline prevents tTAs* from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTAs* binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.

Bacterial Phosphoenolpyruvate-Dependent Phosphotransferase System. Mechanism of the Transmembrane Sugar Translocation and Phosphorylation

The phosphoryl-group transfer from PHPr to glucose or α-methylglucose and from glucose 6-phosphate to these same sugars catalyzed by membrane-bound EIIBGlc of the bacterial phosphoenolpyruvate-dependent phosphotransferase system has been studied in vitro. Kinetic measurements revealed that both the phosphorylation reaction and the exchange reaction proceed according to a ping-pong mechanism in which a phosphorylated membrane-bound enzyme II acts as an obligatory intermediate. The occurrence of a phospho-IIBGlc/IIIGlc has been physically demonstrated by the production of a glucose 6-phosphate burst from membranes phosphorylated by phosphoenolpyruvate, HPr, and EI. The observation of similar second-order rate constants for the production of sugar phosphate starting with different phosphoryl-group donors confirms the catalytic relevance of the phosphoenzyme IIBGlc intermediate. The in vitro results, together with data published by other investigators, have led to a model describing sugar phosphorylation and transport in vivo.

Crystallization and Preliminary X-Ray Analysis of a Lipase from Staphylococcus hyicus

Single crystals of a lipase from Staphylococcus hyicus have been obtained using a combination of 18 to 24% dimethylsulfoxide and 10% isopropanol as a precipitant. The crystals grow at 4°C in 2–3 months. They belong to the orthorhombic space group P212121 with a = 73.31 Å, b = 77.96 Å, and c = 169.81 Å, with two protein molecules per asymmetrical unit. The crystals diffract to at least 2.8 Å resolution and are suitable for an X-ray structure analysis.

The Phosducin-Like Protein PhLP1 Is Essential for Gβγ Dimer Formation in Dictyostelium discoideum

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gβγ subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gβ (GFP-Gβ) and GFP-Gγ subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(−) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Gγ occurs in wild-type cells but not in phlp1(−) and gβ(−) cells. Moreover, Gβγ dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(−) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Gγ proteins are present in a complex in wild-type cells, they are free in phlp1(−) and gβ(−) cells. Collectively, our data strongly suggest the absence of Gβγ dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gβ and Gγ into a functional Gβγ complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions