Mieke Blaauw

Structural evidence for dimerization-regulated activation of an integral membrane phospholipase

Dimerization is a biological regulatory mechanism employed by both soluble and membrane proteins. However, there are few structural data on the factors that govern dimerization of membrane proteins. Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme which participates in secretion of colicins in Escherichia coli. In Campilobacter and Helicobacter pylori strains, OMPLA is implied in virulence. Its activity is regulated by reversible dimerization. Here we report X-ray structures of monomeric and dimeric OMPLA from E. coli. Dimer interactions occur almost exclusively in the apolar membrane-embedded parts, with two hydrogen bonds within the hydrophobic membrane area being key interactions. Dimerization results in functional oxyanion holes and substrate-binding pockets, which are absent in monomeric OMPLA. These results provide a detailed view of activation by dimerization of a membrane protein.Ambitious research agendas should stimulate vigorous demand for investment in broadband networks.

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 (tTA(s)(*)). 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 tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) 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.

Phosducin-like proteins in Dictyostelium discoideum: implications for the phosducin family of proteins.

Retinal phosducin is known to sequester transducin Gβγ, thereby modulating transducin activity. Phos ducin is a member of a family of phosducin‐like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin‐like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin‐I–III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin‐like protein of a different group. Disruption of the phlp1 gene strongly impaired G‐protein signalling, apparently due to mislocalization of Gβγ in phlp1‐null cells. GFP‐Gβ and GFP‐Gγ are membrane associated in wild‐type cells, but cytosolic in phlp1‐null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16–17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role for phosducin‐like proteins in facilitating folding, localization or function of proteins, in addition to modulating G‐protein signalling.

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

ABSTRACT 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.

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum

The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild‐type hormones. We also show that structure‐function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues. Linskens, M. H. K., Grootenhuis, P. D. J., Blaauw, M., Huisman‐de Winkel, B., van Ravestein, A., van Haastert, P. J. M., and Heikoop, J. C. Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum. FASEB J. 13, 639–645 (1999)

Crystallization and preliminary X-ray analysis of outer membrane phospholipase A from Escherichia coli

The outer membrane phospholipase A (OMPLA) of Escherichia coli is one of the few integral outer membrane proteins displaying enzymatic activity. It is encoded as a mature protein of 269 amino acids preceded by a signal sequence of 20 amino acids. There is no sequence homology with water‐soluble lipases and phospholipases. Crystals of the mature enzyme were obtained at 22°C from 24–28% (vlv) 2‐methyl‐2,4‐pentanediol in Bis‐Tris buffer, pH 5.9–6.0, with 1 mM calcium chloride and 1.5% (w/v) β‐octylglucoside. They have the symmetry of the trigonal spacegroup P3121 (or P3221) with cell dimensions of ). Native crystals diffract to a resolution of 2.6 Å.