Functional expression of the yeast alpha-factor receptor in Xenopus oocytes

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431- residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell

Septin Stability and Recycling during Dynamic Structural Transitions in Cell Division and Development

SummarySeptins are conserved proteins found in hetero-oligomeric complexes that are incorporated into distinct structures during cell division and differentiation; yeast septins Cdc3, Cdc10, Cdc11, and Cdc12 form hetero-octamers and polymerize into filaments, which form a “collar” at the mother-bud neck [1]. Posttranslational modifications, nucleotide binding, and protein-protein and protein-lipid interactions influence assembly and disassembly of septin structures [2], but whether individual septins are used repeatedly to build higher-order assemblies was not known. We used fluorescence-based pulse-chase methods to visualize the fate of pre-existing (old) and newly synthesized (new) molecules of two septins, Cdc10 and Cdc12. They were recycled through multiple mitotic divisions, and old and new molecules were incorporated indistinguishably into the collar. Likewise, old and new subunits intermixed within hetero-octamers, indicating that exchange occurs at this organizational level. Remarkably, in meiosis, Cdc10 made during vegetative growth was reutilized to build sporulation-specific structures and reused again during spore germination for budding and during subsequent mitotic divisions. Although Cdc12 also persisted during sporulation, it was excluded from septin structures and replaced by another subunit, Spr3; only new Cdc12 populated the collar of germinating spores. Thus, mechanisms governing septin incorporation are specific to each subunit and to the developmental state of the cell

Septins: molecular partitioning and the generation of cellular asymmetry

During division, certain cellular contents can be distributed unequally; daughter cells with different fates have different needs. Septins are proteins that participate in the establishment and maintenance of asymmetry during cell morphogenesis, thereby contributing to the unequal partitioning of cellular contents during division. The septins themselves provide a paradigm for studying how elaborate multi-component structures are assembled, dynamically modified, and segregated through each cell division cycle and during development. Here we review our current understanding of the supramolecular organization of septins, the function of septins in cellular compartmentalization, and the mechanisms that control assembly, dynamics, and inheritance of higher-order septin structures, with particular emphasis on recent findings made in budding yeast (Saccharomyces cerevisiae)

Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta-nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo

Effects of Bni5 Binding on Septin Filament Organization

AbstractSeptins are a protein family found in all eukaryotes (except higher plants) that have roles in membrane remodeling and formation of diffusion barriers and as a scaffold to recruit other proteins. In budding yeast, proper execution of cytokinesis and cell division requires the formation of a collar of circumferential filaments at the bud neck. These filaments are assembled from apolar septin hetero-octamers. Currently, little is known about the mechanisms that control the arrangement and dynamics of septin structures. In this study, we utilized both Förster resonance energy transfer and electron microscopy to analyze the biophysical properties of the septin-binding protein Bni5 and how its association with septin filaments affects their organization. We found that the interaction of Bni5 with the terminal subunit (Cdc11) at the junctions between adjacent hetero-octamers in paired filaments is highly cooperative. Both the C-terminal end of Bni5 and the C-terminal extension of Cdc11 make important contributions to their interaction. Moreover, this binding may stabilize the dimerization of Bni5, which, in turn, forms cross-filament braces that significantly narrow, and impose much more uniform spacing on, the gap between paired filaments

Potential Tumor Suppressor Role for the c-Myb Oncogene in Luminal Breast Cancer

The transcription factor c-Myb has been well characterized as an oncogene in several human tumor types, and its expression in the hematopoietic stem/progenitor cell population is essential for proper hematopoiesis. However, the role of c-Myb in mammopoeisis and breast tumorigenesis is poorly understood, despite its high expression in the majority of breast cancer cases (60-80%).We find that c-Myb high expression in human breast tumors correlates with the luminal/ER+ phenotype and a good prognosis. Stable RNAi knock-down of endogenous c-Myb in the MCF7 luminal breast tumor cell line increased tumorigenesis both in vitro and in vivo, suggesting a possible tumor suppressor role in luminal breast cancer. We created a mammary-derived c-Myb expression signature, comprised of both direct and indirect c-Myb target genes, and found it to be highly correlated with a published mature luminal mammary cell signature and least correlated with a mammary stem/progenitor lineage gene signature.These data describe, for the first time, a possible tumor suppressor role for the c-Myb proto-oncogene in breast cancer that has implications for the understanding of luminal tumorigenesis and for guiding treatment

Absence of erythrocyte sequestration in a case of babesiosis in a splenectomized human patient

BACKGROUND: The importance of vascular occlusion in the pathogenesis of human haemoprotozoal disease is unresolved. METHODS: Giemsa-stained tissue sections from a human case of Babesia microti infection in a splenectomized patient with chronic lymphocytic leukaemia and colon cancer were examined to ascertain the distribution of parasitized erythrocytes within the vascular lumen. RESULTS: No evidence of sequestration was observed. CONCLUSION: This first report on the vascular location of B. microti in human tissue suggests that severe multi-organ failure due to babesiosis is independent of sequestration of parasitized erythrocytes. A similar pathogenesis may also cause multi-organ failure in other intraerythrocytic protozoal infections, including falciparum malaria

Detection of circulating tumour DNA is associated with inferior outcomes in Ewing sarcoma and osteosarcoma: a report from the Children's Oncology Group.

BackgroundNew prognostic markers are needed to identify patients with Ewing sarcoma (EWS) and osteosarcoma unlikely to benefit from standard therapy. We describe the incidence and association with outcome of circulating tumour DNA (ctDNA) using next-generation sequencing (NGS) assays.MethodsA NGS hybrid capture assay and an ultra-low-pass whole-genome sequencing assay were used to detect ctDNA in banked plasma from patients with EWS and osteosarcoma, respectively. Patients were coded as positive or negative for ctDNA and tested for association with clinical features and outcome.ResultsThe analytic cohort included 94 patients with EWS (82% from initial diagnosis) and 72 patients with primary localised osteosarcoma (100% from initial diagnosis). ctDNA was detectable in 53% and 57% of newly diagnosed patients with EWS and osteosarcoma, respectively. Among patients with newly diagnosed localised EWS, detectable ctDNA was associated with inferior 3-year event-free survival (48.6% vs. 82.1%; p = 0.006) and overall survival (79.8% vs. 92.6%; p = 0.01). In both EWS and osteosarcoma, risk of event and death increased with ctDNA levels.ConclusionsNGS assays agnostic of primary tumour sequencing results detect ctDNA in half of the plasma samples from patients with newly diagnosed EWS and osteosarcoma. Detectable ctDNA is associated with inferior outcomes

Prolactin

During an oral glucose tolerance test (OGTT) glucose and insulin levels were measured in 26 patients with prolactin-producing pituitary tumours without growth hormone excess. Basal glucose and insulin levels did not differ from the values of an age-matched control group. After glucose load the hyperprolactinaemic patients showed a decrease in glucose tolerance and a hyperinsulinaemia. Bromocriptine (CB 154), which suppressed PRL, improved glucose tolerance and decreased insulin towards normal in a second OGTT. — Human PRL or CB 154 had no significant influence on insulin release due to glucose in the perfused rat pancreas. — These findings suggest a diabetogenic effect of PRL. CB 154 might be a useful drug in improving glucose utilization in hormone-active pituitary tumours