Analysis of COPII Vesicles Indicates a Role for the Emp47–Ssp120 Complex in Transport of Cell Surface Glycoproteins

Coat protein complex II (COPII) vesicle formation at the endoplasmic reticulum (ER) transports nascent secretory proteins forward to the Golgi complex. To further define the machinery that packages secretory cargo and targets vesicles to Golgi membranes, we performed a comprehensive proteomic analysis of purified COPII vesicles. In addition to previously known proteins, we identified new vesicle proteins including Coy1, Sly41 and Ssp120, which were efficiently packaged into COPII vesicles for trafficking between the ER and Golgi compartments. Further characterization of the putative calcium‐binding Ssp120 protein revealed a tight association with Emp47 and in emp47Δ cells Ssp120 was mislocalized and secreted. Genetic analyses demonstrated that EMP47 and SSP120 display identical synthetic positive interactions with IRE1 and synthetic negative interactions with genes involved in cell wall assembly. Our findings support a model in which the Emp47–Ssp120 complex functions in transport of plasma membrane glycoproteins through the early secretory pathway.      

TgZFP2 is a novel zinc finger protein involved in coordinating mitosis and budding in Toxoplasma

Zinc finger proteins (ZFPs) are one of the most abundant groups of proteins with a wide range of molecular functions. We have characterised a Toxoplasma protein that we named TgZFP2, as it bears a zinc finger domain conserved in eukaryotes. However, this protein has little homology outside this region and contains no other conserved domain that could hint for a particular function. We thus investigated TgZFP2 function by generating a conditional mutant. We showed that depletion of TgZFP2 leads to a drastic arrest in the parasite cell cycle, and complementation assays demonstrated the zinc finger domain is essential for TgZFP2 function. More precisely, whereas replication of the nuclear material is initially essentially unaltered, daughter cell budding is seriously impaired: to a large extent newly formed buds fail to incorporate nuclear material. TgZFP2 is found at the basal complex in extracellular parasites and after invasion, but as the parasites progress into cell division, it relocalises to cytoplasmic punctate structures and, strikingly, accumulates in the pericentrosomal area at the onset of daughter cell elongation. Centrosomes have emerged as major coordinators of the budding and nuclear cycles in Toxoplasma, and our study identifies a novel and important component of this machinery.     

The influence of ethylene on proliferation and growth of rose shoot cultures

Ethylene accumulation in four different rose in vitro culture containers was evaluated. Multiplication rate was the highest, and axes most elongated, in the two containers where ethylene accumulation was limited. Pulse treatments of ethylene at various concentrations enhanced proliferation depending on concentration (5 ppm generally was the most favourable) and time of application, while reducing elongation of the shoots. An ethylene trap in the flask atmospheres of the cultures reduced rose shoot proliferation rate but increased elongation of the axes. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (CoCl₂), increased multiplication rate by providing a higher number of axes of a suitable size for subculture. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) had a beneficial effect on multiplication rate, although reducing longitudinal growth of the axes.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BA benzyladenine - GA₃ gibberellic acid - IBA indolyl-3-butyric acid     

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

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C‐terminal juxtamembrane region of full‐length M2 protein forms a membrane surface associated amphipathic helix

The influenza A M2 protein is a 97‐residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full‐length protein. Here, the structure of the C‐terminal juxtamembrane region (sites 50–60) is investigated in the full‐length M2 protein using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50–60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C‐terminal juxtamembrane region in full‐length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full‐length and truncated form of the protein.     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

In vitro assays of vesicular transport

Movement of proteins and lipids between the various compartments of eukaryotic cells is fundamental to the maintenance of cellular homeostasis, and an understanding of the molecular mechanisms that govern these processes remains a key goal of cell biological research. This aim has been greatly facilitated by the development of assays that recapitulate specific events in vitro. In the following article we provide an overview of some of the currently used assays that measure the movement of proteins within the exocytic and endocytic pathways, and provide a starting point for those wishing to establish their own systems to study other vesicular transport steps.     

Budding Yeast Protein Extraction and Purification for the Study of Function,Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.     

Scanning electron microscopic evidence for a budding mode of chloroplast multiplication in higher plants

Scanning electron microscopic observations of leaf sections of certain plants show two distinct types of chloroplast multiplication. In the normal mode, chloroplast division is achieved by binary fission. In Bryophyllum pinnatum Kurz. the chloroplast multiplication occurs by a process similar to the budding of yeast. The advantages of using scanning electron microscopy to follow closely the different stages of chloroplast division are demonstrated.