The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

THE SHOOT-GROWTH RHYTHM OF A TROPICAL TREE,THEOBROMA CACAO

The shoot-growth rhythm of tropical trees is a little understood phenomenon. Correlations of tree growth with environment, determined from field studies in the tropics, have been largely inconclusive, and few studies have been done under controlled environmental conditions. As an initial part of a project to study shoot-growth rhythms in tropical trees this paper describes the rhythm in Theobroma cacao L. An individual shoot passes through alternate periods of growth and dormancy. The growth period is characterized by the expansion of leaves and elongation of the shoot. During dormancy the length of the shoot remains constant, and no new leaves expand. Shoot-growth rhythm was divided into phases. Dissection of shoot tips from the various phases shows that the total number of leaves and leaf primordia in the shoot apex remains constant during the dormant period and does not increase until the onset of the growth period. This indicates that activity of the apical meristem as well as leaf expansion and shoot elongation are rhythmic. We found that the rhythm of shoot growth persists under controlled environmental conditions and that growth under these conditions is asynchronous, as it appears to be in the field. Our data strongly suggest endogeneity.     

Determining the overall merit of protein identification data sets: rho-diagrams and rho-scores

This paper described a simple heuristic method for determining the merit of a set of peptide sequence assignments made using tandem mass spectra. The method involved comparing a prediction based on the known stochastic behavior of a sequence assignment algorithm with the assignments generated from a particular data set. A particular formulation of this comparison was defined through the construction of a plot of the data, the rho-diagram, as well as a parameter derived from this plot, the rho-score. This plot and parameter were shown to be able to readily characterize the relative quality of a set of peptide sequence assignments and to allow the straightforward determination of probability threshold values for the interpretation of proteomics data. This plot is independent of the algorithm or scoring scheme used to estimate the statistical significance of a set of experimental results; rather, it can be used as an objective test of the correctness of those estimates. The rho-score can also be used as a parameter to evaluate the relative merit of protein identifications, such as those made across proteome species taxonomic categories.     

REQUIREMENTS FOR THE MAINTENANCE AND GROWTH OF APHANIZOMENON FLOS-AQUAE IN CULTURE1,2

Aphanizomenon flos-aquae has been introduced into unialgal culture in a completely defined medium and maintained for more than 3 years in the colonial flake form found in nature. The color and size of the flake and general appearance of the cells are substantially improved over those found in the original material at the time of collection from Upper Klamath Lake, Klamath County, Oregon. The alga readily forms both akinetes and heterocysts in culture Bacteria-free cultures have not been obtained. A precipitate-free medium designated ASM No. 8a has been developed from the ASM of McLachlan and Gorham. Their medium was altered by modifying the concentration of certain elements and by the addition of sodium bicarbonate, a 1/25 dilution of Arnon's trace element solutions B₇ and C₁₃, hydrogen ferric ethylenediamine di-o-hydroxyphenylacetate (EDDHA), and disodium ethylene dinitrilotetra-acetate (Na₂EDTA).     

The Significance of Protein Maturation by Plastidic Type I Signal Peptidase 1 for Thylakoid Development in Arabidopsis Chloroplasts

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here, we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids.Capture of light energy and subsequent electron transport reactions are key steps in photosynthesis. In eukaryotic cells, these reactions occur in thylakoids, the internal membrane systems of chloroplasts. Thylakoid constituents, i.e. membrane lipids, photosynthetic pigments, and various proteins, have been well defined (Schröder and Kieselbach, 2003; Peltier et al., 2004; Benning and Ohta, 2005; DellaPenna and Pogson, 2006; Masuda and Fujita, 2008), and genetic disruptions of their biosynthesis result in a severe reduction of thylakoid development (e.g. Dörmann et al., 1995; Jarvis et al., 1995; Hsieh and Goodman, 2005). Thylakoids depend for their lipid supply on the envelope membranes (Dörmann and Benning, 2002). Early cytological studies suggested that thylakoids derive from the inner envelope membrane by invagination followed by vesicle traffic and/or lateral transfer of membrane constituents (e.g. Mühlethaler and Frey-Wyssling, 1959; von Wettstein, 1959; Hoober et al., 1991). Several putative components for the vesicle trafficking have been identified (Hugueney et al., 1995; Kroll et al., 2001; Wang et al., 2004), although the mode of their functions remains elusive. Overall, molecular details of thylakoid development are not yet fully understood.Proteins found in thylakoid membranes are encoded in either the chloroplast or nuclear genome, whereas lumenal proteins are encoded exclusively in the nucleus (Robinson and Mant, 2005). All of the nuclear-encoded thylakoid proteins identified so far are synthesized on cytosolic ribosomes with N-terminal extensions called transit peptides. These precursor proteins first traverse the double-membrane envelope via the general import machinery called the TOC and TIC complexes (for translocon at the outer- and inner-envelope-membranes of chloroplasts; Schnell et al., 1997), and their transit peptides are removed by a soluble metalloprotease, stromal processing peptidase (Richter et al., 2005). Extensive biochemical and genetic studies have identified four distinct pathways in the stroma to mediate sorting of these nuclear-encoded proteins to thylakoids (Robinson and Mant, 2005; Cline and Dabney-Smith, 2008). The cpSec (for chloroplast Sec) and cpTat (for chloroplast twin-arginine translocation) pathways catalyze protein targeting to the lumen. The cpSRP (for chloroplast signal recognition particle) pathway mediates sorting of the most abundant membrane proteins, LHCPs (for light-harvesting chlorophyll binding proteins), whereas a nonassisted spontaneous pathway targets several other integral membrane proteins in thylakoids. All known nuclear-encoded substrates of cpSec (e.g. OE33 [for oxygen evolving complex subunit 33] and plastocyanin), cpTat (e.g. OE23, OE17, and PsaN), and some spontaneous (e.g. CFoII, PsbW, and PsbX) pathways carry a cleavable thylakoid-targeting signal peptide that follows the stroma-targeting transit peptide in their N termini (Robinson and Mant, 2005). The signal peptides are necessary for the intraorganellar targeting and are removed in the lumen by a type I signal peptidase (SPase I) called thylakoidal processing peptidase (TPP).SPases I are membrane-bound Ser proteases responsible for cleavage of intracellular and/or intraorganellar targeting sequences (signal peptides) from numerous proteins in both prokaryotic and eukaryotic cells (Paetzel et al., 2002). In bacteria, it was shown that signal peptide cleavage is required for some proteins to be released from the plasma membrane during or after their translocation from the cytoplasm (Dalbey and Wickner, 1985), and SPases I are essential for viability (Date, 1983; Cregg et al., 1996; Zhang et al., 1997; Zhbanko et al., 2005). In thylakoids, by contrast, removal of the signal peptide was shown in vivo to be dispensable for the function and assembly of a chloroplast-encoded substrate, cytochrome f, of the green alga Chlamydomonas reinhardtii (Kuras et al., 1995; Baymann et al., 1999). In addition, the precursor form of spinach (Spinacia oleracea) OE33 that contains both the stroma- and thylakoid-targeting sequences was able to reconstitute oxygen evolution activity in vitro (Popelkova et al., 2002). Nonetheless, the importance of signal peptide cleavage for thylakoid biogenesis remains largely unexplored. In 1998, the first plant TPP cDNA was identified from Arabidopsis (Arabidopsis thaliana) seedlings based on similarity of the encoded protein to a cyanobacterial SPase I in its sequence (Chaal et al., 1998). An immunoblot assay showed thylakoid localization of this protein (At2g30440), and an in vitro assay confirmed its processing activity against wheat (Triticum aestivum) OE23 (Chaal et al., 1998). More recent genetic and immunological studies showed that a second TPP isoform in Arabidopsis, named plastidic type I signal peptidase 1 (Plsp1), is involved in processing of an envelope protein, Toc75, and a lumenal protein, OE33 (Inoue et al., 2005; Shipman and Inoue, 2009). The genetic study also showed that Plsp1 is necessary for proper thylakoid development (Inoue et al., 2005), although the mode of its action remains elusive.In this work, we conducted a more extensive characterization of plsp1-1 plastids to demonstrate the significance of protein maturation for thylakoid development. Disruption of the PLSP1 gene led to a reduction in the level of thylakoid proteins and to accumulation of improperly processed forms of multiple lumenal proteins, in addition to those of OE33 and Toc75. Plastids lacking Plsp1 accumulated stroma-localized vesicles of various sizes, at which OE33 was present. Finally, we used a genetic complementation system to show that complete maturation of the envelope protein Toc75 is dispensable for proper plant development. These results suggest that the maturation of lumenal proteins may be necessary for proper development of thylakoids.     

Global analysis of protein palmitoylation in yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.     

Gibberellin Biosynthesis: Genetic Studies in Gibberella fujikuroi

Biochemical genetic studies on the production of gibberellins by the fungus Gibberella fujikuroi have identified two genes which control different steps in the biosynthetic pathway. One gene (g₁) controls the production of all of the gibberellins: the second gene (g₂) controls the production of GA₁ and GA₃ only. Ascospores are not ordered in the ascus of this fungus. This apparent spore slippage precludes mapping of these genes to their respective centromeres.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.