Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.

With rapid advances in sequencing technologies and precipitous decreases in cost, public sequence databases have increased in size apace. However, experimental characterization of novel genes and their products remains prohibitively expensive and time consuming. For these reasons, bioinformatics approaches have become increasingly necessary to generate hypotheses of biological function. Phylogenomic approaches use phylogenetic methods to place genes, chromosomes, or whole genomes within the context of their evolutionary history and can be used to predict the function of encoded proteins. In this thesis, two new phylogenomic methods and software implementations are presented that address the problems of subcellular localization prediction and functional divergence prediction within protein families respectively. Most of the widely used programs for subcellular localization prediction have been trained on model organisms and ignore phylogenetic information. As a result, their predictions are not always reliable when applied to phylogenetically divergent eukaryotes, such as unicellular protists. To address this problem, PhyloPred-HMM, a novel phylogenomic method was developed to predict sequences that are targeted to mitochondria or mitochondrion-related organelles (hydrogenosomes and mitosomes). This method was compared to existing prediction methods using an existing test dataset of mitochondrion-targeted sequences from well-studied groups, sequences from a variety of protists, and the whole proteomes of two protists: Tetrahymena thermophila and Trichomonas vaginalis. PhyloPred-HMM performed comparably to existing classifiers on mitochondrial sequences from well-studied groups such as animals, plants, and Fungi and better than existing classifiers on diverse protistan lineages. FunDi, a novel approach to the prediction of functional divergence was developed and tested on 11 biological datasets and two large simulated datasets. On the 11 biological datasets, FunDi appeared to perform comparably to existing programs, although performance measures were compromised by a lack of experimental information. On the simulated datasets, FunDi was clearly superior to existing methods. FunDi, and two other prediction programs, was then used to characterize the functional divergence in two groups of plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (GAPDH) adapted to roles in the Calvin cycle. FunDi successfully identified functionally divergent residues supported by experimental data, and identified cases of potential convergent evolution between the two groups of GAPDH sequences.

Sugarcane holds great potential as a biofactory for the tailored production of novel products of commercial value. In many cases however, the accumulation of an alien product within the cytoplasm interferes with essential cell metabolism. To avoid potential interference, targeting the accumulation of biofactory products into vacuoles may be beneficial. Vacuoles represent one endpoint of the plant endomembrane system where proteins destined for inclusion must contain appropriate targeting peptide signals. However, targeting peptide signals used previously to direct heterologous proteins to the vacuole have not yet been shown to function efficiently in sugarcane. The emphasis of the work described in this thesis was first to characterise the diversity of vacuole types in selected sugarcane tissues, and second to identify and test the function of putative vacuolar targeting signals identified in vacuolar proteins of sugarcane. In order to investigate vacuole diversity in sugarcane cells, a series of membrane-permeable fluorescent probes were used to assess both the acidity and proteolytic properties of vacuolar compartments. It is clear that even from early in development, large vacuoles filled most of the volume of storage parenchyma cells within the developing sugarcane stem. These vacuoles were intensely acidic and contained active aminopeptidases. In leaf cells, vacuoles labelled by chromogenic indicators and enzyme substrates appear much more diverse in pH and proteolytic intensity owing to the multiple functions that leaf cells participate in. As the predominant sugarcane vacuole in vegetative tissues appears to be proteolytic, sugarcane sequences showing homology to proteases and protease inhibitors in other plant species were aligned and compared to identify potential vacuolar targeting signals. This analysis revealed the presence of several candidate vacuolar targeting motifs which displayed high conservation across plant homologues. One such motif, represented by the sequence IRLPS, was identified in the N-terminal region of a legumain protein from sugarcane, which was homologous to known vacuolar processing enzymes in other species. To test the efficacy of the legumain targeting signal and to compare with other motifs, a series of GFP reporter constructs was synthesised and expressed in sugarcane. The sugarcane legumain vacuole targeting signal was particularly efficient at directing an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP fusion proteins in the vacuole appeared to be dependent on cell type, suggesting that the vacuolar environment can vary in its ability to degrade introduced proteins. The legumain vacuole targeting signal was further tested for its ability to direct an avidin protein and a fructosyltransferase enzyme into the lytic vacuole of transgenic sugarcane plants. Avidin, derived initially from chicken egg white, is a glycoprotein that displays a high affinity to the vitamin biotin. For this reason it has been investigated for use in sugarcane as a biocontrol agent against cane grub species. For the production of avidin in planta careful targeting to an appropriate subcellular location is required to avoid a detrimental depletion of available plant cell biotin reserves. When the legumain targeting signal was fused to avidin and expressed as a stably integrated transgene, the avidin protein was detected by immunoblotting but appeared to be proteolytically cleaved within the lytic vacuole in all sugarcane tissues analysed. These plants were phenotypically indistinguishable from controls, indicating that avidin did not appreciably deplete cellular biotin reserves while in transit through the endomembrane system. In contrast, when avidin was designed for either retention in the endoplasmic reticulum or for transit to a different type of vacuole using a heterologous targeting signal, stably transformed plants exhibited a biotin deficient phenotype. This suggests that the legumain vacuole targeting signal was efficient at directing heterologous proteins to a lytic type vacuole where they can be degraded and inactivated if susceptible to proteolysis. When the fructosyltransferase (ftf) gene from Streptococcus salivarius was stably transformed into sugarcane and directed into the lytic vacuole using the legumain vacuole targeting signal, no fructan product could be detected. The low pH and proteolytic environment of this vacuole together with low expression of this bacterial transgene most likely resulted in minimal Ftf activity. Taken together, evidence is presented that the legumain vacuolar targeting signal functions efficiently in directing transgene products such as GFP, avidin and a fructosyltransferase enzyme into a lytic type vacuole. This vacuole has been demonstrated to be both acidic and proteolytic and therefore strategies to improve the stability of heterologous proteins targeted to this vacuolar environment are required and may be specific to the product in question.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-22Bitstream added on 2014-06-13T19:49:29Z : No. of bitstreams: 1 martins_pmm_me_rcla.pdf: 7124946 bytes, checksum: 0e5fc6b0551611dda7e75caf8cd99fad (MD5) / O cancro cítrico é uma doença causada pela bactéria Xanthomonas axonopodis pv. citri (Xac), e que afeta plantas de citros por todo o mundo. O genoma de Xac foi completamente seqüenciado, o que revelou grandes quantidades de ORFs (~30%) codificando para produtos com função desconhecida (proteínas hipotéticas). Baseando-se no princípio de que muitos eventos bioquímicos acontecem em sítios específicos no interior celular, a localização de proteínas em fusão com GFP tem sido amplamente utilizada para a obtenção de informações valiosas a respeito de suas funções. Para iniciarmos estudos de localização de proteínas hipotéticas em Xac, construímos um vetor integrativo capaz de expressá-las em fusão com o polipeptídio GFP, pPM2a. O vetor de expressão para Xac carrega um cassete promotor/repressor de xilose (xylR/pxyl), o gene gfp, um RBS sintético e um fragmento do gene de α-amilase de Xac, para direcionar a integração do sistema de expressão no lócus amy do cromossomo bacteriano. Mostramos aqui a integração estável do vetor no lócus amy de Xac. Além disso, mutantes de Xac expressando o polipeptídio GFP não apresentam nenhuma alteração em seu fenótipo de patogenicidade para o hospedeiro (laranja doce). Mutantes de Xac expressando versões marcadas com GFP para as proteínas ParB e ZapA, ambas codificadas por Xac, foram utilizadas para a padronização dos estudos de localização subcelular. GFP-ZapAXac apresentou um padrão de localização análogo ao de seu ortólogo presente em Bacillus subtilis: uma estrutura semelhante a uma barra, posicionada no meio do bacilo, onde o septo se desenvolve, orientado perpendicularmente com relação ao eixo longitudinal da célula. Este é o primeiro relato de um estudo de localização realizado em Xac. Ao contrário de GFP-ZapAXac, ParBXac-GFP não mostrou nenhum padrão de localização, apesar de a fusão... / Citrus canker is a disease caused by the bacterium Xanthomonas axonopodis pv. citri (Xac), which affects citrus plants worldwide. The genome of Xac was completely sequenced, which unveiled an expressive amount of ORFs (~30%) coding for products of unknown function (hypothetical proteins). Based on the principle that many biochemical events happen at specific sites within the cells, protein localization studies have been extensively used to gather valuable information about function. In order to start subcellular localization studies of hypothetical proteins encoded by Xac using fluorescent microscopy, we constructed an integrative expression vector for GFP-tagging of proteins in this bacterium, pPM2a. The expression vector for Xac carries a xylose repressor/promoter cassette (xylR/pxyl), the gfp gene, a synthetic Ribosome Binding Site (RBS), and a fragment of the α-amylase gene of Xac, to drive the integration of the whole expression system into the amy locus of the bacterial chromosome. We show here stable integration of the expression vector into the amy locus of Xac. Furthermore, Xac mutants expressing the polypeptide GFP do not exhibit any alteration in pathogenicity to the host plant sweet orange. Mutants of Xac expressing GFPtagged versions of ParB and ZapA proteins, both encoded by Xac, were used to standardize the subcellular localization studies. GFP-ZapAXac showed a localization pattern analogous to its ortholog encoded by Bacillus subtilis: a bar-like structure positioned in the middle of the rods, where the septum develops, oriented perpendicularly to the longitudinal axis of the cell. This is the first report of a protein localization study performed in Xac Unlike GFP-ZapAXac, ParBXac-GFP did not display any detectable localization pattern, despite the fact that we were able to detect the production of the fusion ParBXac-GFP in Western blot experiments... (Complete abstract click electronic access below)

Salicylic Acid (SA) is an important plant hormone which acts as a therapeutic agent in the plant in response to biotic and abiotic stress. It plays a significant role in growth and development. SABP2, a methyl salicylate esterase is a key player in SA mediated defense signaling. It catalyzes the conversion of mobile methyl salicylate to salicylic acid. During infection, accumulation of salicylic acid in the distal organ in response to the primary infection elsewhere primes the plant to defend against subsequent infection by the mechanism known as Systemic Acquired Resistance (SAR). SIP68, one of the interacting proteins of SABP2 is a glucosyltransferase protein. Glucosyltransferase protein catalyzes the formation of the glycosidic bond by transferring glucose molecule from donor to acceptor molecules. Plant glucosyltransferase is widely distributed in nature playing the dual role of activating and inactivating enzymes. They are also associated with changing the protein stability and solubility of compounds. Since SABP2 has a role in SA mediated defense signaling and glucosyltransferase proteins are associated with physiological function thus, there is a possibility of SIP68 associated with the major or supportive role in either or both functions. The purified recombinant SIP68 protein was tested for glucosyltransferase activity using radioactive method. The purified SIP68 glucosylates various artificially available flavonoid compounds with highest activity detected with Kaempferol (flavonol) followed by quercetin but negligible activity with SA. HPLC based glucosyltransferase assay further verified SIP68 as a flavonoid UDP-glucosyltransferase, not SA glucosyltransferase. Our interest is to further characterize SIP68 and assess its role in plant defense mechanism. Knowing its expression pattern inside plant cell will help us to assess its activity pattern inside the cell. For this enhanced Green Fluorescent Protein (eGFP) tagged SIP68 was transiently expressed inside the plant cell. Confocal microscopy imaging suggests SIP68 likely to be localized in the cytoplasm which will be further confirmed by subcellular fractionation. To assess the role of SIP68 in plant defense mechanism transgenic line expressing altered SIP68 gene was generated using CRISPR Cas9 technique. Verified transgenic line challenged under different biotic and abiotic stress will help us to understand the role of SIP68 in plant defense mechanism. Our research will help us to understand defense mechanism in tobacco model system enabling us to use the knowledge to develop the resistant varieties of crops that are capable of withstanding the adverse condition of pathogenic as well environmental challenges.

Glucosyltransferases catalyze the transfer of glucose molecules from an active donor to acceptor molecules and are involved in many plant processes. SIP68, a tobacco glucosyltransferase protein, is a SABP2-interacting protein. It was identified in a yeast two-hybrid screen using SABP2 as bait and tobacco proteins as prey. SABP2, converts methyl salicylate to salicylic acid (SA) as a part of the signal transduction pathways in SA-mediated defense signaling. Subcellular localization is a crucial aspect of protein functional analysis to assess its biological function. The recombinant SIP68 tagged with eGFP was expressed transiently in Nicotiana benthamiana and observed under confocal microscopy. Fluorescent signals were observed in the epidermal cells. Subcellular fractionation of the tobacco leaves transiently expressing SIP68-+eGFP confirmed that SIP68 is localized in the cytosol. To study the role of SIP68 in plant stress signaling, transgenic lines with altered SIP68 expression were generated using RNAi and CRISPR Cas9 and analyzed.

<p> Preliminary experiments indicated that lysosomes are present in rat liver and onion embryos. A differential centrifugation study was made of the intracellular distribution of acid phosphatase in pea embryo tissue in an attempt to show that this enzyme is enclosed by a membrane forming granules similar to the lysosomes of hepatic tissue. The results reveal that acid phosphatase is soluble under the conditions employed, but it is believed that this may well have resulted from excessive damage to the subcellular bodies during homogenization.</p> / Thesis / Master of Science (MSc)

No description available.

Molecular cell biology has been an intensively studied interdisciplinary field with the rapid development of experimental techniques and fast upgrade of computational hardware and numerical tools. Recent technological developments have led to single-cell experiments which allow us to probe the role of stochasticity in cellular processes. Stochastic modeling of the corresponding processes is thus an essential ingredient for the understanding and interpretation of cellular systems of interest. In this thesis, we explore several nucleation-based stochastic cellular processes, i.e. Min protein oscillation in Escherichia coli, pausing phenomena in DNA transcription, and single-molecule enzyme kinetics. We focus on the key experimental results and build up stochastic models accordingly to provide quantitative insights to the underlying physical mechanisms for the corresponding biological processes. We utilize specific mathematical methods and computational algorithms to gain a better understanding and make predictions for further experimental explorations in the relevant fields. / Ph. D.