Divergent contribution of the MVA and MEP pathways to the formation of polyprenols and dolichols in Arabidopsis.

Isoprenoids, including dolichols (Dols) and polyprenols (Prens), are ubiquitous components of eukaryotic cells. In plant cells, there are two pathways that produce precursors utilized for isoprenoid biosynthesis: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. In this work, the contribution of these two pathways to the biosynthesis of Prens and Dols was addressed using an in planta experimental model. Treatment of plants with pathway-specific inhibitors and analysis of the effects of various light conditions indicated distinct biosynthetic origin of Prens and Dols. Feeding with deuteriated, pathway-specific precursors revealed that Dols, present in leaves and roots, were derived from both MEP and MVA pathways and their relative contributions were modulated in response to precursor availability. In contrast, Prens, present in leaves, were almost exclusively synthesized via the MEP pathway. Furthermore, results obtained using a newly introduced here 'competitive' labeling method, designed so as to neutralize the imbalance of metabolic flow resulting from feeding with a single pathway-specific precursor, suggest that under these experimental conditions one fraction of Prens and Dols is synthesized solely from endogenous precursors (deoxyxylulose or mevalonate), while the other fraction is synthesized concomitantly from endogenous and exogenous precursors. Additionally, this report describes a novel methodology for quantitative separation of 2H and 13C distributions observed for isotopologues of metabolically labeled isoprenoids. Collectively, these in planta results show that Dol biosynthesis, which uses both pathways, is significantly modulated depending on pathway productivity, while Prens are consistently derived from the MEP pathway.

Impact of C-terminal truncations in the Arabidopsis Rab escort protein (REP) on REP-Rab interaction and plant fertility.

Lipid anchors are common post-translational modifications for proteins engaged in signaling and vesicular transport in eukaryotic cells. Rab proteins are geranylgeranylated at their C-termini, a modification which is important for their stable binding to lipid bilayers. The Rab escort protein (REP) is an accessory protein of the Rab geranylgeranyl transferase (RGT) complex and it is obligatory for Rab prenylation. While REP-Rab interactions have been studied by biochemical, structural, and genetic methods in animals and yeast, data on the plant RGT complex are still limited. Here we use hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the structural basis of plant REP-Rab binding. The obtained results show that the interaction of REP with Rabs is highly dynamic and involves specific structural changes in both partners. In some cases the Rab and REP regions involved in the interaction are molecule-specific, and in other cases they are common for a subset of Rabs. In particular, the C-terminus of REP is not involved in binding of unprenylated Rab proteins in plants, in contrast to mammalian REP. In line with this, a C-terminal REP truncation does not have pronounced phenotypic effects in planta. On the contrary, a complete lack of functional REP leads to male sterility in Arabidopsis: pollen grains develop in the anthers, but they do not germinate efficiently and hence are unable to transmit the mutated allele. The presented data show that the mechanism of action of REP in the process of Rab geranylgeranylation is different in plants than in animals or yeast.

The functions of Rab proteins in plants - from cellular to organismal level / Funkcje bialek Rab w roslinach - na poziomie komórki i organizmu.

Rab proteins are necessary for membrane fusion and fission and as such are key regulators of intracellular transport in eukaryotic cells. They also control other aspects of cell functioning, including the cytoskeleton rearrangements, determination of cell polarity or signal transduction. Rab proteins exert their control both indirectly, because they decide whether all necessary proteins and other cargo reach their correct destinations in the cell, and directly, through interactions of their active forms with effector proteins. Therefore, the results of Rab dysfunctions manifest themselves on all levels of biological organization â from cells, through tissues and organs, to whole organisms. In plants, Rab-dependent processes are important for cell architecture, differentiation, reactions to biotic and abiotic stress, as well as for the efficiency of agricultural production.

Global pentapeptide statistics are far away from expected distributions.

The relationships between polypeptide composition, sequence, structure and function have been puzzling biologists ever since first protein sequences were determined. Here, we study the statistics of occurrence of all possible pentapeptide sequences in known proteins. To compensate for the non-uniform distribution of individual amino acid residues in protein sequences, we investigate separately all possible permutations of every given amino acid composition. For the majority of permutation groups we find that pentapeptide occurrences deviate strongly from the expected binomial distributions, and that the observed distributions are also characterized by high numbers of outlier sequences. An analysis of identified outliers shows they often contain known motifs and rare amino acids, suggesting that they represent important functional elements. We further compare the pentapeptide composition of regions known to correspond to protein domains with that of non-domain regions. We find that a substantial number of pentapeptides is clearly strongly favored in protein domains. Finally, we show that over-represented pentapeptides are significantly related to known functional motifs and to predicted ancient structural peptides.

Cell-to-Cell Communication Circuits: Quantitative Analysis of Synthetic Logic Gates.

One of the goals in the field of synthetic biology is the construction of cellular computation devices that could function in a manner similar to electronic circuits. To this end, attempts are made to create biological systems that function as logic gates. In this work we present a theoretical quantitative analysis of a synthetic cellular logic-gates system, which has been implemented in cells of the yeast Saccharomyces cerevisiae (Regot et al., 2011). It exploits endogenous MAP kinase signaling pathways. The novelty of the system lies in the compartmentalization of the circuit where all basic logic gates are implemented in independent single cells that can then be cultured together to perform complex logic functions. We have constructed kinetic models of the multicellular IDENTITY, NOT, OR, and IMPLIES logic gates, using both deterministic and stochastic frameworks. All necessary model parameters are taken from literature or estimated based on published kinetic data, in such a way that the resulting models correctly capture important dynamic features of the included mitogen-activated protein kinase pathways. We analyze the models in terms of parameter sensitivity and we discuss possible ways of optimizing the system, e.g., by tuning the culture density. We apply a stochastic modeling approach, which simulates the behavior of whole populations of cells and allows us to investigate the noise generated in the system; we find that the gene expression units are the major sources of noise. Finally, the model is used for the design of system modifications: we show how the current system could be transformed to operate on three discrete values.

LTR retrotransposons in fungi.

Transposable elements with long terminal direct repeats (LTR TEs) are one of the best studied groups of mobile elements. They are ubiquitous elements present in almost all eukaryotic genomes. Their number and state of conservation can be a highlight of genome dynamics. We searched all published fungal genomes for LTR-containing retrotransposons, including both complete, functional elements and remnant copies. We identified a total of over 66,000 elements, all of which belong to the Ty1/Copia or Ty3/Gypsy superfamilies. Most of the detected Gypsy elements represent Chromoviridae, i.e. they carry a chromodomain in the pol ORF. We analyzed our data from a genome-ecology perspective, looking at the abundance of various types of LTR TEs in individual genomes and at the highest-copy element from each genome. The TE content is very variable among the analyzed genomes. Some genomes are very scarce in LTR TEs (<50 elements), others demonstrate huge expansions (>8000 elements). The data shows that transposon expansions in fungi usually involve an increase both in the copy number of individual elements and in the number of element types. The majority of the highest-copy TEs from all genomes are Ty3/Gypsy transposons. Phylogenetic analysis of these elements suggests that TE expansions have appeared independently of each other, in distant genomes and at different taxonomical levels. We also analyzed the evolutionary relationships between protein domains encoded by the transposon pol ORF and we found that the protease is the fastest evolving domain whereas reverse transcriptase and RNase H evolve much slower and in correlation with each other.

[The p24 family proteins--regulators of vesicular trafficking]. / Bialka z rodziny p24-regulatory transportu pecherzykowego.

The trafficking of proteins in the secretory pathway is mediated by vesicles. Proteins of the p24 family are present on the membranes of secretory pathway organelles (ER, Golgi, COPI and COPII vesicles). Evidence exists showing that p24 proteins play a role in the development of Alzheimer disease, making them an interesting research subject. Their presence in the secretory pathway and their tissue-dependent expression levels suggest that p24 proteins are involved in secretion. However, their molecular function is not clear. Several potential functions have been proposed for p24 proteins: (1) that they function as receptors for selected cargo; (2) that they regulate vesicle biogenesis; (3) that they perform structural and morphogenetic functions in the secretory pathway; (4) that they are responsible for quality control of transported proteins. In this article, we provide a critical review of the postulated functions of p24 proteins.

Mutations in the Saccharomyces cerevisiae vacuolar fusion proteins Ccz1, Mon1 and Ypt7 cause defects in cell cycle progression in a num1Delta background.

The proteins Ccz1 and Mon1 are known to function together with the Rab-GTPase Ypt7 in membrane fusion reactions at the yeast vacuole. In a genome-wide analysis they have also been found to interact genetically with the nuclear-migration protein Num1. In this study we analyze these synthetic effects and we show that the mutants ccz1Delta num1Delta, mon1Delta num1Delta and ypt7Delta num1Delta exhibit severe defects in cell cycle progression. A large fraction of the mutant cells enter a new cell division cycle without having completed mitotic exit, leading to the accumulation of multinuclear, anuclear and multibudded cells. The double deletion strains display also increased sensitivity to calcium ions. The cell-cycle defects are only weakly observed if deletions of other vacuolar protein sorting genes are combined with num1Delta or if other nuclear-migration genes are deleted together with CCZ1, whereas the calcium sensitivity is characteristic for a large subset of the tested double mutants. Further, the cell-cycle defects of the ccz1Delta num1Delta strain can be partially rescued by overproduction of either the calcium pump Pmc1 or the nuclear-migration factors Kar9 and Bim1. Together, these results indicate that deregulation of the cell cycle in these mutants results from two separate mechanisms, one of which is related to calcium homeostasis.

The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery.

The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1Delta mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45, results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12. These results suggest that the Ccz1p-Mon1p-Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.

The yeast protein sorting pathway as an experimental model for lysosomal trafficking.

Lysosomes are conserved organelles that are present in all eukaryotic cells. They are part of a complicated network of intracellular trafficking routes - the lysosomal transport system. Lysosomes are necessary for the maintenance of cellular homeostasis and for many specialized functions, including the activity of many components of the mammalian immune system. Dysfunctions of the lysosomal system are associated with numerous diseases, such as storage disorders, neuro- and myopathies, cancer and some types of albinism and immunological deficiencies. High conservation of the processes of lysosomal biogenesis and transport enables the use of yeast as a model for studying the mechanisms that underlie these diseases. In this review, we discuss several examples of such models in an attempt to present an overview of the most important experimental methods available in yeast research.

The CHiPS Domain--ancient traces for the Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is a rare disorder caused by malfunctions of lysosomes and specialized lysosome-related organelles, resulting primarily in oculocutaneous albinism and bleeding diathesis. The majority of the HPS genes have been described as novel, but herein we report the identification of a conserved protein family which includes human HPS4, as well as distant homologs for other HPS genes. Our results suggest that the cellular machinery involved in the HPS syndrome is ancient.

Multiple functions of the vacuolar sorting protein Ccz1p in Saccharomyces cerevisiae.

The CCZ1 (YBR131w) gene encodes a protein required for fusion of various transport intermediates with the vacuole. Ccz1p, in a complex with Mon1p, is a close partner of Ypt7p in the processes of fusion of endosomes to vacuoles and homotypic vacuole fusion. In this work, we exploited the Ca(2+)-sensitivity of the ccz1Delta mutant to identify genes specifically interacting with CCZ1, basing on functional multicopy suppression of calcium toxicity. The presented results indicate that Ccz1p functions in the cell either in association with Mon1p and Ypt7p in fusion at the vacuolar membrane, or--separately--with Arl1p at early steps of vacuolar transport. We also show that suppression of calcium toxicity by the calcium pumps Pmr1p and Pmc1p is restricted only to the subset of mutants defective in vacuole morphology. The mechanisms of Ca(2+)-pump-mediated suppression also differ from each other, since the action of Pmr1p, but not Pmc1p, appears to require Arl1p function.