Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in <it>Arabidopsis thaliana </it>and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.</p> <p>Results</p> <p>In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.</p> <p>Conclusion</p> <p>Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.</p

Programmed Autophagy in the Fat Body of Aedes aegypti Is Required to Maintain Egg Maturation Cycles

Autophagy plays a pivotal role by allowing cells to recycle cellular components under conditions of stress, starvation, development and cancer. In this work, we have demonstrated that programmed autophagy in the mosquito fat body plays a critical role in maintaining of developmental switches required for normal progression of gonadotrophic cycles. Mosquitoes must feed on vertebrate blood for their egg development, with each gonadotrophic cycle being tightly coupled to a separate blood meal. As a consequence, some mosquito species are vectors of pathogens that cause devastating diseases in humans and domestic animals, most importantly malaria and Dengue fever. Hence, deciphering mechanisms to control egg developmental cycles is of paramount importance for devising novel approaches for mosquito control. Central to egg development is vitellogenesis, the production of yolk protein precursors in the fat body, the tissue analogous to a vertebrate liver, and their subsequent specific accumulation in developing oocytes. During each egg developmental cycle, the fat body undergoes a developmental program that includes previtellogenic build-up of biosynthetic machinery, intense production of yolk protein precursors, and termination of vitellogenesis. The importance of autophagy for termination of vitellogenesis was confirmed by RNA interference (RNAi) depletions of several autophagic genes (ATGs), which inhibited autophagy and resulted in untimely hyper activation of TOR and prolonged production of the major yolk protein precursor, vitellogenin (Vg). RNAi depletion of the ecdysone receptor (EcR) demonstrated its activating role of autophagy. Depletion of the autophagic genes and of EcR led to inhibition of the competence factor, betaFTZ-F1, which is required for ecdysone-mediated developmental transitions. Moreover, autophagy-incompetent female mosquitoes were unable to complete the second reproductive cycle and exhibited retardation and abnormalities in egg maturation. Thus, our study has revealed a novel function of programmed autophagy in maintaining egg maturation cycles in mosquitoes

An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endo-membrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF-defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)-Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development

Tissue- and Stage-specific Expression of Two Lipophorin Receptor Variants with Seven and Eight Ligand-binding Repeats in the Adult Mosquito

We identified two splice variants of lipophorin receptor (LpR) gene products specific to the mosquito fat body (AaLpRfb) and ovary (AaLpRov) with respective molecular masses of 99.3 and 128.9 kDa. Each LpR variant encodes a member of the low density lipoprotein receptor family with five characteristic domains: 1) ligand recognition, 2) epidermal growth factor precursor, 3) putative O-linked sugar, 4) single membrane-spanning domains, and 5) the cytoplasmic tail with a highly conserved internalization signal FDNPVY. Proposed phylogenetic relationships among low density lipoprotein receptor superfamily members suggest that the LpRs of insects are more closely related to vertebrate low density lipoprotein receptors and very low density lipoprotein receptor/vitellogenin receptor than to insect vitellogenin receptor/yolk protein receptors. Two mosquito LpR isoforms differ in their amino termini, the ligand-binding domains, and O-linked sugar domains, which are generated by differential splicing. Polymerase chain reaction and Southern blot hybridization analyses show that these two transcripts originated from a single gene. Significantly, the putative ligand-binding domain consists of seven and eight complement-type, cysteine-rich repeats inAaLpRfb and AaLRov, respectively. Seven cysteine-rich repeats in AaLpRfb are identical to the second through eighth repeats of AaLpRov. Previous analyses (1) have indicated that the AaLpRov transcript is present exclusively in ovarian germ-line cells, nurse cells, and oocytes throughout the previtellogenic and vitellogenic stages, with the peak at 24–30 h after blood meal, coincident with the peak of yolk protein uptake. In contrast, the fat body-specific AaLpRfb transcript expression is restricted to the postvitellogenic period, during which yolk protein production is terminated and the fat body is transformed to a storage depot of lipid, carbohydrate, and protein

Effects of Feeding Level and Diet Energy Density on Cattle Exposed to Heat

Under hot environment conditions, individually fed steers maintained lower body temperatures and grater intakes when limit fed when compared to steers fed the same diet ad libitum