Month: December 2018

Then, membrane fusion of the two apposing bilayers is accomplished by a special set of proteins called SNAREs, and the two previously separate organelles now have a direct community. We focused on the involvement of Ann5 in endomembrane trafficking. Ann5 likely promotes vesicle transport following BFA stimulation by binding to negatively charged phospholipids in a Ca2+-dependent manner and to actin filaments that served as molecular tracks in pollen grains and tubes. Annexin A2 is involved in the biogenesis of multivesicular transport intermediates that are L-Ascorbyl 6-palmitate destined for late endosomes in mammals. In Arabidopsis, AnnAt3, 4, 5 and 8 are more closely related to human annexins based on phylogenetic analysis. RNA interference–mediated knockdown of the annexin AnnAt3 caused the trans-Golgi network and multivesicular body markers to colocalize and block vacuolar transport in tobacco mesophyll protoplasts. It has been shown that AnnAt3 is required for the final step of MVBs as a transport carrier from the TGN to the vacuole. The Ann5-overexpressing pollen cell exhibited a higher velocity for cytoplasmic streaming and vesicle transport than control pollen cells following BFA treatment,BMS-378806 further suggesting that Ann5 promoted endomembrane trafficking. Cytoplasmic streaming is fundamentally under the control of myosin motors moving along actin filament bundles that are involved in the intracellular transport of organelles and vesicles. In addition to the fact that actin filaments serve as molecular tracks for Ann5 in vesicle trafficking, Ann5 can bind to F-actin in vitro and may be involved in the dynamic rearrangement of actin through an unknown mechanism. Alterations in the arrangement of actin cables greatly influence the pattern and velocity of cytoplasmic streaming. Ann5 binds to actin and associates with phospholipid membranes in a Ca2+-dependent manner, making it an ideal regulator of endomembrane trafficking where the cell membrane and cytoskeleton interact and are modulated by Ca2+ within pollen cells. The growth rates of pollen tubes and the cytosolic Ca2+ concentration also synchronously oscillate with the same period and phase.

After the first mitotic division, a large vacuole is split into smaller vacuoles by invaginations of the tonoplast at several points from one side to the opposite side of the vacuole. The small vacuoles then disperse throughout the cytoplasm, and some fuse with the plasma membrane, which becomes intensely convoluted. The observation that many membrane-bound structures appeared between the convoluted plasma membrane and the intine implicated that exocytosis seems to occur in pollen. Our previous data demonstrated that Ann5 is predominantly expressed in pollen after the bicellular stage and pollen tube formation. In addition, downregulation of the function of Ann5 in transgenic RNAi lines led to severely sterile pollen grains at the bicellular stage of pollen development. However,Pancuronium dibromide the mechanisms underlying the function of Ann5 in pollen remain largely unclear. In this study, biochemical analyses indicated that Ann5 binds to phospholipid membrane and this association is stimulated by Ca2+. Moreover, Ann5 interacts with actin filaments in vitro. The pollen germination, pollen tube growth and cytoplasmic streaming of plants overexpressing Ann5 were more resistant to BFA treatment in a Ca2+-regulated manner in vivo. The defects observed in pollen grains of the Ann5UTR-RNAi lines could be only partially recovered by Ann5 mutants at Ca2+-binding sites, which further indicated that the Ann5 Ca2+-dependent phospholipid-binding activity is essential for pollen development. In plant cells, annexins represent approximately 0.1% of the total protein. Despite many years of investigation since the identification of the first plant annexin from tomato,Tolterodine tartrate the main physiological significance of plant annexins remains largely unknown, and most of our knowledge about plant annexins is focused on their structure and in vitro protein function. Ann5 is expressed abundantly in mature pollen grains and elongating pollen tubes. Furthermore, downregulation of Ann5 leads to a sterile pollen grain phenotype. Here, we provide both biochemical and physiological evidence to interpret the molecular mechanisms underlying the function of Ann5 in plant pollen development and pollen tube growth.

These proteins are involved in sterol homeostasis. Among these C. populi ABCG sequences, Cpabc55 showed the most elevated transcript level. Its deduced protein is homologous to TcABCG4C whose involvement in the transport of lipids to the cuticle has been suggested and, thus, that it is required for the formation of a waterproof barrier in the epicuticle. Cpabc55 is also highly Mesatlantin C expressed in glands and fat body tissue but not in the Malpighian tubules. The expression of Tcabcg-4c was higher in intestinal/excretory tissues than in carcass tissue. The function of the other four ABCG transporters cannot be predicted from our analyses. However, it has been demonstrated recently that an ABCG1-homolog in the fungus Grossmannis clavigera confers tolerance to monoterpenes which contributes to the fungus’ ability to cope with the chemical defence of its host plant. Therefore, the ABCG proteins’ specificity in insects may not be limited to sterols or lipids but may have a broader substrate spectrum – that is not known to date. Insect Malpighian tubules are critical for osmoregulation. Moreover, the tubules have the capability to excrete actively a broad range of organic solutes and xenobiotics,Pulchinenoside A such as insecticides. Recently, we have shown a role of the excretion system in the homeostasis of phytochemicals in the larval body of leaf beetles. Additionally, the tubules play a significant role in immunity by sensing bacterial infections and mounting an effective killing response by secretion of antimicrobial peptides. We found 21 transcripts abundant in the Malpighian tubules of C. populi encoding members of the following subfamilies known to contain multidrug resistance proteins: four of ABCB, 14 of ABCC, three of ABCG. Among the four predicted ABCB members displaying a high mRNA level in the Malpighian tubules, two, CpABC7 and 12, were already described in the gut section above. The third candidate, CpABC8, is most similar to human mitochondrial ABCB10. For ABCB10 different roles have been suggested, including protection against toxic reactive oxygen species, heme synthesis, or peptide transport. For this tissue, we speculate that it is involved in antimicrobial peptide transfer.

In D. melanogaster, the ABCE homolog Pixie plays a catalytic role in the assembly of protein complexes required for translation initiation. All genomes of multicellular eukaryotes analyzed to date possess one ABCE gene. In the transcript catalogue of C. populi,Saikosaponin D one complete ABCE protein has been predicted. The NBDs of CpABC45 are highly conserved with the respective NBDs of the human ABCE1 and T. castaneum TcABCE-3A. Among the subfamily ABCF involved in translation initiation and elongation in humans, we found in C. populi three putative members each with two NBDs that are highly similar to the transporters of human and T. castaneum suggesting functional proteins used in similar physiological processes in the cell. The ABCG subfamily in humans is comprised of five half transporters. While the homodimer ABCG2 is a multidrug transporter with a wide substrate specificity,Tetrahydropalmatine the homodimers ABCG1 and ABCG4 and the heterodimer ABCG5:ABCG8 translocate cholesterol and other sterole derivatives. In insects, ABCG transporters are essential for the translocation of ommochromes for the pigmentation of eyes and body coloration. In D. melanogaster, for example, the half transporter White forms heterodimers with Scarlet or Brown, each of which is responsible for the transport of another type of ommochrome precursor to pigment granules. In silkworms, White-orthologs are responsible for the translocation of uric acid for accumulation in urate granules in epidermal cells, resulting in opaque white coloration of the larval skin. In D. melanogaster, E23 encodes a transporter capable of modulating the ecdysone response with consequences for the circadian transcription of clock genes. To link the above suggested functions for the C. populi ABC proteins to those which are differentially expressed in the larval tissues of C. populi, we carried out a comprehensive transcriptome sequencing of different tissues dissected from the poplar leaf beetle. All raw sequence data are listed in Table S3 and S4. The resulting expression patterns of all identified ABC transporters in intestinal tissue, Malpighian tubules, fat body and defensive glands is depicted in Figure 6.

Thus, the activity of EBC5-16 is dependent on expression of the EPOR and specific for the human as opposed to the murine version of the receptor. To test whether the transmembrane domain of the hEPOR is required for EBC516 activity, we introduced EBC5-16 into cells expressing an HAtagged hEPOR mutant in which the transmembrane domain of the hEPOR was replaced with that of the murine PDGFbR. We previously showed that BaF3 cells expressing HA-hEPOR proliferated in response to EPO, which binds to the extracellular domain of the receptor retained in the chimera,Benzoylaconine but did not respond to TC2-3 because of the foreign transmembrane domain. As shown in Figure 2D, EBC5-16 also failed to cooperate with HA-hEPOR to induce growth factor independence, indicating that EBC5-16 requires the hEPOR transmembrane domain for activity. To determine whether EBC5-16 causes biochemical activation of the hEPOR, we immunoprecipitated the HA-tagged hEPOR from BaF3/HA-hEPOR cells expressing EBC5-16 or TC2-3 from the low expression vector, RVY-puro, and immunoblotted with an anti-phosphotyrosine antibody. As shown in Figure 3A, EBC5-16 induced tyrosine phosphorylation of the hEPOR. Interestingly, EBC5-16 and TC2-3 induced a similar level of hEPOR tyrosine phosphorylation, despite the enhanced biological activity of EBC516. Similarly,Licochalcone-C EBC5-16 induced tyrosine phosphorylation of JAK2 and STAT5, major downstream signaling partners of the hEPOR, at levels similar to that induced by TC2-3. These experiments demonstrated that we have isolated a TC2-3 mutant with enhanced biological activity in murine cells, but the basis for enhanced signaling has yet to be determined. A fraction of TC2-3 forms a disulfide bond-linked homodimer mediated by the cysteines at the C-terminus of the protein. To determine if EBC5-16 also forms a homodimer, cell extracts were prepared from BaF3/HA-hEPOR cells expressing either EBC5-16 or TC2-3, and replicate samples were immunoprecipitated with aE5, which recognizes the fixed C-terminus of TC2-3 and EBC516.