The ceramide-activated protein phosphatase Sit4p controls lifespan,mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H₂O₂ resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.     

Assessment of chronological lifespan dependent molecular damages in yeast lacking mitochondrial antioxidant genes

The free radical theory of aging states that oxidative damage to biomolecules causes aging and that antioxidants neutralize free radicals and thus decelerate aging. Mitochondria produce most of the reactive oxygen species, but at the same time have many antioxidant enzymes providing protection from these oxidants. Expecting that cells without mitochondrial antioxidant genes would accumulate higher levels of oxidative damage and, therefore, will have a shorter lifespan, we analyzed oxidative damages to biomolecules in young and chronologically aged mutants lacking the mitochondrial antioxidant genes: GRX2, CCP1, SOD1, GLO4, TRR2, TRX3, CCS1, SOD2, GRX5, and PRX1. Among these mutants, ccp1Δ, trx3Δ, grx5Δ, prx1Δ, mutants were sensitive to diamide, and ccs1Δ and sod2Δ were sensitive to both diamide and menadione. Most of the mutants were less viable in stationary phase. Chronologically aged cells produced higher amount of superoxide radical and accumulated higher levels of oxidative damages. Even though our results support the findings that old cells harbor higher amount of molecular damages, no significant difference was observed between wild type and mutant cells in terms of their damage content.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.     

Sec22 Regulates Endoplasmic Reticulum Morphology but Not Autophagy and Is Required for Eye Development in Drosophila

The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.     

The ceramide activated protein phosphatase Sit4 impairs sphingolipid dynamics,mitochondrial function and lifespan in a yeast model of Niemann-Pick type C1

The Niemann-Pick type C is a rare neurodegenerative disease that results from loss-of-function point mutations in NPC1 or NPC2, which affect the homeostasis of sphingolipids and sterols in human cells. We have previously shown that yeast lacking Ncr1, the orthologue of human NPC1 protein, display a premature ageing phenotype and higher sensitivity to oxidative stress associated with mitochondrial dysfunctions and accumulation of long chain bases. In this study, a lipidomic analysis revealed specific changes in the levels of ceramide species in ncr1Δ cells, including decreases in dihydroceramides and increases in phytoceramides. Moreover, the activation of Sit4, a ceramide-activated protein phosphatase, increased in ncr1Δ cells. Deletion of SIT4 or CDC55, its regulatory subunit, increased the chronological lifespan and hydrogen peroxide resistance of ncr1Δ cells and suppressed its mitochondrial defects. Notably, Sch9 and Pkh1-mediated phosphorylation of Sch9 decreased significantly in ncr1Δsit4Δ cells. These results suggest that phytoceramide accumulation and Sit4-dependent signaling mediate the mitochondrial dysfunction and shortened lifespan in the yeast model of Niemann-Pick type C1, in part through modulation of the Pkh1-Sch9 pathway.     

The stacked over-expression of FPS, CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.

Artemisinin is an endoperoxide sesquiterpene lactone isolated from the aerial parts of Artemisia annua L., and is presently the most potent anti-malarial drug. Owing to the low yield of artemisinin from A. annua as well as the widespread application of artemisinin-based combination therapy recommended by the World Health Organization, the global demand for artemisinin is substantially increasing and is therefore rendering artemisinin in short supply. An economical way to increase artemisinin production is to increase the content of artemisinin in A. annua. In this study, three key genes in the artemisinin biosynthesis pathway, encoding farnesyl diphosphate synthase, amorpha-4, 11-diene C-12 oxidase and its redox partner cytochrome P450 reductase, were over-expressed in A. annua through Agrobacterium-mediated transformation. The transgenic lines were confirmed by Southern blotting and the over-expressions of the genes were demonstrated by real-time PCR assays. The HPLC analysis showed that the artemisinin contents in transgenic lines were increased significantly, with the highest one found to be 3.6-fold higher (2.9 mg/g FW) than that of the control. These results demonstrate that multigene engineering is an effective way to enhance artemisinin content in A. annua.     

Dexamethasone Inhibits Mitogen-dependent Synthesis of Histamine Due to Histidine Decarboxylase Induced in Cultured Mouse Spleen Cells

Schizosaccharomyces pombe has four α-amylase homologs (Aah1p-Aah4p) with a glycosylphosphatidylinositol (GPI) modification site at the C-terminal end. Disruption mutants of aah genes were tested for mislocalization of vacuolar carboxypeptidase Y (CPY), and aah3Δ was found to secrete CPY. The conversion rate from pro- to mature CPY was greatly impaired in aah3Δ, and fluorescence microscopy inidicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. These results indicate that aah3Δ had a defect in the retrograde transport of Vps10p, and that Aah3p is the first S. pombe specific protein required for vacuolar protein sorting.     

AtSec20 is involved in osmotic stress tolerance and AtSec20 mutation unaffects the integrity of intracellular organelles and the anterograde biosynthetic trafficking

The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play essential roles in intracellular trafficking. However, few experimental data have clarified their roles in the stress responses and the early secretary pathway in Arabidopsis. The AtSec20 gene encodes a protein that is homologous to yeast Sec20p and mammalian BNIP1, which are involved in the Golgi-to-ER retrograde trafficking in yeast and mammalian cells. In this study, AtSec20 is found to be required for the responses to salt stress, osmotic stress and gibberellin (GA) during seed germination and early seedling establishment. Mutation of AtSec20 unaffects the morphology of intracellular organelles, such as endoplasmic reticulum (ER), trans-Golgi network, and peroxisome, and vacuolar protein trafficking is normal in sec20 mutants. Collectively, these results imply that the AtSec20 is involved in abiotic stress tolerance, potentially via roles in retrograde vesicle fusion process in Arabidopsis.     

Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

A proteomic analysis of secretory proteins of a pre-vacuolar mutant of Candida albicans

S. cerevisiae mutants lacking VPS4 missort several vacuolar proteins to the extracellular space, including carboxypeptidase (CPY), vacuolar protease A (PrA), and vacuolar protease B (PrB). In addition, certain soluble secretory proteins, such as invertase and acid phosphatase, are missorted from the pre-vacuolar compartment (PVC) to the general secretory pathway prior to exocytosis. Although little is known about sorting of proteins via the PVC in Candida albicans, we have previously demonstrated that the C. albicans vps4Δ null mutant missorts PrA and CPY extracellularly, but fails to secrete the aspartyl proteases Sap2p and Sap4–6p. To further define the role of C. albicans VPS4 in the trafficking of pre-vacuolar proteins, we have used 2 dimensional gel electrophoresis (2-DE) and mass spectrometry techniques to study soluble proteins in the supernatants of planktonic cultures obtained from the C. albicans vps4Δ mutant compared to control strain DAY185. Results indicated that lack of VPS4 results in a decrease of canonically secreted proteins whilst having a limited effect on non-canonically secreted extracellular proteins. Four canonically secreted proteins (Cht3p, Pra1p, Mp65p and Sun41p) were identified as reduced in the supernatants from the mutant strain. We also indentified two other major consequences of lack of VPS4, likely associated with secretion defects: altered branching and biofilm formation.     

The ER-Membrane Transport System Is Critical for Intercellular Trafficking of the NSm Movement Protein and Tomato Spotted Wilt Tospovirus

Plant viruses move through plasmodesmata to infect new cells. The plant endoplasmic reticulum (ER) is interconnected among cells via the ER desmotubule in the plasmodesma across the cell wall, forming a continuous ER network throughout the entire plant. This ER continuity is unique to plants and has been postulated to serve as a platform for the intercellular trafficking of macromolecules. In the present study, the contribution of the plant ER membrane transport system to the intercellular trafficking of the NSm movement protein and Tomato spotted wilt tospovirus (TSWV) is investigated. We showed that TSWV NSm is physically associated with the ER membrane in Nicotiana benthamiana plants. An NSm-GFP fusion protein transiently expressed in single leaf cells was trafficked into neighboring cells. Mutations in NSm that impaired its association with the ER or caused its mis-localization to other subcellular sites inhibited cell-to-cell trafficking. Pharmacological disruption of the ER network severely inhibited NSm-GFP trafficking but not GFP diffusion. In the Arabidopsis thaliana mutant rhd3 with an impaired ER network, NSm-GFP trafficking was significantly reduced, whereas GFP diffusion was not affected. We also showed that the ER-to-Golgi secretion pathway and the cytoskeleton transport systems were not involved in the intercellular trafficking of TSWV NSm. Importantly, TSWV cell-to-cell spread was delayed in the ER-defective rhd3 mutant, and this reduced viral infection was not due to reduced replication. On the basis of robust biochemical, cellular and genetic analysis, we established that the ER membrane transport system serves as an important direct route for intercellular trafficking of NSm and TSWV.