Showing papers on "Cellular compartment published in 2018"

Direct electrochemical observation of glucosidase activity in isolated single lysosomes from a living cell.

Abstract: The protein activity in individual intracellular compartments in single living cells must be analyzed to obtain an understanding of protein function at subcellular locations. The current methodology for probing activity is often not resolved to the level of an individual compartment, and the results provide an extent of reaction that is averaged from a group of compartments. To address this technological limitation, a single lysosome is sorted from a living cell via electrophoresis into a nanocapillary designed to electrochemically analyze internal solution. The activity of a protein specific to lysosomes, β-glucosidase, is determined by the electrochemical quantification of hydrogen peroxide generated from the reaction with its substrate and the associated enzymes preloaded in the nanocapillary. Sorting and assaying multiple lysosomes from the same cell shows the relative homogeneity of protein activity between different lysosomes, whereas the protein activity in single lysosomes from different cells of the same type is heterogeneous. Thus, this study for the analysis of protein activity within targeted cellular compartments allows direct study of protein function at subcellular resolution and provides unprecedented information about the homogeneity within the lysosomal population of a single cell.

Molecular basis for sterol transport by StART-like lipid transfer domains.

Abstract: Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non‐vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a‐c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo‐bound and sterol‐bound forms, at 2.0 A resolution. It folds into a truncated version of the steroidogenic acute regulatory protein‐related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3‐hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.

Virus Assembly and Egress of HSV.

Abstract: The assembly and egress of herpes simplex virus (HSV) is a complicated multistage process that involves several different cellular compartments and the activity of many viral and cellular proteins. The process begins in the nucleus, with capsid assembly followed by genome packaging into the preformed capsids. The DNA-filled capsids (nucleocapsids) then exit the nucleus by a process of envelopment at the inner nuclear membrane followed by fusion with the outer nuclear membrane. In the cytoplasm nucleocapsids associate with tegument proteins, which form a complicated protein network that links the nucleocapsid to the cytoplasmic domains of viral envelope proteins. Nucleocapsids and associated tegument then undergo secondary envelopment at intracellular membranes originating from late secretory pathway and endosomal compartments. This leads to assembled virions in the lumen of large cytoplasmic vesicles, which are then transported to the cell periphery to fuse with the plasma membrane and release virus particles from the cell. The details of this multifaceted process are described in this chapter.

Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles

Abstract: Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles. IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.

Hydroxypropyl-beta and -gamma cyclodextrins rescue cholesterol accumulation in Niemann–Pick C1 mutant cell via lysosome-associated membrane protein 1

Abstract: Niemann–Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-β-cyclodextrin (HPβCD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HPβCD is known to bind cholesterol; however, the mechanisms how HPβCD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-γ-cyclodextrin (HPγCD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HPβCD and HPγCD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HPβCD and HPγCD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.

Homologous expression of lipid droplet protein-enhanced neutral lipid accumulation in the marine diatom Phaeodactylum tricornutum

Abstract: Lipid droplets are ubiquitous cellular compartments that store neutral lipids and specific proteins localize on their surface. These proteins work as a scaffold in maintaining the lipid droplet structure or as regulators of lipogenesis or lipolysis. Previously, the most abundant lipid droplet protein, namely stramenopile-type lipid droplet protein (StLDP), was identified in the marine diatom Phaeodactylum tricornutum; however, its function remains unclear because StLDP does not reveal homology with known lipid droplet proteins and lacks a predictable domain. In this study, P. tricornutum was transformed to express a homologous StLDP gene under an fcpA promoter in order to determine its function. StLDP expression was strongly enhanced in the mutant (H8), especially in nitrogen-sufficient conditions; however, it was attenuated in nitrogen-deficient conditions. Despite the strong expression, no significant difference was observed in the lipid composition between the wild type (WT) and H8 under nitrogen-sufficient conditions. After cultivation in nitrogen-free medium for 6 days, neutral lipid content significantly increased in H8 than in WT. After 2 days of cultivation in nitrogen-free medium, 97.0% of single cells in WT formed one or two lipid droplets, whereas in H8, this proportion decreased to 78.8%, and the proportion of cells forming three or four lipid droplets increased. Thus, the function of StLDP was speculated to sequester triacylglycerol on the initial lipid droplet formation.

Functional Significance of the Adcy10-Dependent Intracellular cAMP Compartments.

Abstract: Mounting evidence confirms the compartmentalized structure of evolutionarily conserved 3′–5′-cyclic adenosine monophosphate (cAMP) signaling, which allows for simultaneous participation in a wide variety of physiological functions and ensures specificity, selectivity and signal strength. One important player in cAMP signaling is soluble adenylyl cyclase (sAC). The intracellular localization of sAC allows for the formation of unique intracellular cAMP microdomains that control various physiological and pathological processes. This review is focused on the functional role of sAC-produced cAMP. In particular, we examine the role of sAC-cAMP in different cellular compartments, such as cytosol, nucleus and mitochondria.

Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry

Abstract: The regulation of pH is essential for proper organelle function, and organelle-specific changes in pH often reflect the dynamics of physiological signaling and metabolism. For example, mitochondrial energy production depends on the proton gradient maintained between the alkaline mitochondrial matrix and neutral cytosol. However, we still lack a quantitative understanding of how pH dynamics are coupled between compartments and how pH gradients are regulated at organelle boundaries. Genetically encoded pH sensors are well suited to address this problem because they can be targeted to specific subcellular locations and they facilitate live, single-cell analysis. However, most of these pH sensors are derivatives of green and yellow fluorescent proteins that are not spectrally compatible for dual-compartment imaging. Therefore, there is a need for ratiometric red fluorescent protein pH sensors that enable quantitative multicolor imaging of spatially resolved pH dynamics. In this work, we demonstrate that the I158E/Q160A mutant of the red fluorescent protein mCherry is an effective ratiometric pH sensor. It has a pKa of 7.3 and a greater than 3-fold change in ratio signal. To demonstrate its utility in cells, we measured activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells. Furthermore, we were able to image pH changes simultaneously in the cytosol and mitochondria by using the mCherryEA mutant together with the green fluorescent pH sensor, ratiometric-pHluorin. Our results demonstrate the feasibility of studying interorganelle pH dynamics in live cells over time and the broad applicability of these sensors in studying the role of pH regulation in metabolism and signaling.

Plastid Transient and Stable Interactions with Other Cell Compartments.

Abstract: Plastids are organelles delineated by two envelopes that play important roles in different cellular processes such as energy production or lipid biosynthesis. To regulate their biogenesis and their function, plastids have to communicate with other cellular compartments. This communication can be mediated by signaling molecules and by the establishment of direct contacts between the plastid envelope and other organelles such as the endoplasmic reticulum, the mitochondria, the plasma membrane, the peroxisomes and the nucleus. These interactions are highly dynamic and respond to different biotic and abiotic stresses. However, the mechanisms involved in the formation of plastid-organelle contact sites and their functions are still enigmatic. In this chapter, we summarize our current knowledge about plastid contact sites and their role in the regulation of plastid biogenesis and function.

Septin localization and function during autophagy

Abstract: Autophagy is a vital conserved recycling process where eukaryotic cells remove unwanted proteins and organelles via lysosomal degradation and in turn, generate nutrients for the cells. The special feature of autophagy process is the formation of double-membrane vesicles called autophagosomes that engulf cellular cargo and deliver them to the vacuole or lysosomes for degradation. Inspite of more than 40 AuTophaGy (ATG) proteins and several organelles as known membrane source, autophagosome biogenesis is not entirely understood. We recently have discovered that septins contribute to autophagosome biogenesis. Septins are GTP-binding proteins, usually localized at the bud neck region and are involved in cytokinesis. Here, we show that during autophagy prevalent conditions, septins traffic between different cellular compartments such as Golgi, mitochondria, endosomes, plasma membrane, and vacuolar membranes.

Endoplasmic reticulum, Golgi, and lysosomes are disorganized in lung fibroblasts from chronic obstructive pulmonary disease patients.

Abstract: Chronic Obstructive Pulmonary Disease (COPD) is often caused by smoking and other stressors. This causes oxidative stress, which induces numerous changes on both the transcriptome and proteome of the cell. We aimed to examine if the endomembrane pathway, including the endoplasmic reticulum (ER), Golgi, and lysosomes, was disrupted in fibroblasts from COPD patients as opposed to healthy ever-smokers or never-smokers, and if the response to stress differed. Different cellular compartments involved in the endomembrane pathway, as well as mRNA expression and apoptosis, were examined before and after the addition of stress in lung fibroblasts from never-smokers, ever-smokers, and patients with COPD. We found that the ER, Golgi, and lysosomes were disorganized in fibroblasts from COPD patients under baseline conditions. After a time course with ER stress inducing chemicals, changes to the phenotypes of cellular compartments in COPD patient fibroblasts were observed, and the expression of the ER stress-induced gene ERP72 was upregulated more in the COPD patient's cells compared to ever-smokers or never-smokers. Lastly, a tendency of increased active Caspase-3 was observed in COPD fibroblasts. Our results show that COPD patients have phenotypic changes in the lung fibroblasts endomembrane pathway, and respond differently to stress. Furthermore, these fibroblasts were cultured for several weeks outside the body, but they were not able to regain proper ER structure, indicating that the internal changes to the endomembrane system are permanent in smokers. This vulnerability to cellular stress might be a cause as to why some smokers develop COPD.

Fluorescence Lifetime-Resolved Ion-Selective Nanospheres for Simultaneous Imaging of Calcium Ion in Mitochondria and Lysosomes.

Abstract: In this paper, novel calcium-selective nanospheres incorporating Pluronic F127 and (4-carboxybutyl) triphenylphosphonium bromide (TPP) as shell layers were designed to monitor the level of free calcium ion in mitochondria and lysosomes at living cells simultaneously. TPP as a target for mitochondria drove the nanospheres to bind intracellular mitochondria, while the lipophilic F127 layer resulted in the partial accumulation of nanospheres in lysosomes. This dual feature of the shell layer led to the colocation of nanospheres in both mitochondria and lysosomes. Chromoionophore III (ETH 5350) was chosen as the chromoionophore in the nanospheres that had different fluorescence lifetimes in either mitochondria or lysosomes, and therefore, the locations of the nanospheres at these two cellular compartments were identified. After the stimulation of cells using ionomycin, a burst of calcium concentration in mitochondria was observed that was associated with almost constant calcium concentration in lysosomes. The simultaneous recording of calcium ions in both of the compartments using fluorescence lifetime-solved nanospheres offered a special strategy to spatially monitor subcellular fluctuation of ions in living cells.

Transport-exclusion pharmacology to localize lactate dehydrogenase activity within cells

Abstract: Recent in vitro and in vivo work has shown that lactate provides an important source of carbon for metabolic reactions in cancer cell mitochondria. An interesting question is whether lactate is oxidized by lactate dehydrogenase (LDH) in the cytosol and/or in mitochondria. Since metabolic processes in the cytosol and mitochondria are affected by redox balance, the location of LDH may have important regulatory implications in cancer metabolism. Within most mammalian cells, metabolic processes are physically separated by membrane-bound compartments. Our general understanding of this spatial organization and its role in cellular function, however, suffers from the limited number of techniques to localize enzymatic activities within a cell. Here, we describe an approach to assess metabolic compartmentalization by monitoring the activity of pharmacological inhibitors that cannot be transported into specific cellular compartments. Oxamate, which chemically resembles pyruvate, is transported into mitochondria and inhibits LDH activity in purified mitochondria. GSK-2837808A, in contrast, is a competitive inhibitor of NAD, which cannot cross the inner mitochondrial membrane. GSK-2837808A did not inhibit the LDH activity of intact mitochondria, but GSK-2837808A did inhibit LDH activity after the inner mitochondrial membrane was disrupted. Our results are consistent with some mitochondrial LDH that is accessible to oxamate, but inaccessible to GSK-2837808A until mitochondria are homogenized. This strategy of using inhibitors with selective access to subcellular compartments, which we refer to as transport-exclusion pharmacology, is broadly applicable to localize other metabolic reactions within cells.

Integration of the Endocytic System into the Network of Cellular Functions

Abstract: Maintenance of physiologic cellular functions and homeostasis requires highly coordinated interactions between different cellular compartments. In this regard, the endocytic system, which plays a key role in cargo internalization and trafficking within the cell, participates in upkeep of intracellular dynamics, while communicating with multiple organelles. This chapter will discuss the function of endosomes from a standpoint of cellular integration. We will present examples of different types of interactions between endosomes and other cellular compartments, such as the endoplasmic reticulum (ER), mitochondria, the plasma membrane (PM), and the nuclear envelope. In addition, we will describe the incorporation of endocytic components, such as endosomal sorting complexes required for transport (ESCRT) proteins and Rab small GTPases, into cellular processes that operate outside of the endolysosomal pathway. The significance of endosomal interactions for processes such as signaling regulation, intracellular trafficking, organelle dynamics, metabolic control, and homeostatic responses will be reviewed. Accumulating data indicate that beyond its involvement in cargo transport, the endocytic pathway is comprehensively integrated into other systems of the cell and plays multiple roles in the complex net of cellular functions.

Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis.

Abstract: Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogenous surface charge allows multi-'omic analyses to be performed on the ER. This facilitates investigations into structure-function relationships within the ER.

Spatial control of irreversible protein aggregation.

Abstract: Liquid cellular compartments spatially segregate from the cytoplasm and can regulate aberrant protein aggregation, a process linked to several medical conditions, including Alzheimer's and Parkinson's diseases. Yet the mechanisms by which these droplet-like compartments affect protein aggregation remain unknown. Here, we combine kinetic theory of protein aggregation and liquid-liquid phase separation to study the spatial control of irreversible protein aggregation in the presence of liquid compartments. We find that, even for weak interactions between the compartment constituents and the aggregating monomers, aggregates are strongly enriched inside the liquid compartment relative to the surrounding cytoplasm. We show that this enrichment is caused by a positive feedback mechanism of aggregate nucleation and growth which is mediated by a flux maintaining the phase equilibrium between the compartment and the cytoplasm. Our model predicts that the compartment volume that maximizes aggregate enrichment in the compartment is determined by the reaction orders of aggregate nucleation. The underlying mechanism of aggregate enrichment could be used to confine cytotoxic protein aggregates inside droplet-like compartments suggesting potential new avenues against aberrant protein aggregation. Our findings could also represent a common mechanism for the spatial control of irreversible chemical reactions in general.

Study of the Cellular Role of GRP78/BiP mono-ADP-ribosylation in UPR and Cancer

Abstract: Mono-ADP-ribosylation is a reversible post-translational protein modification that modulates the function of proteins involved in different cellular processes, including signal transduction, protein transport, transcription, cell cycle regulation, DNA (deoxyribonucleic acid) repair and apoptosis. In mammals, mono-ADPribosylation is catalyzed by three different classes of enzymes: ARTCs, ARTDs, and members of the sirtuin family. In the present study, hARTC1-mediated mono-ADP-ribosylation was investigated in terms of the cellular compartments involved, target(s) and roles. The collected results demonstrated that hARTC1 protein and enzymatic activity is mainly localized to the endoplasmic reticulum (ER), in contrast to other ARTCs, which are either typically GPI-anchored enzymes in the plasma membrane, or secreted enzymes. Previous studies in my laboratory demonstrated that a protein macro domain was useful for the study of APD-ribosylation. The data reported here indicate, for the first time, that the macro domain can be used for immunofluorescence, allowing visualization of ADP-ribosylated proteins in intact cells, and in far-Western Blotting, allowing the detection of specific ADPribosylated targets. These methodologies were employed to demonstrate that the ER-localized chaperone, GRP78/BiP, was a prime target of hARTC1. A doubly mutated hARTC1 mutant was designed, and used as a specific control for hARTC1 expression. The mutant enzyme localized to the ER, but did not catalyze GRP78/BiP ADP-ribosylation. The demonstration that GRP78/BiP was mono-ADP-ribosylated by hARTC1 suggested that hARTC1 could be a key regulator of GRP78/BiP-mediated functions. Consistent with the key role of GRP78/BiP in the ER stress response, it was found that hARTC1 was activated during short-term cell treatment with ER stressors, resulting in acute GRP78/BiP ADP-ribosylation. However, the monoADP-ribosylation of the chaperone did not trigger an unfolded protein response. Recently, hARTC1 has been associated with cancer, suggesting a possible role in cell proliferation. In line with these findings, the results presented here demonstrated that hARTC1 over-expression inhibited cell proliferation.

FRET Flow Cytometry-Based High Throughput Screening Assay To Identify Disrupters of Glucose Levels in Trypanosoma brucei

Abstract: Trypanosoma brucei, which causes human African typanosomiasis (HAT), derives cellular ATP from glucose metabolism while in the mammalian host. Targeting glucose uptake or regulation in the parasite has been proposed as a potential therapeutic strategy. However, few methods have been described to identify and characterize potential inhibitors of glucose uptake and regulation. Here, we report development of a screening assay that identifies small molecule disrupters of glucose levels in the cytosol and glycosomes. Using an endogenously expressed fluorescent protein glucose sensor expressed in cytosol or glycosomes, we monitored intracellular glucose depletion in the different cellular compartments. Two glucose level disrupters were identified, one of which only exhibited inhibition of glycosomal glucose and did not affect cytosolic levels. In addition to inhibiting glucose uptake with relatively high potency (EC50 = 700 nM), the compound also showed modest bloodstream form parasite killing activity. Expandi...

Calcium in plant peroxisomes. What for

Abstract: Peroxisome organelles have a versatile metabolism whose enzymatic content can be modulated by physiological and environment-dependent cellular conditions. They are characterized by a highly active nitro-oxidative metabolism and basic elements (H2O2 and nitric oxide (NO)) with signaling properties. However, new elements have increased our understanding of the connections between peroxisomes and other cellular compartments. Furthermore, the presence of calcium (Ca2+) intensifies communication between different signaling molecules and the relationship of Ca2+ itself with NO and H2O2.

Mechanisms of Longevity Extension by Caloric Restriction and Lithocholic Acid in the Yeast Saccharomyces Cerevisiae

Abstract: The objective of studies described in this thesis was to elucidate molecular and cellular mechanisms by which caloric restriction (CR) and lithocholic acid (LCA) extend longevity of the budding yeast Saccharomyces cerevisiae. Recent studies of how CR influences a pattern of metabolism and organelle dynamics in the chronologically aging yeast S. cerevisiae have revealed that this low-calorie diet alters age-related dynamics of ethanol metabolism, lipid synthesis and degradation, trehalose metabolism, ROS homeostasis maintenance, mitochondrial morphology control, mitochondrial functionality preservation, stress response control, cell cycle regulation, quiescence maintenance, and apoptotic and liponecrotic death subroutines. Our hypothesis was that CR may delay yeast chronological aging by altering the age-related dynamics of some or all these cellular processes. Findings presented here support this hypothesis. Indeed, we found that CR slows yeast chronological aging by mechanisms that coordinate the spatiotemporal dynamics of various cellular processes before entry into a non-proliferative state and after such entry. CR causes a stepwise establishment of an aging-delaying cellular pattern by tuning a network that assimilates the following: 1) pathways of carbohydrate and lipid metabolism; 2) communications between the endoplasmic reticulum, lipid droplets, peroxisomes, mitochondria and the cytosol; and 3) a balance between the processes of mitochondrial fusion and fission. Through different phases of the aging process, the CR-dependent remodeling of this intricate network 1) postpones the age-related onsets of apoptotic and liponecrotic modes of regulated cell death; and 2) actively increases the chance of cell survival by supporting the maintenance of cellular proteostasis. Because CR decreases the risk of cell death and actively increases the chance of cell survival throughout chronological lifespan, this dietary intervention extends longevity of chronologically aging yeast. We also used a mass spectrometry-based quantitative analysis of the water-soluble cellular metabolome for the investigation of how CR and the longevity-extending tor1Δ mutation (which eliminates the Tor1 protein kinase known to orchestrate the nutrient- and energy-sensing TOR [target of rapamycin] pro-aging signaling pathway) influence the concentrations of various water- soluble metabolites at consecutive stages of the chronological aging process in S. cerevisiae. Our investigation provided the first evidence that both the longevity-extending diet CR and the- longevity extending mutation tor1Δ establish a similar pattern of relative concentrations of methionine metabolism intermediates through the entire process of chronological aging in S. cerevisiae. We proposed a hypothesis that the observed redirection of metabolite flow from the biosynthesis of methionine and spermidine to the biosynthesis of cysteine and glutathione may represent an anti-aging pattern characteristic of the ʺmetabolic signatureʺ of longevity extension in chronologically aging yeast cells placed on the CR diet or having the TOR pro-aging signaling pathway being inactivated. Based on recent findings from the Titorenko laboratory, we hypothesized that the LCA- driven changes in mitochondrial lipidome may have a causal role in the age-related remodeling of proteome, thus eliciting changes in mitochondrial functionality and delaying yeast chronological aging. To test this hypothesis, we used a mass spectrometry-based quantitative analysis to investigate how certain mutations that eliminate enzymes involved in mitochondrial phospholipid metabolism influence the mitochondrial proteome and how they affect the geroprotective efficiency of LCA in chronologically aging yeast. Our investigation provided the first evidence that LCA-driven specific changes in the composition of mitochondrial membrane lipids cause a distinct remodeling of mitochondrial proteome by decreasing and increasing concentration of many mitochondrial proteins. These proteins have been implicated in such vital mitochondrial functions as the ETC and respiration, the TCA cycle, ribosome assembly, amino acid metabolism, carbohydrate metabolism, protein import, proteostasis, metabolite synthesis, protein synthesis, ATP synthesis, metabolite transport, lipid metabolism, contact sites and cristae maintenance, redox homeostasis, mtDNA maintenance, stress response, mRNA synthesis and processing, the maintenance of contact sites between mitochondria and vacuoles, and mitochondrial fusion. We provided evidence that the LCA-dependent remodeling of mitochondrial lipidome and the resulting changes in mitochondrial proteome are essential for the ability of LCA to delay aging. Our recent studies have indicated that under CR conditions LCA influences not only the composition and functionality of mitochondria but also some cellular processes confined to other cellular compartments. We therefore hypothesized that LCA may delay chronological aging of yeast limited in calorie supply also because it affects these other cellular processes taking place in various cellular locations. To test this hypothesis, we investigated mechanisms through which LCA controls the spatiotemporal dynamics of these other cellular processes in different cellular locations under CR conditions. Our investigation provided important new insights into the mechanisms by which LCA delays yeast chronological aging under CR conditions by altering the spatiotemporal dynamics of a cellular network that integrates certain pathways of lipid and carbohydrate metabolism, some intercompartmental communications, specific aspects of mitochondrial morphology and functionality, and liponecrotic and apoptotic modes of regulated cell death. Because LCA triggers major changes in the age-related chronology of several vital processes taking place in mitochondria, we hypothesized that LCA may cause these changes by eliciting a reversible phosphorylation of some mitochondrial proteins. To test this hypothesis, we investigated if an exposure of chronologically aging yeast to exogenous LCA can trigger such phosphorylation. We found that LCA elicits the establishment of a distinct phosphoprotein profile of mitochondria, which significantly differs from the phosphoprotein profile of mitochondria in yeast cells cultured without LCA.

Analysis of the MTL Supercomplex at Contact Sites Between Mitochondria and Plastids.

Abstract: Plastids are organelles playing fundamental roles in different cellular processes such as energy metabolism or lipid biosynthesis To fulfill their biogenesis and their function in the cell, plastids have to communicate with other cellular compartments This communication can be mediated by the establishment of direct contact sites between plastids envelop and other organelles These contacts are dynamic structures that are modified in response to stress As example, during phosphate (Pi) starvation, the number of contact sites between plastids and mitochondria significantly increases In this situation, these contacts play an important role in the transfer of galactoglycerolipids from plastids to mitochondria Recently, Pi starvation stress was used to identify key proteins involved in the traffic of galactoglycerolipids from plastids to mitochondria in Arabidopsis thaliana A mitochondrial lipoprotein complex called MTL (mitochondrial transmembrane lipoprotein complex) was identified This complex contains mitochondrial proteins but also proteins located in the plastid envelope, suggesting its presence at the plastid-mitochondria junction This chapter describes the protocol to isolate the MTL complex by clear-native polyacrylamide gel electrophoresis (CN-PAGE) from the mitochondrial fraction of Arabidopsis cell cultures and the methods to study different features of this complex

pH gradient mitigation in the leaf cell secretory pathway alters the defense response of Nicotiana benthamiana to agroinfiltration

Abstract: Partial neutralization of the Golgi lumen pH by ectopic expression of influenza virus M2 proton channel stabilizes acid-labile and protease-susceptible recombinant proteins in the plant cell secretory pathway. Here, we assessed the impact of M2 channel expression on the proteome of Nicotiana benthamiana leaf tissue infiltrated with the bacterial gene vector Agrobacterium tumefaciens, keeping in mind the key role of pH homeostasis on secreted protein processing and the involvement of protein secretion processes in plant cells upon microbial challenge. The proteomes of leaves agroinfiltrated with an empty vector or with an M2 channel-encoding vector were compared with the proteome of non-infiltrated leaves using a iTRAQ quantitative proteomics procedure. Leaves infiltrated with the empty vector had a low soluble protein content compared to non-infiltrated leaves, associated with a strong decrease of photosynthesis-associated proteins (including Rubisco) and a parallel increase of stress-related secreted proteins (including pathogenesis-related proteins, protease inhibitors and molecular chaperones). M2 expression partly compromised these alterations of the proteome to restore original soluble protein and Rubisco contents, associated with higher levels of translation-associated (ribosomal) proteins and reduced levels of stress-related proteins in the apoplast. Proteome changes in M2-expressing leaves were determined both transcriptionally and post-transcriptionally, to alter the steady-state levels of proteins not only along the secretory pathway but also in other cellular compartments including the chloroplast, the cytoplasm, the nucleus and the mitochondrion. These data illustrate the cell-wide influence of Golgi lumen pH homeostasis on the leaf proteome of N. benthamiana plants responding to microbial challenge. They underline in practice the relevance of carefully considering the eventual off-target effects of accessory proteins used to modulate specific cellular or metabolic functions in plant protein biofactories.

Response and Cytoprotective Mechanisms Against Proteotoxic Stress in Yeast and Fungi

Abstract: Misfolded and/or aggregated proteins are not only functionless but also hazardous for cells. The budding yeast Saccharomyces cerevisiae has been a significant model organism for exploring cellular strategies against protein misfolding. There are various intracellular machineries for sequestering protein aggregates away from other cellular compartments or for disaggregating them. In addition, the ubiquitin-proteasome system functions in degradation of misfolded proteins accumulated in the cytosol, nuclei, and the endoplasmic reticulum. The heat shock response and unfolded protein response are gene expression programs for the induction of factors that contribute to these protein quality control systems in response to an accumulation of misfolded proteins. While, in general, observations obtained through yeast studies can be applied to other eukaryotic species, yeast- and fungal-specific factors or phenomena are also intriguing not only as matters of basic cell biology but also for clinical (pathogenic yeast and fungi) and industrial (fermentation) applications.

Transport of glutathione across the endoplasmic reticulum membrane

Abstract: γ-L-glutamyl-L-cysteinylglycine known as glutathione is tripeptide synthesised in the cytoplasm. Glutathione plays important role in several cellular processes and has many functions, for example detoxification of xenobiotics, oxidative protein folding, protection against reactive oxygen species, modulation of cell proliferation and apoptosis. Glutathione is present in almost all organelles within the cell, however, it is still not known how glutathione is transported from the cytoplasm to other cellular compartments. Here we investigated the transport of glutathione into the ER where glutathione plays an important role in oxidative protein folding. Two assays to monitor glutathione transport across the ER membrane were developer and an attempt to identify the putative glutathione transporter was made. Both assays rely on selective permeability of biological membranes and use microsomes to mimic the ER environment. Microsomes were prepared from HT1080 cells expressing either redox sensitive green fluorescent protein (roGFP-iE) or glutathione S-transferase P1 (GSTP1-1A) inside the ER. By measuring the change in fluorescence of roGFP-iE it was possible to measure the rate at which glutathione is transported inside microsomes. GSTP1-1A is able to conjugate glutathione to various substrates and form a stable product, by measuring the increase in glutathione conjugates it was possible to estimate the transport of glutathione inside microsomes. Using both roGFP-iE and GSTP1-1A based assays we were able to measure glutathione transport into microsomes, both assays provide slightly different information about glutathione transport and both have different limitations. In order to identify the glutathione transporter, we used GSH as an affinity ligand and isolated all ER membrane proteins interacting with glutathione. The first approach using glutathione Sepharose beads allowed to isolate several proteins, however, all of them belonged to GST family. A second approach relied on isolating the transporter using glutathione attached to Mts-Aft-Biotin a photo activated crosslinker. Mts-Aft-Biotin approach did not result in isolation of any proteins binding specifically to GSH, this approach requires more work and needs to be improved. We showed that it is possible to isolate GSH binding proteins using GSH as affinity ligand, using this strategy it might be possible to isolate GSH transporter in the future. Both assays presented in this work are the first assays specific for the transport of glutathione across the ER membrane. In the future these assays can be used to investigate the transport of glutathione even further and contribute to the understanding of redox homeostasis in the ER.

Physiological Functions of Phosphoinositide-Modifying Enzymes and Their Interacting Proteins in Arabidopsis.

Abstract: The integrity of cellular membranes is maintained not only by structural phospholipids such as phosphatidylcholine and phosphatidylethanolamine, but also by regulatory phospholipids, phosphatidylinositol phosphates (phosphoinositides). Although phosphoinositides constitute minor membrane phospholipids, they exert a wide variety of regulatory functions in all eukaryotic cells. They act as key markers of membrane surfaces that determine the biological integrity of cellular compartments to recruit various phosphoinositide-binding proteins. This review focuses on recent progress on the significance of phosphoinositides, their modifying enzymes, and phosphoinositide-binding proteins in Arabidopsis.