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Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.

Macropinocytosis is a regulated form of endocytosis that mediates the non‐selective uptake of solute molecules, nutrients and antigens. It is an actin‐dependent process initiated from surface membrane ruffles that give rise to large endocytic vacuoles called macropinosomes. Macropinocytosis is important in a range of physiological processes; it is highly active in macrophages and dendritic cells where it is a major pathway for the capture of antigens, it is relevant to cell migration and tumour metastasis and it represents a portal of cell entry exploited by a range of pathogens. The molecular basis for the formation and maturation of macropinosomes has only recently begun to be defined. Here, we review the general characteristics of macropinocytosis, describe some of the regulators of this pathway, which have been identified to date and highlight strategies to explore the relevance of this endocytosis pathway in vivo.

Two well-known pathways for the degradation of chloroplast proteins are via autophagy and senescence-associated vacuoles. Here, we describe a third pathway that was activated by senescence-and abiotic stress-induced expression of Arabidopsis thaliana CV (for chloroplast vesiculation). After targeting to the chloroplast, CV destabilized the chloroplast, inducing the formation of vesicles. CV-containing vesicles carrying stronfiai proteins, envelope membrane proteins, and thylakoid membrane proteins were released from the chloroplasts and mobilized to the vacuole for proteolysis. Overexpression of CV caused chloroplast degradation and premature leaf senescence, whereas silencing CV delayed chloroplast turnover and senescence induced by abiotic stress. Transgenic CIA-silenced plants displayed enhanced tolerance to drought, salinity, and oxidative stress. Immunoprecipitation and bimolecular fluorescence complementation assays demonstrated that CV interacted with photosystem II subunit PsbO1 in vivo through a C-terminal domain that is highly conserved in the plant kingdom. Collectively, our work indicated that CV plays a crucial role in stress-induced chloroplast disruption and mediates a third pathway for chloroplast degradation. From a biotechnological perspective, silencing of CV offers a suitable strategy for the generation of transgenic crops with increased tolerance to abiotic stress.

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

Vacuolar acidity in yeast is shown to decline with age, and preventing this decrease suppresses mitochondrial dysfunction and extends the lifespan of yeast. Acid drop a sign of mitochondrial ageing This manuscript identifies mitochondrial dysfunction due to increasing vacuolar pH as a determinant of lifespan in the yeast Saccharomyces cerevisiae . Vacuolar acidity is shown to decline over time, but the pH is reset in newborn cells with full lifespan potential. Calorie restriction increases vacuolar acidity via conserved nutrient-sensing pathways and both promotes lifespan extension and suppresses mitochondrial dysfunction. Alterations in acidity appear to impair mitochondrial function via a reduction of amino acid storage in the vacuolar lumen. These findings point to a potential mechanism by which amino acid and glucose limitation may converge to regulate lifespan. Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it 1 . Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes 2 , but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae , and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways 3 . Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential 4 . Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution 5 , we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.

Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella -containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila .

Key message The stigma papilla cells of Arabidopsis thaliana  control water transport to pollen by regulating the morphology of vacuoles in papilla cells after pollination. Pollen hydration is the first crucial response after pollination for successful fertilization. In the Brassicaceae family, papilla cells on the stigma supply water to pollen. In pollinated papilla cells, cellular responses essential for pollen hydration are induced. However, it remains unclear how papilla cells release water from inside the cells to the pollen. Here, we set up a live-cell imaging system for observing vacuole dynamics in Arabidopsis thaliana papilla cells and investigated the role of vacuole morphology in these cells in the regulation of water transfer to pollen. Before pollination, vacuoles in the papilla cells changed their morphology through fusion and constriction; however, after pollination, they formed larger vacuoles and exhibited reduced movement. Additionally, when the morphological variation of vacuoles in the papilla cells was inhibited by wortmannin treatment, the pollen hydration rate decreased in a concentration-dependent manner. In contrast, the vacuoles tended to be less constricted even before pollination and showed less variation than wild-type after pollination in Rho-like GTPase from plants 2 (ROP2) mutant papilla cells, where the pollen hydration rate is faster. We propose that the regulation of vacuole morphology in papilla cells is involved in water transfer to pollen during pollination.

About the Authors: Lenka Cernikova Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland Carmen Faso Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland ORCID logo http://orcid.org/0000-0002-1831-9365 Adrian B. Hehl * E-mail: adrian.hehl@uzh.ch Affiliation: Laboratory of Molecular Parasitology, Institute of Parasitology, University of Zurich (ZH), Zurich, Switzerland ORCID logo http://orcid.org/0000-0002-2110-4445 Citation: Cernikova L, Faso C, Hehl AB (2018) Five facts about Giardia lamblia. Based on the latest figures provided by WHO, G. lamblia is the third most common agent of diarrheal disease worldwide with over 300 million reported cases per annum, preceded only by rotavirus and Cryptosporidium parvum and hominis in the most vulnerable target group of children under five years of age [2]. ER, endoplasmic reticulum; GILRP, giardial putative low-density lipoprotein receptor; LDL, low-density lipoprotein; PM, plasma membrane; PV, peripheral vacuole; TEM, transmission electron microscopy; VSP, variant surface protein. https://doi.org/10.1371/journal.ppat.1007250.g001 Fact 4: G. lamblia survives in the environment as infectious cysts Completion of the life cycle by transmission of G. lamblia to a new host requires no vectors and is based on the alternation of a vegetative stage, the trophozoite, and an environmentally resistant infectious stage—the cyst. Novel rational design-based vaccination strategies against G. lamblia are yielding encouraging results Currently, treatment of giardiasis in humans is based almost exclusively on administration of antiprotozoals belonging to the family of 5-nitroimidazoles, whereas infected animals are treated with benzimidazoles.

Main conclusion The vacuolar membrane is an essential component in protecting the plant cell from stress factors. Different variations in the tonoplast lipid content, which depend on the type of stress, have been reviewed. The lipid content of vacuolar membranes of beet roots ( Beta vulgaris L.) under hypoosmotic, hyperosmotic and oxidative types of stress has been studied. These types of stress induce variations in the content of almost all the classes of studied lipids (phospholipids, glycoglycerolipids, sterols and fatty acids). The variations, which are characteristic of a single stress, include the variations (i) in the content of individual glycoglycerolipids and in their total content, (ii) in the total content of sterols, and (iii) in the ratio of content of phosphatidylcholine/phosphatidylethanolamine in the scope of tonoplast phospholipids. Variations observed under all of the types of stress under scrutiny include (i) variations in the content of fatty acids of tonoplast lipids, (ii) some decrease in the content of phosphatidic acid and phosphatidylethanolamine, and (iii) variations in the content of individual sterols. Stigmasterol, campesterol, as well as the stigmasterol/sitosterol ratio increased in varying degrees under all of the types of stress. The most substantial variations have been observed in the content of sterols under abiotic stress. This is probably due to role of sterols in regulation of such membrane characteristics as permeability and microviscosity. In our opinion, sterols may represent one of the main components of tonoplast adaptive mechanisms.