Cytoplasmic bacteria can be targets for autophagy

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.     

Glucose repression and autolysis ofSaccharomyces cerevisiae cells: alterations in the cytochemical localization of acid phosphatase

Summary Allerations in the localization of acid phosphatase inSaccharomyces cerevisiae during glucose repression and during autolysis have been studied. Cell morphology becomes distinctly changed after only 2 h in the presence of high glucose concentration while after 3 h of glucose repression the majority of the mitochondirial structures resemble promitochondria. Yeast cells repressed for 6 h contain almost completely degraded mitochondrial structures and numerous lipid droplets in the central vacuole and cytoplasm. Destruction of mitochondria is accompanied by the accumulation of acid phosphatase in these organelles and in the cytoplasm whereas its activity in the central vacuole is lowered, most probably because of the leakage of the enzyme into the cytoplasm.No preferential breakdown of mitochondria is observed during autolysis. On the contrary, mitochondria are apparently the last to be degraded. Digestion of cytoplasmic regions and membranous elements occurs intravacuolarly after sequestration by protrusions of the central vacuole which are formed at the initial stages of autolysis. Acid phosphatase is not released from the central vacuole, suggesting indirectly that vacuole enzymes do not migrate into the cytoplasm during autolysis.     

Topical aspects of vacuolar protein transport: autophagy and prevacuolar compartments

For many plant researchers protein transport to the vacuole is primarily a question of the mechanisms underlying the recognition of vacuolar proteins and their segregation in the Golgi apparatus from other products of the secretory pathway. Autophagy is an alternative process by which proteins can enter the vacuole. Examples of apparent selective autophagy are seen in cereal grains depositing storage proteins, and in rubber particle sequestration. Non-selective autophagy is observed during starvation and in senescing tissues. Another overlooked aspect of vacuolar protein transport involves the participation of prevacuolar sorting compartments, some of which may function as endosomes.     

What to Eat: Evidence for Selective Autophagy in Plants

Autophagy is a macromolecular degradation pathway by which cells recycle their contents as a developmental process, housekeeping mechanism, and response to environmental stress. In plants, autophagy involves the sequestration of cargo to be degraded, transport to the cell vacuole in a double-membrane bound autophagosome, and subsequent degradation by lytic enzymes. Autophagy has generally been considered to be a non-selective mechanism of degradation. However, studies in yeast and animals have found numerous examples of selective autophagy, with cargo including proteins, protein aggregates, and organelles. Recent work has also provided evidence for several types of selective autophagy in plants. The degradation of protein aggregates was the first selective autophagy described in plants, and, more recently, a hybrid protein of the mammalian selective autophagy adaptors p62 and NBR1, which interacts with the autophagy machinery and may function in autophagy of protein aggregates, was described in plants. Other intracellular components have been suggested to be selectively targeted by autophagy in plants, but the current evidence is limited. Here, we discuss recent findings regarding the selective targeting of cell components by autophagy in plants.     

自体吞噬在植物生长发育中的功能

自体吞噬是一种细胞内自我降解系统,它能将植物细胞内溶物运输至液泡并降解.自体吞噬可划分为内溶物的包裹、运输至液泡、内溶物的降解和降解产物的重新利用几个连续步骤.关于细胞自体吞噬的认识主要来源于酵母、人类、小鼠、果蝇和线虫等生物,以拟南芥等为代表的植物细胞自体吞噬的研究虽然刚刚开始,但也取得了一些标志性的成果,且近十几年来已迅速成为植物研究领域的热点之一.自体吞噬在植物体内具有多种生理和病理作用,如对饥饿的适应、细胞内蛋白质和细胞器的清除、种子中贮藏蛋白的积累、抵制微生物、细胞死亡和胁迫响应等.本文在介绍自体吞噬形成过程的基础上,着重探讨了自体吞噬在植物生长发育中的功能,并对植物中自体吞噬的研究方向进行了展望.     

FURTHER OBSERVATIONS ON THE PHENOMENON OF SECONDARY VACUOLATION IN LIVING CELLS

Invagination of the plasma membrane in plant cells forms peripheral or endocytic structures which often contain a complement of membrane-bound vesicles. These structures, or secondary vacuoles, move with the streaming cytoplasm although their velocities are somewhat slower than that for the various organelles within the cytoplasm. They glide over the nucleus or flow from the peripheral cytoplasm onto a transvacuolar strand and continue unabated along the length of a strand. These structures may detach from the plasma membrane as sacs to become positioned in the cytoplasm directly under the tonoplast and project into the primary vacuole. Some endocytic vacuoles may separate from the peripheral cytoplasm and remain free within the primary vacuole; subsequently they can re-associate with the cytoplasm. While the content and function of these vacuoles are yet to be determined, indirect evidence indicates that they are pinocytic in character since the content of an invagination is confined to the sac upon its detachment from the plasma membrane and is subsequently transported throughout the cell by cyclosis.     

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.     

Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors

The Saccharomyces cerevisiae a-factor receptor (STE3) is subject to two modes of endocytosis: a constitutive process that occurs in the absence of ligand and a regulated process that is triggered by binding of ligand. Both processes result in delivery of the receptor to the vacuole for degradation. Receptor mutants deleted for part of the COOH- terminal cytoplasmic domain are disabled for constitutive, but not ligand-dependent internalization. Trans-acting mutants that impair constitutive endocytosis have been isolated. One of these, ren1-1, is blocked at a late step in the endocytic pathway, as receptor accumulates in a prevacuolar endosome-like compartment. REN1 is identical to VPS2, a gene required for delivery of newly synthesized vacuolar enzymes to the vacuole. Based on this identity, we suggest a model in which the transport pathways to the vacuole--the endocytic pathway and the vacuolar biogenesis pathway--merge at an intermediate endocytic compartment. As receptor also accumulates at the surface of ren1 cells, receptor may recycle from the putative endosome to the surface, or REN1 may also be required to carry out an early step in endocytosis.     

The Cvt pathway as a model for selective autophagy

Autophagy is a highly conserved, ubiquitous process that is responsible for the degradation of cytosolic components in response to starvation. Autophagy is generally considered to be non-selective; however, there are selective types of autophagy that use receptor and adaptor proteins to specifically isolate a cargo. One type of selective autophagy in yeast is the cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway is responsible for the delivery of the hydrolase aminopeptidase I to the vacuole; as such, it is the only known biosynthetic pathway that utilizes the core machinery of autophagy. Nonetheless, it serves as a model for the study of selective autophagy in other organisms.     

The molecular mechanism of autophagy

Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions. During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to a degradative organelle, the vacuole in yeast and the lysosome in mammalian cells, for breakdown and recycling. Autophagy is induced under starvation conditions and in mammalian cells is also invoked in response to specific hormones. In yeast, under nutrient-rich conditions, a constitutive biosynthetic pathway, termed the cytoplasm to vacuole targeting (Cvt) pathway, utilizes most of the same molecular machinery and topologically similar vesicles for the delivery of the resident hydrolase aminopeptidase I to the vacuole. Both autophagy and the Cvt pathway have been extensively studied and comprehensively reviewed in the past few years. In this review, we focus on the yeast system, which has provided most of the insight into the molecular mechanism of autophagy and the Cvt pathway, and highlight the most recent additions to our current knowledge of both pathways.     

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.     

Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Vid30 is required for the association of Vid vesicles and actin patches in the vacuole import and degradation pathway

When Saccharomyces cerevisiae is starved of glucose, the gluconeogenic enzymes fructose-1,6-bisphosphatase (FBPase), malate dehydrogenase (MDH2), isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are induced. However, when glucose is added to prolonged starved cells, these enzymes are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. Recent evidence suggests that the Vid pathway merges with the endocytic pathway at actin patches where endocytic vesicles are formed. The convergence of the Vid pathway with the endocytic pathway allows cells to remove intracellular and extracellular proteins simultaneously. However, the genes that regulate this step of the convergence have not been identified previously. Here we show that VID30 plays a critical role for the association of Vid vesicles and actin patches. Vid30 is constitutively expressed and interacts with Vid vesicle proteins Vid24 and Sec28 but not with the cargo protein FBPase. In the absence of SEC28 or VID24, Vid30 association with actin patches was prolonged. In cells lacking the VID30 gene, FBPase and Vid24 were not localized to actin patches, suggesting that Vid30 has a role in the association of Vid vesicles and actin patches. Vid30 contains a LisH and a CTLH domain, both of which are required for FBPase degradation. When these domains were deleted, FBPase trafficking to the vacuole was impaired. We suggest that Vid30 also has a role in the Vid pathway at a later step in a process that is mediated by the LisH and CTLH domains.     

Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non‐selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy‐dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2‐type ribonuclease, generating 3′‐NMPs that were immediately converted to nucleosides by the vacuolar non‐specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re‐assimilated, but excreted from the cell. Bulk non‐selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.     

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.     

Quantitative regulation of vesicle formation in yeast nonspecific autophagy

In eukaryotic cells, autophagy is a degradative pathway necessary for the turnover of bulk cytoplasm. In yeast, this pathway also mediates the specific transport of a vacuolar hydrolase zymogen, precursor aminopeptidase (prApe1), from the cytoplasm to the vacuole. Autophagy is under precise regulation, not only qualitatively but also quantitatively, especially in the steps involved in the vesicle formation process. We have recently used a fluorescence microscopy-based method to study the stoichiometry of autophagy-related (Atg) proteins during different conditions. This analysis shows that increased expression of Atg11 in the cytoplasm to vacuole targeting (Cvt) pathway increases the amount of this protein localized at the phagophore assembly site (PAS). In turn, under nutrient-rich conditions, the increased level of Atg11 causes the recruitment of higher than normal levels of Atg8 and Atg9 to the PAS, resulting in the formation of more Cvt vesicles, whereas the vesicle size is not affected. Combined with results from previous studies in starvation conditions, in this addendum we discuss the possible role of Atg8 and Atg9 in quantitatively regulating the vesicle formation process.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Ordering of compartments in the yeast endocytic pathway

We have characterized the morphology of the yeast endocytic pathway leading from the plasma membrane to the vacuole by following the trafficking of positively charged nanogold in combination with compartment identification using immunolocalization of t-SNARE proteins. The first endocytic compartment, termed the early/recycling endosome, contains the t-SNARE, Tlg1p. The next compartment, the prevacuolar compartment, contains Pep12p. After transport to the prevacuolar compartment, where vacuolar enzymes are seen on their way to the vacuole, endocytic content is delivered to the late endosome and on to the vacuole, both of which are devoid of Pep12p immunolabel. Traffic to the prevacuolar compartment is reduced in strains mutant for the Rab5 homologs, Vps21p, Ypt52p, and Ypt53p and in vps27 mutant cells. On the other hand, traffic to the early recycling endosome is less dependent on Rab5 homologs and does not require Vps27p.