652875
Autophagic Tubes
Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle.
20150695
Mechanisms behind the organization of membranous organelles in cells.
Electron microscopy (EM) promotes the notion that organelles are relatively static entities that occupy definable territories in the cell and have rather specific forms. In this view, transfer of membrane between organelles is thought to occur via shuttle-vesicles that bud from one organelle and move to the other to fuse with it, net membrane balance being achieved by a postulated return of shuttle-vesicles in the opposite direction. The biochemical individuality of the interacting compartments is thought to be maintained by a filter at the point of shuttlevesicle formation, while directionality in membrane flow is achieved by cellular controls on vesicle movement and fusion.
30752342
Coat Proteins and Vesicle Budding
The trafficking of proteins within eukaryotic cells is achieved by the capture of cargo and targeting molecules into vesicles that bud from a donor membrane and deliver their contents to a receiving compartment. This process is bidirectional and may involve multiple organelles within a cell. Distinct coat proteins mediate each budding event, serving both to shape the transport vesicle and to select by direct or indirect interaction the desired set of cargo molecules. Secretion, which has been viewed as a default pathway, may require sorting and packaging signals on transported molecules to ensure their rapid delivery to the cell surface.
535165
Dynamic shape changes of cytoplasmic organelles translocating along microtubules
Transient shape changes of organelles translocating along microtubules are directly visualized in thinly spread cytoplasmic processes of the marine foraminifer. Allogromia laticollaris, by a combination of high- resolution video-enhanced microscopy and fast-freezing electron microscopy. The interacting side of the organelle flattens upon binding to a microtubule, as if to maximize contact with it. Organelles typically assume a teardrop shape while moving, as if they were dragged through a viscous medium. Associated microtubules bend around attachments of the teardrop-shaped organelles, suggesting that they too are acted on by the forces deforming the organelles. An 18-nm gap between the organelles and the microtubules is periodically bridged by 10-nm-thick cross-bridge structures that may be responsible for the binding and motive forces deforming organelles and microtubules.
12519499
The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts
The formation of a dynamic tubulovesicular membrane network that resembles the endoplasmic reticulum (ER) has been observed in extracts of cultured chick embryo fibroblasts (CEF cells) using video-enhanced differential interference contrast microscopy. Initially, membranes in the CEF extracts appeared amorphous and aggregated, but with time, membrane tubules moved out along stationary microtubules. The membrane tubules formed new branches on intersecting microtubules and fused with other branches to form a network of interconnected polygons. The tubulovesicular network was solubilized by detergent and took on a beaded morphology in a hypotonic buffer. Formation of the tubulovesicular network required ATP and microtubules. The network did not contain elements of the plasma membrane, Golgi apparatus, or mitochondria but could be labeled with ER markers. We suggest that the tubulovesicular network contains components from the ER and is formed by membrane associated motors moving upon microtubules in a process we call microtubule-dependent tethering.
20727220
Mitochondrial dynamics in yeast.
Proteins that control mitochondrial dynamics in yeast are being identified at a rapid pace. These proteins include cytoskeletal elements that regulate organelle distribution and inheritance and several outer membrane proteins that are required to maintain the branched, mitochondrial reticulum. Interestingly, three of the high molecular weight GTPases encoded by the yeast genome are required for mitochondrial integrity and are potential regulators of mitochondrial branching, distribution, and membrane fusion. The recent finding that mtDNA mixing is restricted in the mitochondrial matrix has stimulated the hunt for the molecular machinery that anchors mitochondrial nucleoids in the organelle. Considering that many aspects of mitochondrial structure and behavior are strikingly similar in different cell types, the functional analyses of these yeast proteins should provide general insights into the mechanisms governing mitochondrial dynamics in all eukaryotes.
20234969
Lysosomes, a meeting point of proteins, chaperones, and proteases
Abstract Lysosomes, classically considered as nonspecific systems for protein degradation, have recently also been shown to be able selectively to degrade specific intracellular proteins. Here we review this selective pathway of lysosomal protein degradation that involves cytosolic and intralysosomal chaperones and a receptor in the lysosomal membrane. This pathway is highly selective for cytosolic proteins containing a lysosomal targeting signal. The selective lysosomal degradation pathway is active under conditions of nutrient deprivation and plays an important role in the regulation of intracellular protein levels in stress situations. Several physiological and pathological modifications in the activity of this selective lysosomal pathway of protein degradation are discussed.
15776985
Import into and degradation of cytosolic proteins by isolated yeast vacuoles.
In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.
38736746
Protein traffic in the yeast endocytic and vacuolar protein sorting pathways.
Endocytosis is a fundamental membrane trafficking event that occurs in all eukaryotes. The yeast Saccharomyces cerevisiae has been particularly useful in efforts to uncover novel proteins that mediate endocytosis, and many of these factors share similarity with proteins from higher eukaryotes. In the past two years, progress has centered on three major areas: modifications/signaling pathways that initiate or regulate internalization, protein complexes that are implicated in the internalization process, and factors that are involved in regulation of traffic through late endosomal compartments. As the parallels between the mechanisms employed in yeast and higher eukaryotes are further explored, new insights into the complex process of endocytosis should emerge.
6405331
Identification of Novel Vesicles in the Cytosol to Vacuole Protein Degradation Pathway
The key gluconeogenic enzyme, fructose1,6-bisphosphatase (FBPase), is induced when Saccharomyces cerevisiae are starved of glucose. FBPase is targeted from the cytosol to the yeast vacuole for degradation when glucose-starved cells are replenished with fresh glucose. Several vid mutants defective in the glucose-induced degradation of FBPase in the vacuole have been isolated. In some vid mutants, FBPase is found in punctate structures in the cytoplasm. When extracts from these cells are fractionated, a substantial amount of FBPase is sedimentable in the high speed pellet, suggesting that FBPase is associated with intracellular structures in these vid mutants. In this paper we investigated whether FBPase association with intracellular structures also existed in wild-type cells. We report the purification of novel FBPase-associated vesicles from wild-type cells to near homogeneity. Kinetic studies indicate that FBPase association with these vesicles is stimulated by glucose and occurs only transiently, suggesting that these vesicles are intermediate in the FBPase degradation pathway. Fractionation analysis demonstrates that these vesicles are distinct from known organelles such as the vacuole, ER, Golgi, mitochondria, peroxisomes, endosomes, COPI, or COPII vesicles. Under EM, these vesicles are 30–40 nm in diam. Proteinase K experiments indicate that the majority of FBPase is sequestered inside the vesicles. We propose that FBPase is imported into these vesicles before entering the vacuole.
37472218
Delivery of proteins and organelles to the vacuole from the cytoplasm.
The vacuole/lysosome is a primary catabolic site in the eukaryotic cell. One implication of its cellular role is that delivery systems must exist to target both hydrolytic enzymes and substrates destined for degradation to this organelle. A number of nonclassical vacuolar targeting pathways that deliver degradative substrates and at least one resident enzyme from the cytosol to the vacuole have recently been described. The pathways identified so far include cytoplasm to vacuole targeting, macroautophagy, pexophagy and vacuolar import and degradation. Cytological, genetic and molecular genetic approaches have begun to provide insight into the molecular basis of these processes.
10331564
Aminopeptidase I Is Targeted to the Vacuole by a Nonclassical Vesicular Mechanism
The yeast vacuolar protein aminopeptidase I (API) is synthesized as a cytosolic precursor that is transported to the vacuole by a nonclassical targeting mechanism. Recent genetic studies indicate that the biosynthetic pathway that transports API uses many of the same molecular components as the degradative autophagy pathway. This overlap coupled with both in vitro and in vivo analysis of API import suggested that, like autophagy, API transport is vesicular. Subcellular fractionation experiments demonstrate that API precursor (prAPI) initially enters a nonvacuolar cytosolic compartment. In addition, subvacuolar vesicles containing prAPI were purified from a mutant strain defective in breakdown of autophagosomes, further indicating that prAPI enters the vacuole inside a vesicle. The purified subvacuolar vesicles do not appear to contain vacuolar marker proteins. Immunogold EM confirms that prAPI is localized in cytosolic and in subvacuolar vesicles in a mutant strain defective in autophagic body degradation. These data suggest that cytosolic vesicles containing prAPI fuse with the vacuole to release a membrane-bounded intermediate compartment that is subsequently broken down, allowing API maturation.
21017998
Autophagy and other vacuolar protein degradation mechanisms
Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject tofeedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive α1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a ‘salvage compartment’ closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins roturn to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In thetrans-Golgi network some proteins are proteolytically processed by Ca2+-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occurr in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.
22920849
Genetic and Phenotypic Overlap between Autophagy and the Cytoplasm to Vacuole Protein Targeting Pathway*
We have explored the phenotypic and genetic overlap between autophagocytosis and cytoplasm to vacuole targeting in the yeast Saccharomyces cerevisiae. Complementation analysis was performed with mutants in each of these groups (aut and cvt, respectively), and three complementation groups were found to overlap. Also, most of the unique aut mutants accumulated precursor aminopeptidase I in the cytoplasm, while maintaining wild type kinetics and maturation of proteins targeted to the vacuole via the secretory pathway. The majority of the non-overlapping cvt mutants were found to be at least partially defective in autophagy. Some mutants in each group, however, appear to be only marginally affected in the other phenotype, implying that these pathways only partially overlap. We propose that import of aminopeptidase I into the vacuole shares a number of components required for bulk autophagocytosis, but is made specific, saturable, and constitutive by the presence of a receptor or other interacting protein(s).
9971712
Cytoplasm-to-vacuole targeting and autophagy employ the same machinery to deliver proteins to the yeast vacuole.
The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.
6277618
A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole
Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Δ mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Δ mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.
12234779
Degradation and turnover of peroxisomes in the yeast Hansenula polymorpha induced by selective inactivation of peroxisomal enzymes
Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth. After transfer of methanol-grown cells into media containing glucose — a substrate that fully represses alcohol oxidase synthesis — the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein. The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer. This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded. Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment. Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes. The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis. Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment. The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment. The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins. The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle. Peroxisomes of methanolgrown H. polymorpha were degraded individually. In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome. However, fusions of autophagic vacuoles in one cell were frequently observed. After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.
11095691
Selective autophagy of peroxisomes in methylotrophic yeasts.
The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha respond to a methanol substrate by synthesizing peroxisomal enzymes resulting in the formation of large peroxisomes. When the carbon source was changed from methanol to glucose, we observed a rapid loss of peroxisomes. In this comparative study, we utilized biochemical and morphological techniques to characterize the loss of peroxisomes in these yeasts. We used metabolic labeling and chase procedures to evaluate whether this loss was due to suppressed synthesis or enhanced degradation. The synthesis of alcohol oxidase was depressed 10-fold when cultures grown in methanol attained stationary growth. However, no further reduction of synthesis was observed upon transfer of these cultures to glucose medium. In stationary phase cultures maintained in methanol, two peroxisomal proteins, alcohol oxidase and dihydroxyacetone synthase, were degraded with a half-life of over 3 h. However, within 3 h of glucose repression, as much as 80% of the radiolabeled peroxisomal proteins were lost from both yeasts. This glucose-mediated degradative event appeared to be specific for peroxisomal proteins, since mitochondrial proteins were stable. Ultrastructural examination of both yeasts revealed that glucose induced the sequestration of peroxisomes into the yeast vacuole, the presumed site of degradation. These results suggest that peroxisome loss during glucose repression is due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.
26673445
Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris.
The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroautophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.
46017791
Isolation and characterization of autophagy‐defective mutants of Saccharomyces cerevisiae
Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase‐deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild‐type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.
26072787
Isolation of autophagocytosis mutants of Saccharomyces cerevisiae
Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.
37983564
Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha
We have isolated a collection of peroxisome degradation-deficient (Pdd-) mutants of the yeast Hansenula polymorpha which are impaired in the selective autophagy of alcohol oxidase-containing peroxisomes. Two genes, designated PDD1 and PDD2, have been identified by complementation and linkage analyses. In both mutant strains, the glucose-induced proteolytic turnover of peroxisomes is fully prevented. The pdd1 and pdd2 mutant phenotypes were caused by recessive monogenic mutations. Mutations mapped in the PDD1 gene appeared to affect the initial step of peroxisome degradation, namely, sequestration of the organelle to be degraded by membrane multilayers. Thus, Pdd1p may be involved in the initial signalling events which determine which peroxisome will be degraded. The product of the PDD2 gene appeared to be essential for mediating the second step in selective peroxisome degradation, namely, fusion and subsequent uptake of the sequestered organelles into the vacuole. pdd1 and pdd2 mutations showed genetic interactions which suggested that the corresponding gene products may physically or functionally interact with each other.
24520138
Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein.
Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.
38296007
The use of low temperature X‐ray diffraction to evaluate freezing methods used in freeze‐fracture electron microscopy
Two methods of freezing samples for freeze‐fracture electron microscopy have been compared using X‐ray diffraction and freeze‐fracture results from lipid‐water model systems. Perturbations of the molecular organization of hydrocarbon chains and the extent of ice crystal formation have been evaluated for lamellar phases of egg lecithin containing 16% water and egg lecithin‐phosphatidylinositol containing 55% water, both freeze‐quenched in liquid Freon‐22 near its melting temperature (113 K). Very thin samples sandwiched between copper sheets separated by an electron microscope grid show much less freezing induced structural rearrangement than small samples contained on conventional Balzers‐type gold planchettes. These results show that the rate of freezing in the very thin preparations is greater than in the conventional ones, which is probably due in part to the improved dissipation of heat from a poorly conductive sample through highly conductive copper sheets.
15495893
A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast
We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.
565634
Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction
For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.
37396404
Analysis of the membrane structures involved in autophagy in yeast by freeze-replica method.
Under starvation conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components by forming autophagosomes with double membrane in the cytoplasm. The autophagosome then fuses with the vacuolar membrane and delivers its own cytoplasmic components into the vacuole in the form of an autophagic body with a single membrane (Baba, M., Takeshige, K., Baba, N., and Ohsumi, Y. 1994. J. Cell Biol., 124: 903-913). We examined membrane structures involved in the autophagy induced by nitrogen-starvation by using freeze-replica method. The most conspicuous characteristic of the autophagic body is that the intramembrane particles were rarely detected on either the protoplasmic or exoplasmic face of its fracture membrane. This morphological feature of the fractured face was clearly different from other intracellular organelles. Next we examined the autophagosomal membrane. The inner membrane of autophagosome was also intramembrane particle-free, and its morphological feature was identical to the membrane of autophagic body. At the fusion site between autophagosome and vacuole we obtained direct evidence that two different membranes, the outer membrane of autophagosome and vacuolar membrane, became continuous by using freeze-etching technique. From these results we concluded that the autophagic body originated from the inner membrane of the autophagosome, and its membrane reflects an intrinsic feature of autophagosomal membrane. The outer membrane of autophagosome had only a few intramembrane particles and may be differentiated from the inner membrane. In cells under nitrogen-starvation condition, the density of intramembrane particles of vacuolar membrane decreased beyond that in control cells.
12967086
Fab1p PtdIns(3)P 5-Kinase Function Essential for Protein Sorting in the Multivesicular Body
Sorting of signal-transducing cell surface receptors within multivesicular bodies (MVBs) is required for their rapid down-regulation and degradation within lysosomes. Yeast mutants defective in late stages of transport to the vacuole/lysosome accumulate MVBs. We demonstrate that the membrane glycoprotein carboxypeptidase S and the G protein-coupled receptor Ste2p are targeted into the vacuole lumen, and this process requires a subset of VPS gene products essential for normal endosome function. The PtdIns(3)P 5-kinase activity of Fab1p, which converts the product of the Vps34p PtdIns 3-kinase PtdIns(3)P into PtdIns(3,5)P2, also is required for cargo-selective sorting into the vacuole lumen. These findings demonstrate a role for phosphoinositide signaling at distinct stages of vacuolar/lysosomal protein transport and couple PtdIns(3,5)P2 synthesis to regulation of MVB sorting.
161746
Phosphoinositide signaling and turnover: PtdIns(3)P, a regulator of membrane traffic, is transported to the vacuole and degraded by a process that requires lumenal vacuolar hydrolase activities
The Golgi/endosome‐associated Vps34 phosphatidylinositol 3‐kinase is essential for the sorting of hydrolases from the Golgi to the vacuole/lysosome. Upon inactivation of a temperature‐conditional Vps34 kinase, cellular levels of PtdIns(3)P rapidly decrease and it has been proposed that this decrease is due to the continued turnover of PtdIns(3)P by cytoplasmic phosphatases. Here we show that mutations in VAM3 (vacuolar t‐SNARE) and YPT7 (rab GTPase), which are required to direct protein and membrane delivery from prevacuolar endosomal compartments to the vacuole, dramatically increase/stabilize PtdIns(3)P levels in vivo by disrupting its turnover. We find that the majority of the total pool of PtdIns(3)P which has been synthesized, but not PtdIns(4)P, requires transport to the vacuole in order to be turned over. Unexpectedly, strains with impaired vacuolar hydrolase activity accumulate 4‐ to 5‐fold higher PtdIns(3)P levels than wild‐type cells, suggesting that lumenal vacuolar lipase and/or phosphatase activities degrade PtdIns(3)P. Because vacuolar hydrolases act in the lumen, PtdIns(3)P is likely to be transferred from the cytoplasmic membrane leaflet where it is synthesized, to the lumen of the vacuole. Interestingly, mutants that stabilize PtdIns(3)P accumulate small uniformly‐sized vesicles (40–50 nm) within prevacuolar endosomes (multivesicular bodies) or the vacuole lumen. Based on these and other observations, we propose that PtdIns(3)P is degraded by an unexpected mechanism which involves the sorting of PtdIns(3)P into vesicles generated by invagination of the limiting membrane of the endosome or vacuole, ultimately delivering the phosphoinositide into the lumen of the compartment where it can be degraded by the resident hydrolases.
1778700
Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment
A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.
16322524
Ultrastructural analysis of the autophagic process in yeast: detection of autophagosomes and their characterization
Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze- substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane- structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.
27163768
Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway.
Organelle biogenesis and turnover are necessary to maintain biochemical processes that are appropriate to the needs of the eukaryotic cell. Specific degradation of organelles in response to changing environmental cues is one aspect of achieving proper metabolic function. For example, the yeast Saccharomyces cerevisiae adjusts the level of peroxisomes in response to differing nutritional sources. When cells are grown on oleic acid as the sole carbon source, peroxisome biogenesis is induced. Conversely, a subsequent shift to glucose-rich or nitrogen-limiting conditions results in peroxisome degradation. The degradation process, pexophagy, requires the activity of vacuolar hydrolases. In addition, peroxisome degradation is specific. Analyses of cellular marker proteins indicate that peroxisome degradation under these conditions occurs more rapidly and to a greater extent than mitochondrial, Golgi, or cytosolic protein delivery to the vacuole by the non-selective autophagy pathway. To elucidate the molecular mechanism of selective peroxisome degradation, we examined pexophagy in mutants that are defective in autophagy (apg) and the selective targeting of aminopeptidase I to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Inhibition of peroxisome degradation in cvt and apg mutants indicates that these pathways overlap and that peroxisomes are delivered to the vacuole by a mechanism that utilizes protein components of the Cvt/autophagy pathways.
29190629
Apg7p/Cvt2p is required for the cytoplasm-to-vacuole targeting, macroautophagy, and peroxisome degradation pathways.
Proper functioning of organelles necessitates efficient protein targeting to the appropriate subcellular locations. For example, degradation in the fungal vacuole relies on an array of targeting mechanisms for both resident hydrolases and their substrates. The particular processes that are used vary depending on the available nutrients. Under starvation conditions, macroautophagy is the primary method by which bulk cytosol is sequestered into autophagic vesicles (autophagosomes) destined for this organelle. Molecular genetic, morphological, and biochemical evidence indicates that macroautophagy shares much of the same cellular machinery as a biosynthetic pathway for the delivery of the vacuolar hydrolase, aminopeptidase I, via the cytoplasm-to-vacuole targeting (Cvt) pathway. The machinery required in both pathways includes a novel protein modification system involving the conjugation of two autophagy proteins, Apg12p and Apg5p. The conjugation reaction was demonstrated to be dependent on Apg7p, which shares homology with the E1 family of ubiquitin-activating enzymes. In this study, we demonstrate that Apg7p functions at the sequestration step in the formation of Cvt vesicles and autophagosomes. The subcellular localization of Apg7p fused to green fluorescent protein (GFP) indicates that a subpopulation of Apg7pGFP becomes membrane associated in an Apg12p-dependent manner. Subcellular fractionation experiments also indicate that a portion of the Apg7p pool is pelletable under starvation conditions. Finally, we demonstrate that the Pichia pastoris homologue Gsa7p that is required for peroxisome degradation is functionally similar to Apg7p, indicating that this novel conjugation system may represent a general nonclassical targeting mechanism that is conserved across species.
33393088
Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy.
In the yeast Saccharomyces cerevisiae, the Apg12p-Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc-tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p-Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p-Apg5p conjugation.
36646805
Functions of lipid rafts in biological membranes.
Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties. Sphingolipid and cholesterol-rich domains may exist as phase-separated "rafts" in the membrane. We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments. We also discuss possible functions of lipid rafts in membranes. Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts. Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons. Rafts may also function in sorting and trafficking through the secretory and endocytic pathways.
4322776
A lipid associated with the antiphospholipid syndrome regulates endosome structure and function
Little is known about the structure and function of membrane domains in the vacuolar apparatus of animal cells. A unique feature of late endosomes, which are part of the pathway that leads to lysosomes, is that they contain a complex system of poorly characterized internal membranes in their lumen. These endosomes are therefore known as multivesicular or multilamellar organelles,. Some proteins distribute preferentially within these internal membranes, whereas others are exclusively localized to the organelle's limiting membrane. The composition and function of this membrane system are poorly understood. Here we show that these internal membranes contain large amounts of a unique lipid, and thus form specialized domains within endosomes. These specialized domains are involved in sorting the multifunctional receptor for insulin-like growth factor 2 and ligands bearing mannose-6-phosphate, in particular lysosomal enzymes. We also show that this unique lipid is a specific antigen for human antibodies associated with the antiphospholipid syndrome,. These antibodies may act intracellularly by altering the protein-sorting functions of endosomes.
8821036
Cell-Free Reconstitution of Microautophagic Vacuole Invagination and Vesicle Formation
Many organelles change their shape in the course of the cell cycle or in response to environmental conditions. Lysosomes undergo drastic changes of shape during microautophagocytosis, which include the invagination of their boundary membrane and the subsequent scission of vesicles into the lumen of the organelle. The mechanism driving these structural changes is enigmatic. We have begun to analyze this process by reconstituting microautophagocytosis in a cell-free system. Isolated yeast vacuoles took up fluorescent dyes or reporter enzymes in a cytosol-, ATP-, and temperature-dependent fashion. During the uptake reaction, vacuolar membrane invaginations, called autophagic tubes, were observed. The reaction resulted in the transient formation of autophagic bodies in the vacuolar lumen, which were degraded upon prolonged incubation. Under starvation conditions, the system reproduced the induction of autophagocytosis and depended on specific gene products, which were identified in screens for mutants deficient in autophagocytosis. Microautophagic uptake depended on the activity of the vacuolar ATPase and was sensitive to GTPγS, indicating a requirement for GTPases and for the vacuolar membrane potential. However, microautophagocytosis was independent of known factors for vacuolar fusion and vesicular trafficking. Therefore, scission of the invaginated membrane must occur via a novel mechanism distinct from the homotypic fusion of vacuolar membranes.