Unique phenotype of opaque cells in the white-opaque transition of Candida albicans.

Select strains of Candida albicans switch reversibly and at extremely high frequency between a white and an opaque colony-forming phenotype, which has been referred to as the white-opaque transition. Cells in the white phase exhibit a cellular phenotype indistinguishable from that of most standard strains of C. albicans, but cells in the opaque phase exhibit an unusually large, elongate cellular shape. In comparing the white and opaque cellular phenotypes, the following findings are demonstrated. (i) The surface of the cell wall of maturing opaque cells when viewed by scanning electron microscopy exhibits a unique pimpled, or punctate, pattern not observed in white cells or standard strains of C. albicans. (ii) The dynamics of actin localization which accompanies opaque-cell growth first follows the pattern of budding cells during early opaque-bud growth and then the pattern of hypha-forming cells during late opaque-bud growth. (iii) A hypha-specific cell surface antigen is also expressed on the surface of opaque budding cells. (iv) An opaque-specific surface antigen is distributed in a punctate pattern.     

The morphological study of the cysts Pelomyxa palustris Greeff, 1874

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.     

HOPS Interacts with Apl5 at the Vacuole Membrane and Is Required for Consumption of AP-3 Transport Vesicles

Adaptor protein complexes (APs) are evolutionarily conserved heterotetramers that couple cargo selection to the formation of highly curved membranes during vesicle budding. In Saccharomyces cerevisiae, AP-3 mediates vesicle traffic from the late Golgi to the vacuolar lysosome. The HOPS subunit Vps41 is one of the few proteins reported to have a specific role in AP-3 traffic, yet its function remains undefined. We now show that although the AP-3 δ subunit, Apl5, binds Vps41 directly, this interaction occurs preferentially within the context of the HOPS docking complex. Fluorescence microscopy indicates that Vps41 and other HOPS subunits do not detectably colocalize with AP-3 at the late Golgi or on post-Golgi (Sec7-negative) vesicles. Vps41 and HOPS do, however, transiently colocalize with AP-3 vesicles when these vesicles dock at the vacuole membrane. In cells with mutations in HOPS subunits or the vacuole SNARE Vam3, AP-3 shifts from the cytosol to a membrane fraction. Fluorescence microscopy suggests that this fraction consists of post-Golgi AP-3 vesicles that have failed to dock or fuse at the vacuole membrane. We propose that AP-3 remains associated with budded vesicles, interacts with Vps41 and HOPS upon vesicle docking at the vacuole, and finally dissociates during docking or fusion.     

In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transporters and GST

In cells, anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum, and are then transported and finally accumulated inside the vacuole. In Vitis vinifera (grapevine), two kinds of molecular actors are putatively associated with the vacuolar sequestration of anthocyanins: a glutathione-S-transferase (GST) and two MATE-type transporters, named anthoMATEs. However, the sequence of events by which anthocyanins are imported into the vacuole remains unclear. We used MYBA1 transformed hairy roots as a grapevine model tissue producing anthocyanins, and took advantage of the unique autofluorescence of anthocyanins to study their cellular trafficking. In these tissues, anthocyanins were not only visible in the largest vacuoles, but were also present at higher concentrations in several vesicles of different sizes. In the cell, small vesicles actively moved alongside the tonoplast, suggesting a vesicular trafficking to the vacuole. Subcellular localization assays revealed that anthoMATE transporters were closely related with these small vesicles, whereas GST was localized in the cytoplasm around the nucleus, suggesting an association with the endoplasmic reticulum. Furthermore, cells in hairy roots expressing anthoMATE antisense did not display small vesicles filled with anthocyanins, whereas in hairy roots expressing GST antisense, anthocyanins were accumulated in vesicles but not in the vacuole. This suggests that in grapevine, anthoMATE transporters and GST are involved in different anthocyanin transport mechanisms.     

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.     

Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation.

Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.     

ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching

Immunoscreening of a Candida albicans expression library resulted in the isolation of a novel gene encoding a 32.9-kDa polypeptide (288 amino acids), with 27.7% homology to the product of Saccharomyces cerevisiae YGR106c, a putative vacuolar protein. Heterozygous mutants in this gene displayed an altered budding growth pattern, characterized by the formation of chains of buds, decreasingly in size towards the apex, without separation of the daughter buds. Consequently, this gene was designated ABG1. A conditional mutant for ABG1 with the remaining allele under the control of the MET3 promoter did not grow in the presence of methionine and cysteine, demonstrating that ABG1 was essential for viability. Western analysis revealed the presence of a major 32.9-kDa band, mainly in a particulate fraction (P40) enriched in vacuoles, and tagging with green fluorescent protein confirmed that Abg1p localized to the vacuole. Vacuole inheritance has been linked to the regulation of branching frequency in C. albicans. Under repressing conditions, the conditional mutant had an increased frequency of branching under hyphal inducing conditions and an altered sensitivity to substances that interfered with cell wall assembly. Repression of ABG1 in the conditional mutant strain caused disturbance of normal size and number of vacuoles both in yeast and mycelial cells and also in the asymmetric vacuole inheritance associated with the characteristic pattern of germ tubes and branching in C. albicans. These observations indicate that ABG1 plays a key role in vacuole biogenesis, cytokinesis, and hyphal branching.     

FINE STRUCTURE AND HISTOCHEMISTRY OF VESICLE CELLS OF THE RED ALGA ANTITHAMNION DEFECTUM (CERAMIACEAE)1

The ultrastructure and histochemistry of the refractile, vesiculate cells (“blasenzellen,”“cellules secretrices,”“gland cells”) of Antithamnion defectum Kylin were examined. The refringent vacuolar contents disclosed two components of differing density: an electron opaque, proteinaceous matrix material surrounding cores of irregularly shaped, less opaque material. The cores contain less protein and more unknown material than the matrix. Part or all of the vacuolar material is synthesized by abundant rough endoplasmic reticulum (ER) and deposited in smooth surfaced cisternae that swell to form vesicles. Mitochondria are usually associated with stacks of the swelling cisternae. The vesicles enlarge by continued deposition of synthesized material and coalescence with other vesicles. All vesicles eventually coalesce to form the mature vacuole. A crystalline array of fibrils develops in the cytoplasm during later stages of vacuole enlargement. The crystal contains a sulfated, acidic polysaccharidic material. The chloroplasts, if present, and nucleus degenerate at vacuole maturity. Active release of the vacuolar material does not occur, and organelles for extracellular secretion are not present. Structural evidence suggests a storage, rather than secretory, function for the cells.     

Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway

Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins.     

An electron microscopical study of absorbing cells in the posterior caput epididymidis of rabbits

Summary The columnar cells in regions 3 and 4 of the ductus epididymidis in rabbits display ultrastructural features characteristic of absorbing cells. The stereocilia show basal anastomoses and often a fibrillar core continuous with a fibrillar web in the apical cytoplasm. Numerous invaginations of the slightly downy apical cell membrane and many thick-walled apical vesicles and vacuoles contain an opaque substance similar to that seen in the lumen. The vacuoles often contain small vesicles or bodies, probably formed from the vacuolar wall by budding. Numerous bodies or vacuoles with moderately dense contents are seen in the Golgi area and in the supranuclear and intranuclear cytoplasm in region 3. In region 4 they are denser and mainly seen above the nucleus. A high acid phosphatase activity was demonstrated in most dense and some light bodies. India ink introduced by way of the rete testis was taken up from the lumen into apical invaginations, vesicles and vacuoles and slowly transferred to denser bodies below the Golgi apparatus.These observations are interpreted as evidence for a resorption of substances from the lumen by a pinocytotic process, and for their storage and perhaps digestion in the dense bodies, which appear to have a lysosomal character. The Golgi apparatus is large with many vesicles of two types and empty cisternae but few typical Golgi vacuoles. The partly granular endoplasmic reticulum is very well developed and has opaque contents. Microtubules run from the terminal bar region into the Golgi area. Thick-walled vesicles occur throughout the cytoplasm, sometimes in continuity with the cell membrane. The basal parts of the cell borders often interdigitate.Supported by a grant from the Swedish State Medical Research Council.     

The Structure and Cytochemistry of the Pistil of Hypericum calycinum: The Stigma

The pistil of Hypericum calycinum has a pentacarpellary, syncarpousovary with five slender styles, each terminating in a smallstigma. The stigma is dry and papillate with a thin lining ofpellicle. The cuticle is thin and continuous around the papillae.A large vacuole filled with tannins occupies the major partof the papillae and the cytoplasm forms a thin lining aroundthe vacuole. The cell wall of the mature papillae show two distinctlayers - an outer layer of loosely woven fibrils and an innerdenser layer with compact fibrils. A large number of small lipoidalbodies accumulate just below the cuticle. The papillae havefewer organelles than those typical of glandular cells. Dictyosomesobserved occasionally are without associated vesicles. The cytoplasmis rich in ribosomes. The basal portions of the papillae mergeinto the transmitting tissue made up of loosely arranged cells.The intercellular matrix of the transmitting tissue is richin lipids. Pollen grains are deposited between the papillae.Upon pollen germination, pollen tubes enter the stigma throughthe interstices between the papillae Hypericum calycinum, cytochemistry, pistil, pollen-pistil interaction, stigma, ultrastructure     

The mechanics of budding and copulation in saccharomyces

The yeast cell contains a nucleus whose rigid centrosome carries a band of Feulgen-positive chromatin (centrochromatin) on its surface. The first step in budding is the formation of the bud by an extension of the centrosome over which the cell wall persists. Next the nuclear vacuole extends a process into the bud which contains the chromosomes. Finally the centrochromatin divides directly and the cells separate; a plug either of centrosome or cytoplasm sealing the bud pore. The cytoplasm, the centrosome, the centrochromatin and the nuclear wall are autonomous non genic organelles which never originate de novo.Copulation is the reverse of budding. The centrosomes fuse first; the cytoplasms mix; the nuclear vacuoles fuse by processes which travel along the fused centrosomes; and finally the centrochromatins fuse to form a single band.Figures 1–12. Drawings of budding yeast cells fixed in Schaudinn's fluid and stained with iron alum hemotoxylin, mounted in balsam. The cell wall is not visible due to the clearing action of the balsam. Except for Figure 5, the chromosomes and the nucleolus in the nuclear vacuole have been completely destained. The bud scar described by Barton is shown clearly at the end of the cell distal from the centrosome. The nuclear vacuole is usually forced into the extrusion formed by the bud scar. Since the cell wall is not visible, the plug of material connecting bud and mother cell as shown in Figure 12, fits into the cell wall and probably corresponds to the plug in the bud scar described by Barton. The details of the budding process are described in the text.Figures 13–18. Copulating yeast cells stained with Barrett's hemotoxylin and aceto-orcein and mounted in the stain. Chromosomes are visible in the nuclear vacuoles. The centrosome is usually visible and often appears to have a core which stains differentially. Except in Figure 16, the centrochromatin is visible as darkly stained material; in some cases surrounded by a clear zone. The “thick waisted” form of the cells identifies them as derived from recent copulations and distinguishes them from budding cells. The process of copulation is discussed in the text.     

Cytoplasmic deletion processes during spermatogenesis in mosses

Comparative ultrastructural observations reveal that cytoplasmic deletion during spermatogenesis in Sphagnum and other mosses (Bryopsida) has two distinct phases. In young spermatids, Golgi-derived vesicles produce the mucopolysaccharide sheaths in which the gametes are liberated. Golgi bodies, however, play no part in removal of cytoplasm during gamete maturation. Rounding off of the cells during this process results in a 50% reduction in volume. Mid-spermatid stages in Sphagnum are characterised by the sequential loss of Golgi bodies and endoplasmic reticulum (ER) but no further diminution of the cytoplasm. The final stages of nuclear metamorphosis and chromatin condensation, in late spermatids, are marked by the sudden appearance, in the otherwise featureless central cytoplasm, of a membrane vesicle complex (MVC) comprising cisternae, tubules, and smooth and coated vesicles. Following repositioning of the MVC beneath the plasma membrane, rapid shrinkage of the cytoplasm is associated with the presence of vesicle fusion profiles at the cell surface. The MVC is considered to be intimately involved in cytoplasmic breakdown and loss. Acid phosphatase activity can be detected throughout spermatogenesis. Spermatogenous cells and young spermatids possess relatively low levels of the enzyme, restricted to the ER and perinuclear space, but particularly high levels occur in the MVC region of late spermatids of Sphagnum. The deletion process in Bryopsida is much more gradual than that of Sphagnum. Mid-spermatids contain sheets of ER, Golgi with small vesicles, and irregular cisternae associated with coated vesicles. Vacuoles derived either from dilation of the ER or the coated vesicle complexes gradually increase in size and number at the expense of the cytoplasm. During the early stages of chromatin condensation, a large central vacuole opens onto the anterior face of the gametes. Further discharge of vesicles continues throughout gamete maturation. A comparative survey of spermatogenesis in land plants indicates that cytoplasmic deletion is achieved in different ways in different groups. We speculate that the spermatozoids of the common ancestor of archegoniate plants probably possessed large amounts of cytoplasm. The deletion mechanisms may have originated from a contractile vacuole apparatus.     

The Mode of Function of the Cytopharyngeal Basket of the Ciliate Pseudomicrothorax dubius

The ciliate Pseudomicrothorax dubius feeds on filamentous blue-green algae, ingesting them at rates of up to 15 μm per second, by means of a cytopharyngeal basket. The wall of the basket is composed of 22 ± 3 nemadesmata, each of which is a bundle of about 200 microtubules which are cross-linked in a hexagonal pattern. The lumen of the non-feeding basket is filled with cytoplasma into which project the nemadesmal lamellae. Each nemadesmal lamella is attached to a nemadesm and consists of a single row of 20–30 microtubules. Each microtubule of the nemadesmal lamella bears a row of pairs of arm-like projections which are embedded in a filamentous matrix. During feeding, the lumen of the basket is occupied by the developing food vacuole. The nemadesmal lamellae are observed between the vacuole membrane and the nemadesmata, and the arms of the nemadesmal lamellae microtubules are oriented toward the membrane of the food vacuole or of small vesicles. A mechanism for the generation of force for phagocytosis by means of the microtubule arms is proposed.
During food uptake the membrane of the food vacuole increases rapidly at rates up to 270 μm² per second. Vacuole growth results from the fusion of membrane-bound vesicles. During phagocytosis a fast streaming of these vesicles can be observed in the cytoplasm surrounding the basket. The direction of streaming is opposite to that of ingestion of the algal filament. The vesicles enter the lumen of the basket at its anterior end, in a zone where the wall of the basket is perforated.     

Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.     

N-acetylglucosamine induces white-to-opaque switching and mating in Candida tropicalis, providing new insights into adaptation and fungal sexual evolution

Pathogenic fungi are capable of switching between different phenotypes, each of which has a different biological advantage. In the most prevalent human fungal pathogen, Candida albicans, phenotypic transitions not only improve its adaptation to a continuously changing host microenvironment but also regulate sexual mating. In this report, we show that Candida tropicalis, another important human opportunistic pathogen, undergoes reversible and heritable phenotypic switching, referred to as the "white-opaque" transition. Here we show that N-acetylglucosamine (GlcNAc), an inducer of white-to-opaque switching in C. albicans, promotes opaque-cell formation and mating and also inhibits filamentation in a number of natural C. tropicalis strains. Our results suggest that host chemical signals may facilitate this phenotypic switching and mating of C. tropicalis, which had been previously thought to reproduce asexually. Overexpression of the C. tropicalis WOR1 gene in C. albicans induces opaque-cell formation. Additionally, an intermediate phase between white and opaque was observed in C. tropicalis, indicating that the switching could be tristable.     

COPI proteins: A model for their role in vesicle budding

Summary COPI-coated vesicles are involved in intracellular trafficking between the endoplasmic reticulum and the Golgi complex. In the current model for COPI assembly the small GTP-binding protein ADP-ribosylation factor 1 is recruited from the cytoplasm to the Golgi membrane followed by binding of the hetero-oligomeric protein complex coatomer. However, the mechanism of subsequent vesicle budding is discussed controversially. This review summarizes the available experimental data on the COPI coat and discusses a model of how the major coat protein, coatomer, might act in vesicle budding.     

Determination of four sequential stages during microautophagy in vitro

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.     

A cycling protein complex required for selective autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.