Vesicular transport in capillary endothelium: does it occur?

A revised picture of the organization of endothelial plasmalemmal vesicles is presented. Three-dimensional reconstructions of endothelial segments from frog mesenteric capillaries and rat heart capillaries based on ultrathin serial sectioning have shown that plasmalemmal vesicles are not true vesicles but parts of an elaborate system of invaginations of the surface membrane. The revised picture probably applies to capillary endothelia in general. The absence of free cytoplasmic vesicles implies that vesicular transport is unlikely to occur. A reinterpretation of previous studies of vesicular transport shows that they are equally compatible with the present view that plasmalemmal vesicles are static elements of invaginations of the endothelial surface membrane.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

Distribution of endothelial vesicles in the microvasculature of skeletal muscle and brain cortex of the rat,as demonstrated by tannic acid tracer analysis

Summary Ultrathin serial sectioning and labeling with tannic acid have demonstrated that most plasmalemmal vesicles of rat vascular endothelial cells are not free, but rather are conjoined in three dimensions to form racemose invaginations from the cell surfaces. To elucidate the distribution of vesicles in these microvascular endothelial cells, we have examined terminal arterioles, capillaries and post-capillary venules of rat skeletal muscle and brain cortex, using tannic acid labeling and stereological methods, and have determined the proportions of free vesicles and the vesicles of luminal and abluminal invaginations, as well as the numerical density of vesicles. In the case of capillaries, regional differences in distribution have also been studied. The ratio of free vesicles is 6–7% and is constant throughout the muscle microvasculature. The distribution (proportions and numerical densities) of vesicles in the brain and muscle microvascular endothelial cells shows regionally distinctive patterns. In rapid-frozen, freeze-substituted endothelial cells, there are almost as many fused vesicles as seen in chemically fixed cells. Therefore, aldehydes do not seem to induce membrane fusion, and the distribution of vesicles seems to be preserved by chemical fixation. The structure and function of plasmalemmal vesicles are discussed.     

Three-dimensional organization of the plasmalemmal vesicular system in directly frozen capillaries of the rete mirabile in the swim bladder of the eel

Summary Several recent studies comparing chemically fixed and cryofixed endothelium have indicated that glutaraldehyde fixation may result in increases in the population of vesicles in the cytoplasm. Other reports based on ultrathin serial-section reconstruction of chemically fixed endothelium have revealed that the vesicular system is comprised of interconnected membranous compartments, which are ultimately continuous with either cell surface but do not extend across the endothelial cell. In this study, we have investigated the three-dimensional organization of the vesicular system in directly frozen, freeze-substituted capillaries of the rete mirabile from the swim bladder of the eel, specifically using the same block of embedded capillaries in which frozen capillaries had previously been found to contain less vesicles than chemically fixed capillaries. The results show that essentially all vesicles remain inter-connected with each other and are part of two separate sets of invaginations from the luminal and abluminal cell surface like in chemically fixed tissue. Any increase in vesicle number resulting from glutaraldehyde fixation does not affect the overall three-dimensional organization of the vesicular system in these endothelial cells.     

The three-dimensional organization of smooth endoplasmic reticulum in capillary endothelia: its possible role in regulation of free cytosolic calcium

A rise in cytosolic free Ca in capillary endothelia leads to increased permeability. It has been proposed that this Ca(2+)-regulated modulation of junctional permeability of vascular endothelia involves structural elements comparable to those involved in stimulus-contraction coupling in smooth muscle. To explore this analogy the three-dimensional organization of smooth-surfaced cisternae, vesicular membrane profiles, and tight junctions was examined in endothelia of diaphragm and heart capillaries of the rat. Three-dimensional reconstructions, based on consecutive sections of the capillaries, have demonstrated a population of small, irregular membrane profiles, occurring in individual thin sections of the endothelial cytoplasm. These profiles represent an elaborate system of smooth-surfaced cisternae, structurally similar to the sarcoplasmic reticulum (SR) of smooth muscle cells. Slender processes from the cisternae are often situated in parallel to the tight junctions at a distance of about 100 nm. The great majority of the characteristic circular membrane profiles represents caveolae and racemose invaginations of the endothelial plasma membrane, often in close relation to the cisternae. It is hypothesized that the endothelial cisternae and invaginations of the cell membrane are involved in regulation of free cytosolic calcium in the same way as the SR and caveolae in smooth muscle cells. The junction-related cisternal processes may play a role in the Ca(2+)-regulated modulation of junctional permeability.     

Plasma membrane-associated vesicles in retinal capillaries of the rat

The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.     

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

Member-associated changes during erythropoiesis. On the mechanism of maturation of reticulocytes to erythrocytes

The mature mammalian erythrocyte has a unique membranoskeleton, the spectrin-actin complex, which is responsible for many of the unusual membrane properties of the erythrocyte. Previous studies have shown that in successive stages of differentiation of the erythropoietic series leading to the mature erythrocyte there is a progressive increase in the density of spectrin associated with the membranes of these cells. An important stage of this progression occurs during the enucleation of the late erythroblast to produce the incipient reticulocyte, when all of the spectrin of the former cell is sequestered to the membrane of the reticulocyte. The reticulocyte itself, however, does not exhibit a fully formed membranoskeleton. In particular, the in vitro binding of multivalent ligands to specific membrane receptors on the reticulocyte was shown to cause a clustering of some fractions of these ligand-receptor complexes into special mobile domains on the cell surface. These domains of clustered ligand-receptor complexes became invaginated and endocytosed as small vesicles. By immunoelectron microscopic experiments, these invaginations and endocytosed vesicles were found to be specifically free of spectrin on their cytoplasmic surfaces. These earlier findings then raised the possibility that the maturation of reticulocytes to mature erythrocytes in vivo might involve a progressive loss of reticulocyte membrane free of spectrin, thereby producing a still more concentrated spectrin-actin membranoskeleton in the erythrocyte than in the reticulocyte. This proposal is tested experimentally in this paper. In vivo reticulocytes were observed in ultrathin frozen sections of spleens from rabbits rendered anemic by phenylhydrazine treatment. These sections were indirectly immunolabeled with ferritin-antibody reagents directed to rabbit spectrin. Most reticulocytes in a section had one or more surface invaginations and one or more intra-cellular vesicles that were devoid of spectrin labeling. The erythrocytes in the same sections did not exhibit these features, and their membranes were everywhere uniformly labeled for spectrin. Spectrin-free surface invaginations and intracellular vesicle were also observed with reticulocytes within normal rabbit spleens. Based on these results, a scheme for membrane remodeling during reticulocyte maturation in vivo is proposed.     

Functional and morphological studies of protein transcytosis in continuous endothelia

Continuous microvascular endothelium constitutively transfers protein from vessel lumen to interstitial space. Compelling recent biochemical, ultrastructural, and physiological evidence reviewed herein demonstrates that protein transport is not the result of barrier "leakiness" but, rather, is an active process occurring primarily in a transendothelial vesicular pathway. Protein accesses the vesicular pathway by means of caveolae open to the vessel lumen. Vascular tracer proteins appear in free cytoplasmic vesicles within minutes; contents of transport vesicles are rapidly deposited into the subendothelial matrix by exocytosis. Caveolin-1 deficiency eliminates caveolae and abolishes vesicular protein transport; interestingly, exchange vessels develop a compensatory transport mode through the opening of a paracellular permeability pathway. The evidence supports the transcytosis hypothesis and the concept that transcytosis is a fundamental component of transendothelial permeability of macromolecules.     

Spatial distribution of synaptic vesicles in presynaptic terminals: Computer simulation

A procedure for computer simulation is proposed, which allows one to quantitatively characterize the spatial distribution of synaptic vesicles in presynaptic terminals (PST) using ultrathin sections of such terminals. The procedure includes three stages: simulation, topographical analysis, and comparison. At the first stage, the spatial distribution of vesicles within a PST and the process of random sectioning of it are simulated using the corresponding mathematical model. At the second stage, the topographical distribution of vesicle profiles within the plane of PST section is estimated; three respective approaches have been used: (i) nearest neighbor distance distribution; (ii) minimal spanning tree; and (iii) Voronoi paving. At the third stage, the simulated parameters are compared with the parameters of native terminal sections; when the coincidence of these two parameter groups is satisfactory, we believe that the simulated spatial distribution agrees with the real distribution. The software for the procedure is written in C++ programing langage. The results of a pilot study on ultrathin sections of cultured rat hippocampal neurons showed that the method offers broad possibilities for spatial interpretation and quantitative characterization of distributions of synaptic vesicles.     

Endothelial Vesicles in the Blood–Brain Barrier: Are They Related to Permeability?

1. Macromolecules cross capillary walls via large vascular pores that are thought to be formed by plasmalemmal vesicles. Early hypotheses suggested that vesicles transferred plasma constituents across the endothelial wall either by a shuttle mechanism or by fusing to form transient patent channels for diffusion. Recent evidence shows that the transcytotic pathway involves both movement of vesicles within the cell and a series of fusions and fissions of the vesicular and cellular membranes.2. The transfer of macromolecules across the capillary wall is highly specific and is mediated by receptors incorporated into specific membrane domains. Therefore, despite their morphological similarity, endothelial vesicles form heterogeneous populations in which the predominant receptor proteins incorporated in their membranes define the functions of individual vesicles.3. Blood–brain barrier capillaries have very low permeabilities to most hydrophilic molecules. Their low permeability to macromolecules has been presumed to be due to an inhibition of the transcytotic mechanism, resulting in a low density of endothelial vesicles.4. A comparison of vesicular densities and protein permeabilities in a number of vascular beds shows only a very weak correlation, therefore vesicle numbers alone cannot be used to predict permeability to macromolecules.5. Blood–brain barrier capillaries are fully capable of transcytosing specific proteins, for example, insulin and transferrin, although the details are still somewhat controversial.6. It has recently been shown that the albumin binding protein gp60 (also known as albondin), which facilitates the transcytosis of native albumin in other vascular beds, is virtually absent in brain capillaries.7. It seems likely that the low blood–brain barrier permeability to macromolecules may be due to a low level of expression of specific receptors, rather than to an inhibition of the transcytosis mechanism.     

Regulation of synaptic vesicle docking by different classes of macromolecules in active zone material

The docking of synaptic vesicles at active zones on the presynaptic plasma membrane of axon terminals is essential for their fusion with the membrane and exocytosis of their neurotransmitter to mediate synaptic impulse transmission. Dense networks of macromolecules, called active zone material, (AZM) are attached to the presynaptic membrane next to docked vesicles. Electron tomography has shown that some AZM macromolecules are connected to docked vesicles, leading to the suggestion that AZM is somehow involved in the docking process. We used electron tomography on the simply arranged active zones at frog neuromuscular junctions to characterize the connections of AZM to docked synaptic vesicles and to search for the establishment of such connections during vesicle docking. We show that each docked vesicle is connected to 10-15 AZM macromolecules, which fall into four classes based on several criteria including their position relative to the presynaptic membrane. In activated axon terminals fixed during replacement of docked vesicles by previously undocked vesicles, undocked vesicles near vacated docking sites on the presynaptic membrane have connections to the same classes of AZM macromolecules that are connected to docked vesicles in resting terminals. The number of classes and the total number of macromolecules to which the undocked vesicles are connected are inversely proportional to the vesicles' distance from the presynaptic membrane. We conclude that vesicle movement toward and maintenance at docking sites on the presynaptic membrane are directed by an orderly succession of stable interactions between the vesicles and distinct classes of AZM macromolecules positioned at different distances from the membrane. Establishing the number, arrangement and sequence of association of AZM macromolecules involved in vesicle docking provides an anatomical basis for testing and extending concepts of docking mechanisms provided by biochemistry.     

Modulation of solute permeability in microvascular endothelium

Modulation of macromolecular permeability involves creation of venular leaks in response to receptor-operated mechanisms in the endothelial cell membrane elicited by various autacoids (histamine, serotonin, bradykinin). Reversible modulations may occur within seconds in response to specific agents, which indicates receptor-mediated events that act via the endothelial cells' contractile apparatus, leading to subtle changes in junctional microtopography and allowing faster passage of small solutes. This mechanism probably involves activation of the actin-myosin system in endothelial cells. Ca2+ is an important signal substance, as reflected in the permeability-increasing effect of calcium ionophores. The junctional control system may share functional similarities with the contractile system in various types of muscle cells, in particular, smooth muscle. This suggests a function for the extensive vesicular invaginations of the plasmalemmal membrane present in endothelial cells. Rather than being a system to carry macromolecules across the endothelium, its physiological role may be to regulate free cytosolic calcium concentration. It is reminiscent of similar membrane invaginations found in muscle cells. Thus intracellular free calcium may be regulated by a combination of energy-requiring extrusion and passive influx through receptor-operated calcium channels located in the invaginated vesicular membranes, with short diffusion distances to the actin-myosin filaments in the cytoplasm.     

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.     

Compound exocytosis of casein micelles in mammary epithelial cells

Ultrastructure of lactating bovine and rat mammary epithelial cells was studied with emphasis on secretory vesicle interactions. In the apical zone of the cell, adjacent secretory vesicles formed ball and socket configurations at their points of apposition. Similar configurations were formed between plasma membrane and secretory vesicle membrane. These structures may be formed by the diffusion of water between vesicles with different osmotic potentials. Frequently, vesicular chains consisting of 10 or more linked secretory vesicles were observed. Prior to the exocytotic release of casein micelles, adjacent vesicles fused through fragmentation of the ball and socket membrane. These membrane fragments and the casein micelles appeared to be secreted into the alveolar lumen after passing from one vesicle into another and finally through a pore in the apical plasma membrane. Emptied vesicular chains appeared to collapse and fragmentation of their membrane was observed. Based on these observations, we suggest that most vesicular membrane does not directly contact or become incorporated into the plasma membrane during secretion of the nonfat phase of milk.     

The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin

Strong evidence implicates clathrin-coated vesicles and endosome-like vacuoles in the reformation of synaptic vesicles after exocytosis, and it is generally assumed that these vacuoles represent a traffic station downstream from clathrin-coated vesicles. To gain insight into the mechanisms of synaptic vesicle budding from endosome-like intermediates, lysed nerve terminals and nerve terminal membrane subfractions were examined by EM after incubations with GTP gamma S. Numerous clathrin-coated budding intermediates that were positive for AP2 and AP180 immunoreactivity and often collared by a dynamin ring were seen. These were present not only on the plasma membrane (Takei, K., P.S. McPherson, S.L.Schmid, and P. De Camilli. 1995. Nature (Lond.). 374:186-190), but also on internal vacuoles. The lumen of these vacuoles retained extracellular tracers and was therefore functionally segregated from the extracellular medium, although narrow connections between their membranes and the plasmalemma were sometimes visible by serial sectioning. Similar observations were made in intact cultured hippocampal neurons exposed to high K+ stimulation. Coated vesicle buds were generally in the same size range of synaptic vesicles and positive for the synaptic vesicle protein synaptotagmin. Based on these results, we suggest that endosome-like intermediates of nerve terminals originate by bulk uptake of the plasma membrane and that clathrin- and dynamin-mediated budding takes place in parallel from the plasmalemma and from these internal membranes. We propose a synaptic vesicle recycling model that involves a single vesicle budding step mediated by clathrin and dynamin.     

甜菊愈伤组织细胞中的液泡膜内突和液泡内囊泡

对生长在分化培养基上的甜菊愈伤组织分生区细胞的液泡膜内突和液泡内囊泡,进行了超微结构和酸性磷酸酶细胞化学研究。在不同液泡化时期的细胞中,都存在不同大小和形态的液泡膜内突,它们有的缺乏明显的内含物;有的含有许多小泡或复杂膜系;有的含有一个较大的具许多小泡或复杂膜系的膜束缚囊泡。在液泡内还存在一些游离的液泡内囊泡,它们通常具有两层紧贴的界膜或为多层同心膜,推测它们来自液泡膜内突。ＡｃＰａｓｅ定位结果显     

Ultrastructural distribution of terbium across capillary endothelium: detection by electron spectroscopic imaging and electron energy loss spectroscopy

We used terbium as an intravital tracer of permeability pathways across the walls of capillaries in the rete mirabile of the eel swimbladder and in frog mesentery. Terbium was detected in unstained ultra-thin sections by electron density using electron spectroscopic imaging (ESI) and by electron energy loss spectroscopy (EELS). Enhancement of intrinsic contrast in zero loss images (elastically scattered electrons) permitted imaging of membrane-bound compartments and terbium within them which might otherwise have been undetected in counterstained sections. Element-selective imaging with EELS indicated that terbium was associated with heavy electron-dense deposits, but the terbium mass:volume of sections in areas of lighter deposition was insufficient to obtain a terbium signal. In the rete capillaries, terbium was deposited on the luminal surface, throughout vesicular profiles, and in the interstitium, but could not be traced through interendothelial junctions. Fine terbium deposits were detectable throughout apparent vesicular connections across the endothelium. In the frog mesentery, terbium penetrated some but not all interendothelial clefts, and was detectable in small quantities within luminal and abluminal vesicular profiles and in the interstitium. The results indicate that in the rete capillaries, terbium permeates the capillary via a transcellular route. This route may be provided by transient fusions of luminal and abluminal vesicular compartments.     

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.