We investigated structural changes of the yeast vacuole by fluorescence light microscopy. After staining the vacuoles with the vital dye FM4-64 (Vida and Emr 1995
), we detected large invaginations of the vacuolar membrane. Typically, there was only one invagination per vacuole, though in some cases up to three. The invaginations could also be seen under Nomarski optics, but they were much more difficult to detect than by fluorescence. The invaginations were tubular, variable in length, sometimes branched, and often expanded into a bubble-like structure at the tip (). They were dynamic, dangling back and forth in the lumen of the vacuole. The base of the invagination was more stationary. It was able to move along the boundary membrane of the vacuole, but the movement was much slower than that of the tip in the lumen of the vacuole. Extended tubes were remarkably slim. The membrane lining them usually was not able to be resolved from the lumen of the tube by fluorescence microscopy, due to the limited resolution of this method.
Tubular invaginations of vacuoles in living yeast cells. (A) CRY1 cells were grown logarithmically in YPD medium, stained with the vital dye FM4-64, and starved in SD(−N) for 1.5 h. The cells were viewed under a confocal microscope. The left images show the fluorescently stained vacuole; the right images show the same cell in transmitted light. Arrows indicate the position of the invagination. (B) Schematic summary of different shapes of tubes that can be observed. Bar, 3 μm.
The invaginations were not only laterally mobile, but also able to grow and shrink rapidly (). We observed the formation and disappearance of tubes within as little as 10–20 s. The tubes were able to pinch off vesicles from their tip, which were then released into the lumen of the vacuole. Released vesicles moved around freely in the vacuolar lumen and at a much higher pace than the tubes, indicating that they had become fully separated from the tubes. Formation and scission of a vesicle were completed in <40 s (). Formed vesicles were initially labeled with FM4-64, but after pinching off into the lumen, they lost the dye rapidly. Stable staining with FM4-64 may depend on a pH or ion gradient over the membrane. Due to the lack of an energy source, it is not likely that such gradients can be maintained by vesicles inside the vacuoles, resulting in loss of the dye.
Figure 2 Budding of vesicles into the lumen of the vacuole. (A and B) CRY1 cells were grown logarithmically in YPD medium. Their vacuoles were stained with FM4-64 and the cells were starved in SD(−N) medium with 1 mM PMSF at an OD600 of 1 at 25°C. (more ...)
The invaginations were also investigated by EM (). Cells were starved for nitrogen to induce autophagocytosis. They were quick-frozen in liquid propane, freeze-substituted, embedded, and analyzed by thin section EM. Autophagic bodies, which are the vesicular products of autophagocytosis, accumulated inside the vacuoles of Pep4
mutants ( and ; compare with results from Takeshige et al. 1992
). Vacuoles of such cells have reduced hydrolytic activity, which delays the degradation of autophagic bodies. The invaginations had an average diameter of 200–300 nm and frequently showed a constriction at the neck of the tube (, A–D, arrowheads), that is, at the interface between the invaginating membrane and the vacuolar boundary membrane. The lumen of the invaginations, as well as that of nascent vesicles at their tips, was continuous with the cytosol. Its ultrastructure was identical to that of bulk cytosol and to the lumen of autophagic bodies (arrows). Thus, budding of vesicles from the tip of the invagination must coincide with the uptake of cytosol into the lumen of the vacuole, establishing a microautophagic pathway for S. cerevisiae
. In fluorescence analyses, we found that the frequency of vacuolar invaginations depends on the nutrition state. In rich media, only 17% of the cells showed invaginations. In this and in the other assays described below, we observed quantitative variations of the percentage of cells that showed invaginations between different wild-type strains, but no qualitative differences (not shown). Under starvation conditions, which induce autophagocytosis (Takeshige et al. 1992
), the frequency of invaginations increased to 63% ( A). Thus, starvation induced autophagocytosis and the formation of the tubular membrane invagination of vacuoles.
Figure 3 Tubular invaginations are filled with cytosol. Yeast cells were grown logarithmically in YPD medium, transferred to SD(−N) medium, and starved. The cells were quick-frozen in liquid propane, freeze-substituted, and embedded. Thin sections were (more ...)
Figure 4 Frequency of autophagic tubes is influenced by starvation and the autophagy pathway. (A) DBY5734 cells were grown logarithmically overnight and then transferred to rich medium (YPD) or starved for nitrogen in SD(−N) medium. After 4 h of incubation (more ...)
Many mutants are known to affect macroautophagocytosis in yeast. We tested the effect of such mutations on the membrane invagination of vacuoles. Mutations in Vam3
, two components involved in multiple pathways of vesicular trafficking to the vacuole (Darsow et al. 1997
) and in maintenance of vacuolar integrity (Wada et al. 1992
), allow the formation of macroautophagosomes, but block their fusion with vacuoles (Darsow et al. 1997
). We tried to analyze autophagic tubes in temperature-sensitive mutants for Vam3
after shifting to a restrictive temperature. In starvation media at 37°C, the majority of the cells showed rapid (already 15 min after the temperature shift) fragmentation of vacuoles into smaller vesicles, making it impossible to quantitate the frequency of vacuolar invaginations (not shown). Similar behavior was observed for temperature-sensitive Sec18
mutants. The Aut
mutants were isolated based on their inability to transfer cytosolic proteins into the vacuole and accumulate autophagic bodies inside this organelle (Tsukada and Ohsumi 1993
; Thumm et al. 1994
). A priori, the screens did not distinguish whether these autophagic bodies emanated from macro- or microautophagy. Several mutants are defective in autophagosome formation, demonstrating a function in macroautophagy. Their influence on microautophagy of soluble proteins has not been analyzed because this pathway has not been characterized in Saccharomyces
to date. We found that in contrast to the situation in wild-type cells, invaginations were rare in mutants with deletions of genes involved in autophagocytosis, such as Apg5, Aut1, Aut7,
( B). Furthermore, cytosols from these mutants showed a significantly lower activity in an in vitro assay reconstituting microautophagic vacuole invagination and uptake (Sattler and Mayer 2000
, this issue). We conclude that macroautophagocytosis and the microautophagic invaginations of yeast vacuoles are both controlled via the Aut/Apg pathway. In light of its unique structure and its functional connection to autophagocytosis, we therefore term this specialized vacuolar invagination an “autophagic tube.”
What determines the formation and structure of autophagic tubes? To address this, we also detected autophagic tubes in vacuoles that had been extracted from the cells, floated in density gradients, fast frozen, and freeze–fractured (). The tubes found were indistinguishable from those seen in vivo. They had the same diameter, were sometimes branched, and carried expanded termini. Also, the sharp bending of the membrane at the neck of the tube and its constriction were maintained (). Since authentic autophagic tubes were able to be detected after extracting the organelle from the cell, their maintenance cannot depend on an intact surrounding cytoskeletal framework. This is further supported by the observation that purified vacuoles can even form new tubes in a cell-free system (Sattler and Mayer 2000
, this issue). Therefore, we conclude that autophagic tubes are formed and maintained by the vacuolar membrane autonomously, independent of an intact cellular environment.
Figure 5 Tubular invaginations are maintained on isolated vacuoles. Vacuoles were isolated from logarithmically growing cells by flotation through a Ficoll gradient (Sattler and Mayer 2000, this issue). The purified organelles were quick frozen in liquid propane, (more ...)
We extended the freeze–fracture analysis of vacuoles to compare the membrane structure of autophagic tubes with that of the vacuolar boundary membrane. In brief, this reveals either the extraplasmic fracture face (EF) or the protoplasmic fracture face (PF) of the split membrane bilayers, that is, the leaflet adhering to the vacuolar lumen or to the cytosol, respectively. Intramembranous particles, which may show up as corresponding holes on one fracture face, are known to be integral membrane proteins (Plattner and Zingsheim 1983
). There was a striking gradient of intramembranous particles along the autophagic tubes (). At the base of the tube, their density was high, resembling that of the vacuolar boundary membrane (asterisk). We found 510 particles/μm2
on the EF face of the vacuolar membrane (determined from ten independent vacuoles; SD = 190 particles/μm2
). The particle density dropped significantly towards the vacuolar lumen (arrows), often resulting in a smooth, particle-free appearance at the tip of the invaginations (arrowheads). Here, we found only 17 particles/μm2
(determined as described above; SD = 15 particles/μm2
). Areas with few or no particles were often expanded into bubble-like structures, suggesting that they were sites of vesicle formation. Autophagic bodies, the vesicular products of autophagocytosis, accumulate in cells deficient for vacuolar hydrolases (Takeshige et al. 1992
). In our freeze–fracture analysis, the membrane of autophagic bodies also appeared particle free (; compare with results from Baba et al. 1995
). The smooth membrane structure of autophagic bodies matched the situation seen on the terminal bubbles of autophagic tubes. This suggests that autophagic tubes are sites of autophagic body formation and that autophagic bodies arise from micro- as well as from macroautophagocytosis (Takeshige et al. 1992
Figure 6 Distribution of transmembrane particles along autophagic tubes. (A, B, and C) Three vacuoles from DBY5734 cells processed for freeze–fracture analysis. The boxed areas are shown at higher magnifications in D, E, and F. Note the higher density (more ...)
Figure 7 Autophagic bodies are also devoid of transmembrane particles. Yeast cells with a deletion of the PEP4 gene (DBY5734-16) were starved on SD(−N) medium for 3 h and then processed for freeze–fracture analysis as described in the legend to (more ...)
Further examination of the vacuolar surface in freeze–fracture replicas revealed small, particle-free areas on the vacuolar boundary membrane that often bulged inward (, arrows). and , shows a concave PF face and a convex EF face, respectively. Therefore, the particle-free area in B is an invagination. This indicates that particle-free zones may be early stages of autophagic tube formation. It suggests that the exclusion of large transmembrane proteins from these areas, which is probably accompanied by an enrichment of lipids in these zones, may be causal for invagination and vesicle formation.
Figure 8 Smooth areas on the vacuolar membrane as precursors of tubular invaginations. Vacuoles from strain DBY5734 are shown after freeze–fracture analysis as described in the legend to . The arrows point to well-defined, particle-free areas. (A) (more ...)
In conclusion, we propose that lateral segregation of lipids, with local exclusion of large transmembrane proteins in the plane of the vacuolar membrane, may lead to the invagination of this membrane. The resulting autophagic tubes are organized structures serving as templates for a budding reaction into the lumen of the organelle. Budding is restricted to the laterally segregated zones depleted of transmembrane particles. It results in the formation of autophagic bodies, which are also poor in transmembrane proteins.