On hypertonic treatment, vacuoles shrink within seconds, probably to compensate for the water efflux from the cytosol to the surrounding medium. Shrinking is accompanied by tubular invaginations of the vacuole. Vesicles are formed from the finger-like protrusions remaining between them. These observations raise several interesting questions. First, why do vacuoles fragment at all in an active, protein- and lipid-dependent manner? It appears that many vacuolar functions, such as hydrolytic degradation or the storage of polyphosphates, amino acids, and polyamines, might also work in a shrunken organelle that is not round. A major difference between a deflated and an inflated state of an organelle is the tension of its membrane. Shrinking changes the surface-to-volume ratio and eliminates membrane tension. Fragmenting the organelle into multiple smaller copies readjusts the surface-to-volume ratio and hence allows reestablishment of tension of the vacuolar boundary membrane. Membrane tension can influence the activity of channel and transport proteins (Hamill and Martinac, 2001
). Vacuoles contain numerous channels and transporters, which are crucial for its function in storage and release of various compounds. Some of them might be affected by membrane tension, for example, the vacuolar Ca2+
channel Yvc1p, which releases Ca2+
upon hypertonic shock (Chang et al., 2010
). In addition, membrane tension may be necessary to allow vesicular traffic to the organelle. Fusion between vacuoles and vesicular transport to them depend on the Rab-GTPase Ypt7p (Wada et al., 1992
; Wichmann et al., 1992
; Haas et al., 1995
; Mayer and Wickner, 1997
), and the function of Ypt7p is influenced by membrane tension (Brett and Merz, 2008
). Thus it is likely that vacuolar membrane tension needs to be maintained to sustain vacuolar membrane trafficking routes.
A second interesting aspect is the fact that fragmentation happens asymmetrically. It immediately produces fragmentation products of the final size rather than proceeding through a series of equal divisions to generate vesicles of increasingly smaller size (). Separating small vesicles with a high surface-to-volume ratio should permit more rapid readjustment of this ratio and the regaining of functionality of the compartment because already the first fragmentation products will possess a drastically increased surface-to-volume ratio.
A third interesting aspect is the involvement of the different fragmentation factors at different phases of the process (). Osmotically induced invaginations of the vacuolar membrane might be taken as a passive shape change dictated by the efflux of water and loss of volume, but this seems not to be the case. Invagination can be suppressed by deletion of either the V-ATPase or the dynamin-like GTPase Vps1p. Salt stress stimulates rapid assembly of the V1
sectors of the V-ATPase (Li et al., 2012
). The resulting augmented electrochemical gradient across the vacuolar membrane might directly affect distribution and properties of vacuolar lipids in order to support its large-scale deformations. Changes in the electrochemical membrane potential can directly induce transbilayer lipid asymmetry (Farge and Devaux, 1992
; Mui et al., 1995
; Sackmann and Feder, 1995
) and lateral phase separations of lipids (Schaffer and Thiele, 2004
). Such changes are sufficient not only to tubulate pure two-phase lipid systems, but also to allow vesicle scission from them (Julicher and Lipowsky, 1993
; Lipowsky, 1995
Schematic representation of the phases of hypertonically induced vacuole fragmentation and the involvement of various fragmentation factors at different phases.
A deficiency in proton pumping could also affect the vacuolar membrane by influencing the turnover of vacuolar contents. The major vacuolar compounds are polyphosphates, which are synthesized by the vacuolar VTC complex (Hothorn et al., 2009
) and can form up to 30% of the dry weight of yeast (Liss and Langen, 1962
). Polyphosphates influence vacuolar membrane dynamics, as illustrated by their roles in vacuolar invagination during microautophagy (Uttenweiler et al., 2007
) and in vacuole fusion (Muller et al., 2002
). Their turnover depends on an endopolyphosphatase that must be matured by vacuolar hydrolases, a process that probably depends on vacuolar acidification (Sethuraman et al., 2001
; Shi and Kornberg, 2005
). Polyphosphates contain up to hundreds of phosphate residues, are highly negatively charged, and form complexes with Ca2+
, basic amino acids, and other monovalent cations (Rao et al., 2009
). It is conceivable that polyphosphates might also associate with and cluster charged lipids, either by direct binding or by ionic bridges via bivalent cations. Because uptake and turnover of most vacuolar compounds depend on H+
-driven transporters (Kane, 2007
), perturbation of the proton gradient could interfere with vacuolar invagination by affecting vacuolar ion balance and lipid distribution.
We observed an unexpected early role for Vps1p in fragmentation since Δvps1 vacuoles do not show the large invaginations that can be observed in wild-type cells. The membrane in invaginated areas is negatively curved, but dynamin-like proteins bind to membrane areas of high positive curvature and can thereby promote tubulation and scission of membranes (Roux et al., 2010
; Schmid and Frolov, 2011
). If the role of Vps1p for forming the invagination was related to its binding to positively curved regions, it could only affect the rim of a forming indentation of the vacuolar boundary membrane. Here the membrane is positively curved. Vps1p might thus stabilize the rims of the invaginating structures. In this way, Vps1p should also be enriched at the tips of the remaining finger-like structures that can be observed between invaginations, that is, at the sites where scission of the final fragmentation products occurs. We could not test this model directly by microscopy because we were not able to produce tagged versions of Vps1p that showed a normal invagination pattern, although our tagged versions were functional for other aspects of Vps1p activity, such as endocytosis or vacuole fusion (Peters et al., 2004
; Smaczynska-de Rooij et al., 2010
). Attempts to localize Vps1p by immuno–electron microscopy have not succeeded. Our observation of a role of Vps1 in the formation of invaginations is consistent with observations of Hyams and coworkers in Schizosaccharomyces pombe
, who ascribed to Vps1p a function in tubulating vacuoles (Rothlisberger et al., 2009
). In S. pombe
, vacuole scission required an additional dynamin-like GTPase, Dnm1p. In S. cerevisiae
, however, we observed that vacuole fragmentation in a Δdnm1 mutant occurs normally (unpublished data).
The locally appearing tubules are probably accompanied by changes in the lipid phase in those areas. Our study illustrates this for one lipid, PI(3)P. On hypertonic shock, the amounts of PI(3,5)P2
on the vacuole increases 10- to 20-fold (Dove et al., 1997
; Bonangelino et al., 2002
). In addition, the levels of PI(3)P rise, although more moderately. Live-cell imaging of a strain deleted for the PI(3)P 5-kinase Fab1p shows that the mutant vacuoles invaginate even more vigorously than those of wild-type cells, whereas the actual formation of new vesicles is drastically reduced and delayed. Instead, the deep invaginations evolve into spherical structures that accumulate inside the vacuole. We consider those as degenerated or “frustrated” invaginations. They show a high level of PI(3)P. Because cells lacking Fab1p accumulate PI(3)P, these spherical invaginated structures may result from the hyperaccumulation of PI(3)P due to the inability to convert it into PI(3,5)P2
. In line with this, a Δvps34 strain that no longer produces PI(3)P does not show this increased invagination activity and does not accumulate intravacuolar spherical structures. We hypothesize that PI(3)P and PI(3,5)P2
could act sequentially in vacuole fragmentation. PI(3)P, produced from PI 3-kinase complex II, might stabilize invaginations, and its conversion to PI(3,5)P2
might induce the subsequent fission of vesicles from the membrane protrusions remaining between the invaginations. A surplus in PI(3)P might recruit proteins that induce negative curvature and stabilize the invaginations, eventually leading to the observed spherical structures if PI(3)P is not converted into PI(3,5)P2
The formation of PI(3)P and PI(3,5)P2
from PI might itself influence membrane curvature, but the change in the head group is rather small. We consider it as more likely that these lipids operate by recruiting lipid-binding proteins, which then help to shape the membranes. A candidate for such a factor is Atg18p, a PI(3,5)P2
-binding protein that regulates Fab1p activity (Dove et al., 2004
; Efe et al., 2007
). Atg18p is recruited to the vacuolar membrane after hypertonic shock. Δatg18 cells fragment their vacuoles less well than wild-type cells, although they have even more PI(3,5)P2
. In other mutants affecting the Fab1 complex the situation is inverse, that is, their fragmentation defects correlate to strong reductions in PI(3,5)P2
levels. The fragmentation defect of Δatg18 cells might result from the perturbations caused by the increased PI(3,5)P2
level. This, however, seems unlikely because fab1-5 mutants, which show a similar increase in PI(3,5)P2
as Δatg18 cells, have hyperfragmented vacuoles (Gary et al., 2002
; Efe et al., 2007
). Thus it is more likely that Atg18p supports the transition from invaginated to fragmented vacuoles independent of its influence on the conversion of PI(3)P to PI(3,5)P2
, perhaps via its interaction with PI(3,5)P2
and resulting influences on membrane curvature.
Fragmentation of vacuoles happens not only during adaptation to changes in the osmotic environment of the yeast, but also during the cell cycle. The vacuole in the mother cell forms an elongated structure, which extends into the bud and can pinch off tubulovesicular structures (Weisman, 2003
). When the bud neck closes, driven by the septins and an actin–myosin ring, these structures are separated from the mother vacuoles, where they fuse again to form the vacuole of the daughter cell (Weisman, 2003
). Lack of Fab1p delays this process, whereas cells lacking Vps1p or a functional V-ATPase appear not to be deficient for vacuole inheritance (unpublished observation). The independence of vacuole inheritance from two factors implicated in salt-induced fragmentation suggests that the rather slow fragmentation during cell division may not require all of the factors necessary for the fast adaptation to hyperosmotic shock. Inversely, there are factors necessary for vacuole inheritance that do not influence osmotically induced vacuole fragmentation. In vacuole inheritance, a major force-providing factor for the formation of the thin segregation structures growing out of the vacuole and their migration toward the bud is the myosin-driven transport of vacuoles along actin cables (Hill et al., 1996
; Catlett and Weisman, 1998
). This factor most likely does not play an active role during osmolarity-induced fragmentation, since we observed that this process is insensitive to the actin depolymerizing drug latrunculin B, as well as to various mutations interfering with actin function (unpublished data).
Careful examination of the morphological changes of the vacuole during salt-induced fragmentation allowed us to dissect the process into two distinct phases with nonoverlapping requirements for the known fragmentation factors. This dissection and the fact that vesiculation happens in an asymmetrical manner at sites that are identifiable in the light microscope provides an important tool for future identification of additional proteins involved in vacuole fragmentation and for studying how they shape the membrane.