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Review

Vacuole dynamics in fungi

Andrea RICHARDS, Veronica VESES, Neil A. R. GOW*

The Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen,

Aberdeen AB25 2ZD, UK

a r t i c l e i n f o

Article history:

Received 29 January 2010

Received in revised form

7 April 2010

Accepted 7 April 2010

Keywords:

Biogenesis

Dynamic

Fission

Fungi

Fusion

Hyphae

Vacuole

* Corresponding author. Tel.: þ44 1224 55587E-mail address: n.gow@abdn.ac.uk (N. A.

1749-4613/$ e see front matter ª 2010 The Bdoi:10.1016/j.fbr.2010.04.002

a b s t r a c t

Yeast-like and filamentous fungi contain vacuoles which are similar to mammalian lyso-

somes and plant vacuoles, and have a variety of important functions in solute storage,

protein turnover, ion homeostasis and apoptosis. Fungal vacuoles are unusual in their

wide variety of architectures and roles in different species and in different cell types e their

morphology and dynamics reflecting their ecological specialisation. Filamentous fungi have

a network of interconnected spherical and tubular vacuole structures which may form the

basis of a solute transport system that acts as nutrient transport pipelines. Some filamen-

tous fungi fill entire cellular compartments with vacuole, which reduces the metabolic

demands for cytoplasm biosynthesis and markedly affects cell cycle timing. Vacuoles are

also highly dynamic, undergoing a continuous balance of fusion and fission reactions to

allow changes in size, shape and number during cell division and in response to osmotic

stress. This article summarises recent developments in our understanding of the dynamics,

regulation and functions of fungal vacuoles.

ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction yeast-like and filamentous fungi highlight the diversity of

Fungal vacuoles share similar features with both mammalian

lysosomes and plant vacuoles. They are complex organelles

with a range of functions including degradation and turnover

of cellular components, storage of ions and metabolites,

cellular homeostasis, nutrient transport, the regulation of

growth via the cell cycle and death via apoptosis (reviewed

in Klionsky et al., 1990; Li and Kane, 2009; Veses et al., 2008;

Weber, 2002). Vacuoles are critical for cell viability and

mutants that do not inherit a vacuole have to generate one

before they can divide (Banta et al., 1988; Weisman, 2003;

Weisman et al., 1987). While most current knowledge about

fungal vacuoles originates from studies in the budding yeast

Saccharomyces cerevisiae recent studies in a variety of other

9; fax: þ44 1224 555844.R. Gow).ritish Mycological Societ

form and function of these essential organelles.

2. Vacuole dynamics

Vacuoles change shape, size and copy number in response to

external stimuli through a balance of membrane fission and

fusion. This can easily be affected by growth, morphogenesis

and experimental conditions (Cakar et al., 2000). For example

Bone et al. (1998) found the process of vacuole fission to be

slower than fusion inSchizosaccharomycespombecellsehowever

a recent study found the opposite result (Rothlisberger et al.,

2009). Thepresence of at least one vacuole seems to beessential

for viability and cell division of yeast (Weisman, 2003). Vacuole

y. Published by Elsevier Ltd. All rights reserved.

94 A. Richards et al.

morphology ismodulated in almost all fungi during the growth

and maturation of cells and colonies. Sometimes they fuse to

form vesicles that can occupy a significant fraction of the total

cell volume of a fungus.

Filamentous fungi grow by apical extension and lateral

branching to form mycelial colonies. Vacuoles are one of the

most prominent organelles in hyphal compartments, and

are characteristically pleiomorphic (Fig. 1). Spherical and

tubular vacuoles of varying size have been shown to form

functionally interconnected networks, almost like a somatic

vasculature, within hyphae of fungi such as Pisolithus tinctorius

(Hyde et al., 2002; Shepherd et al., 1993). Other fungi such as

Neurospora crassa and Aspergillus species contain larger, more

heterogeneous vacuoles, that may or may not be intercon-

nected, in the older distal hyphal compartments and smaller,

numerous vacuoles in the apical region (Bagar et al., 2009;

Bowman et al., 2009; Penalva, 2005; Seiler et al., 1999; Vaughn

and Davis, 1981). Sometimes vacuoles seem to be captured

and retained at septal regions (Veses and Gow, 2008). Despite

the knowledge of vacuolar functions in filamentous fungi, the

molecular mechanisms underlying vacuolar expansion and

Fig. 1 e Patternofvacuoledistributioninyeast-likeand

filamentousfungi.Vacuolesareindicatedinwhite,nucleiinred.

(A)Networkofsphericalandtubularvacuolesfoundinmany

filamentousfungie.g.Pisolithustinctorius,Phanerochaetevelu-

tina,Aspergillusoryzae,Paxillusinvolutus,Gigasporamargarita.

(B)MigratorycytoplasminhyphaeofCandidaalbicans.Asimilar

patternoccursinBasidiobolusranarum,Schizosaccharomyces

japonicus,Schizophyllumcommune.(C)Binucleatedtipcelland

empty-lookingvacuolatedsub-apicalcompartments

ofUstilagomaydis.B.ranarumformssimilarhighlyvacuolated

anucleatedcells.(DandE)Oval/sphericalvacuolesofbudding

yeaste.g.Saccharomycescerevisiae,C.albicans,andthevacuole

segregationstructureextendingfrommothertodaughter

duringvacuoleinheritance.(F)Numeroussmallvacuolesof

fissionyeastSchizosaccharomycespombe.Nottoscale.Adapted

fromVesesetal.(2008).

movement in filamentous fungi have not been fully

elucidated.

In most eukaryotes movements of organelles are based on

distalmicrotubules and their associatedmotor proteins that is

coordinated with actin at the growing cell apex (Steinberg,

2007a, 2007b; Weisman, 2006). Fungal vacuoles are similar to

mammalian lysosomes which are associated with microtu-

bule-dependent movement (Heuser, 1989; Swanson et al.,

1987). Studies using drugswhich inhibitmicrotubule and actin

activity, and mutants that are defective in microtubule func-

tion or in various motor proteins coupled with microscopy

have allowed insight into mechanisms of vacuole movement.

Motility and morphology of tubular vacuoles in the ectomy-

corrhizal fungus P. tinctorius were demonstrated to require

microtubules but not actin microfilaments (Hyde et al., 1999).

Vacuoles in the cowpea rust fungus Uromyces phaseoli var.

vignae, N. crassa, and Ustilago maydis also exhibit microtubule

dependent movement (Herr and Heath, 1982; Steinberg,

2000, 2007b; Steinberg and Schliwa, 1993). In contrast, in yeast

cells of S. cerevisiae and yeast and hyphae of the human path-

ogen Candida albicans actin cables are required for vacuole

movement and segregation rather than microtubules

(Steinberg, 2000; Veses and Gow, 2008; Weisman, 2006).

Studies with mutants of kinesin and dynein in N. crassa,

molecules which transport cellular organelles by movement

along microtubules, revealed an important role for dynein in

vacuole movement. In a dynein mutant, ro-1, vacuoles accu-

mulated near the tip and were less dynamic than in the wild

type as tubular projections rarely formed (Seiler et al., 1999).

Mutations in the multi-subunit complex dynactin (Dro-2,

Dro-7, and Dro-12) which regulates dynein activity also lead

to reduced vesicle trafficking and defects in vacuole distribu-

tion (Lee et al., 2001). These results highlight the role of dynein,

in association with microtubules, in retrograde vacuolar

traffic and vacuolar dynamics (Lee et al., 2001; Seiler et al.,

1999; Steinberg, 2007a,b).

Hyphae of P. tinctorius contain a dynamic network of spher-

ical and tubular vacuoles (Fig. 1A). The hyphal tip cell contains

a mixture of small spherical and tubular vacuoles. Large

spherical vacuoles are the predominant form in sub-apical

cells and they may be interconnected by tubular vacuoles.

Material is moved along tubules via peristaltic-like move-

ments. This is potentially a solute transport system but

without the membrane transfer required in vesicle based

transport systems. Studies on P. tinctorius revealed that the

different forms of the vacuole system may be differentially

regulated along a hypha (Hyde et al., 2002; Shepherd et al.,

1993). Similar networks of spherical and tubular vacuoles

have been identified in a range of fungi including the wood

rotting plant pathogen Phanerochaete velutina, the commer-

cially important fungusAspergillus oryzae, the ectomycorrhizal

species Paxillus involutus and arbuscular mycorrhizal fungus

Gigaspora margarita (Darrah et al., 2006; Rees et al., 1994; Shoji

et al., 2006a,b; Tuszynska, 2006; Uetake et al., 2002; Zhuang

et al., 2009). Hyphae of N. crassa contain spherical vacuoles

of various sizes in older portions and a dynamic tubular

network in growing tip cells. Hyphae tend to be highly vacuo-

lated in the older portions of the mycelium with less vacuolar

structures near the hyphal tip (Bowman et al., 2009; Vaughn

and Davis, 1981).

Fungal vacuoles 95

Some filamentous fungi exhibit a pattern of migratory

cytoplasm towards the growing apex and become more vacu-

olated in sub-apical compartments e.g. C. albicans (Fig. 1B; Gow

andGooday, 1982b; Veses andGow, 2008), Basidiobolus ranarum

(Robinow, 1963), the hyphal form of dimorphic fission yeast

Schizosaccharomyces japonicus (Sipiczki et al., 1998), the plant

pathogen U. maydis (Steinberg et al., 1998) and thewood decay-

ing fungus Schizophyllum commune (Inselman et al., 1999).

C. albicans hyphae branch less frequently and have longer

cell compartments in low nutrient environments and it has

been suggested that space-filling by vacuoles may be an

important energy-saving mechanism and foraging response

in poor nutrient environments that minimises requirements

for protein synthesis (Barelle et al., 2003; Veses et al., 2009).

Similarly many of the fungi that generate vacuole-filled distal

hyphae do so under nutrient limited conditions where hyphal

translocation without branching can be seen as an appro-

priate adaptive growth strategy on barren substrates.

Hyphae of the maize plant pathogen U. maydis consist of

a dikaryotic tip cell containing large basal vacuoles adjacent

to the last formed septum. As a hypha extends septa are laid

down adjacent to the basal vacuoles, forming empty-looking

sub-apical compartments (Fig. 1C). Deletion of the gene

encoding kinesin, kin2, resulted in almost no formation of

the empty vacuole-rich compartments and vacuoles in the

tip cell were small, numerous and scattered throughout the

cell. This suggests that kinesin is involved in formation and

positioning of vacuoles in tip cells of U. maydis and due to

this perhaps interrupts the formation of the characteristic

empty-looking sub-apical compartments (Steinberg et al.,

1998).

3. Participation of the vacuole inmorphogenesis

Vacuoles play important roles in spore germination, appresso-

rium function, hyphal growth and in apoptosis. For example,

an enlarging vacuole pushes cytoplasm into developing

conidium of the frog pathogen B. ranarum. Eventually the

wall breaks and propels the conidium in to the air (Weber,

2002). In the appressorium of the rice blast fungus Magna-

porthe grisea vacuoles also have a role in generating the

massive turgor pressure in the appressorium cell that is

required for plant invasion. This fungus is able to penetrate

plant tissue using the mechanical force applied via the infec-

tion peg underlying the appressorium from turgor pressure

generated by the uptake of water following the accumulation

of glycerol (de Jong et al., 1997; Howard et al., 1991). Glycerol

may be derived from degradation of lipid droplets which

were shown to be mobilised to developing appressoria and

are taken up by the large vacuoles in mature appressoria

(Weber et al., 2001). The turgor is so large that the infection

peg can penetrate gold leaf or the plasticware of Petri

dishes.

Germ tube growth from a spore of a fungus is characteris-

tically autocatalytic e as it increases in length an increasing

volume of cytoplasm and vesicles supports the growing tip

which therefore increases its extension rate (Prosser and

Tough, 1991; Prosser and Trinci, 1979; Trinci, 1969, 1974). In

contrast, germ tubes of C. albicans exhibit linear growth

kinetics rather than exponential growth. Also, in C. albicans

lateral hyphal branching does not normally occur immedi-

ately after septation as it does in other fungi, instead there

is a delay of several cell cycles before a new branch forms

(Gow and Gooday, 1982a; Gow et al., 1986; Veses et al., 2009).

Microscopical analyses of developing C. albicans germ tubes

revealed that a volume of cytoplasm derived from the parent

yeast cell migrates forwards with the growing tip while a large

vacuole occupies most of the volume of the mother cell. As

hyphal extension occurs, the cytoplasm moves forward with

the tip leaving behind sub-apical compartments containing

large vacuoles occupying the majority of the space with little

cytoplasmic content (Fig. 1B). Parent cells and sub-apical

compartments regenerate cytoplasmic content at the expense

of the vacuole before they are able to support formation of

lateral branches. Thismay explain why branches of C. albicans

do not form directly after septation. These observations of

extensive germ tube vacuolation also account for the linear,

rather than exponential growth kinetics. In this fungus,

hyphal extension is supported by a fixed volume of cytoplasm

originating from the parental cell, therefore growth is not

autocatalytic and therefore linear and not exponential

(Barelle et al., 2003; Gow and Gooday, 1982b, 1984; Gow et al.,

1986; Veses and Gow, 2008).

C. albicans is polymorphic, able to switch between budding

yeast cells, chains of constricted pseudohyphal cells, and true

hyphae (Berman and Gow, 2004; Sudbery et al., 2004). These

morphogenetic changes are cell cycle regulated (Berman,

2006). The vacuole content of the sub-apical compartments

has an effect on the timing of branching and thus entry of

a hyphal compartment into the cell cycle, perhaps by influ-

encing cell size threshold measurements at specific check-

points within the cell cycle. A study using mutants with

defects in the vacuole inheritance pathway showed a correla-

tion between the extent of vacuolation and hyphal branching.

A series of tetracycline-repressible conditional mutants were

constructed in which the transcription of vacuole inheritance

genes was repressed during hyphal growth. Three mutants

(vac7, vac8 and fab1) had more symmetrically distributed

vacuoles and an increased branching frequency and three

mutants showed the reverse phenotype (vac1, vam2 and

vam3) with enlarged vacuoles and sparse hyphal branching.

Therefore the hyphal cell cycle of C. albicans was modulated

by genes that controlled the volume of vacuole, and coinci-

dently the relative cytoplasmic volume in hyphal compart-

ments (Fig. 2; Barelle et al., 2006; Veses et al., 2009).

Corroborating these findings it has been shown that many

cell cycle mutants that alter cell cycle timing also have

changes in the relative vacuole volume of cellular compart-

ments (Richards, Veses and Gow, unpublished observations).

These observations suggest that in eukaryotes in general the

size of the vacuole, and other membrane bound organelles,

may affect cell cycle timing by influencing cell size regulation

of the cycle. The large volume of the vacuole may not be rele-

vant to the mechanism that couples cell size to the execution

of key check-point controls, such as that occurring at G1/Start

and at the G2-M transition. Hence large but highly vacuolated

cells may behave as though they are small in terms of cell

cycle regulation.

Fig. 2 e Influence of vacuole distribution in cell cycle

progression of Candida albicans hyphae. The ratio of vacuole

and cytoplasmic volumes influences progression through

cell cycle checkpoint Start. The cells on the right hand side

indicate the net cytoplasmic volume after subtracting the

vacuole volume from the total cells shown on the left hand

side. Adapted from Veses et al. (2009).

96 A. Richards et al.

4. Regulation of vacuolar dynamicsin yeast-like fungi

In budding yeast cells, vacuoles are usually oval or spherical in

shape and may have one to several vacuoles depending on

environmental conditions and stage of growth (Fig. 1D and

E). For example, in the yeast form of the polymorphic fungus

C. albicans vacuole content occupies approximately 23 % of

a cell, while in sub-apical hyphal cells vacuole may represent

39 % of cell compartments (Veses et al., 2009). Studies in

S. cerevisiae have identified many genes involved in vacuole

inheritance, dynamics and vacuole-related biochemical

processes. Genes designated VPS are involved in sorting and

transport of proteins to the vacuole (Bankaitis et al., 1986;

Bonangelino et al., 2002; Robinson et al., 1988; Rothman and

Stevens, 1986; Rothman et al., 1989), VAM genes are involved

in vacuole morphology and biogenesis (Wada et al., 1992;

Wang et al., 1996), and VAC genes are involved in vacuole

inheritance (Weisman et al., 1990; Weisman and Wickner,

1992). These are divided into classifications A to F dependent

on vacuole morphology of the mutants (Raymond et al., 1992).

Cellular organelles, including the vacuole, compartmen-

talise diverse intracellular functions. When a cell divides,

a portion of the organelles is inherited by the daughter cell.

Distinct mechanisms of inheritance exist for different organ-

elles and aspects of their mechanism are widely conserved.

For example the spatial regulation of organelle movement is

invariably tightly coupled with the cell cycle (Warren and

Wickner, 1996; Weisman, 2006). Vacuole inheritance during

cell division has been particularly well characterised in the

budding yeast S. cerevisiae and involves segregation of the

mother cell vacuole, movement into the daughter cell and

fusion of vacuole vesicles (reviewed in Catlett and Weisman,

2000; Fagarasanu and Rachubinski, 2007; Weisman, 2003,

2006). In yeast cells, vacuoles can be large or consist of clusters

of smaller vesicles. During interphase these smaller vacuoles

then fuse to form one to five large vacuoles during most of

the yeast cell cycle. Vacuole inheritance starts early in the

cell cycle when a string of vacuole vesicles and tubules called

the segregation structure, extends from mother to daughter

cell (Fig. 1E; Conradt et al., 1992; Gomes de Mesquita et al.,

1991; Weisman and Wickner, 1988). In many yeast most

organelles move solely on actin (Weisman, 2006). During cell

division the larger vacuoles are broken down into a stream

of smaller spherical and tubular vacuoles that are transported

into the bud via the actomyosin cytoskeleton (Veses and Gow,

2008; Weisman, 2006). Vacuole inheritance in S. cerevisiae is

actin based and driven by the myosin-V motor protein Myo2

(Fig. 3; Bretscher, 2003; Hill et al., 1996). Vac17, a vacuole

specific Myo2 receptor protein binds simultaneously with

Myo2 and vacuole membrane protein Vac8 (Fig. 3; Ishikawa

et al., 2003). This Myo2eVac17eVac8 complex travels along

actin cables moving the vacuole from mother to daughter

cell. Oncewithin the daughter cell Vac17 is degraded, breaking

down the Myo2eVac17eVac8 complex, and depositing the

vacuole near the centre of the cell (Fig. 3; Tang et al., 2003).

Once deposited in the daughter cell vacuole vesicles fuse to

form a larger vacuole (Conradt et al., 1992).

The vacuole specific Myo2 receptor Vac17 (Ishikawa et al.,

2003) appears to play a central role in coordinating vacuole

inheritance with the cell cycle in S. cerevisiae through cell-

cycle dependent changes in synthesis and turnover. Levels

of the Vac17 protein oscillate with the cell cycle (Tang et al.,

2003). Vac17 is phosphorylated by the cyclin dependent kinase

(Cdk) Cdc28 in coordination with the cell cycle. Levels of phos-

phorylation correspond with arrival of an inherited vacuole in

the bud (Peng and Weisman, 2008). These findings reinforce

the role of Vac17 in the tightly coordinated cell cycle regula-

tion of vacuolar inheritance in the budding yeast.

Findings of a recent study suggest that the two main p21

activated kinases (PAKs) in yeast (Cla4 and Ste20) are involved

in resolving the segregation structure and degrading Vac17 in

the bud. Mutant cells lacking PAK function were unable to

resolve segregation structures. Over expression of Cla4 or

Ste20 inhibited vacuole inheritance, but this could be over-

come by expressing a non-degradable form of Vac17

(Bartholomew and Hardy, 2009). PAK activity requires

Rho-type small GTPase Cdc42 and its regulator Bem1. Bem1

is phosphorylated in a Cdc28 dependent manner (Han et al.,

2005) again linking vacuole inheritance with the cell cycle.

Bem1 and Cdc42 have already been shown to coordinate

vacuole fusion with the cell cycle through cyclin Cln3 (Han

et al., 2005). In addition, a 14-3-3 protein in filamentous fungi

U. maydis, Pdc1, has been shown to be involved in both cell

cycle regulation and also regulation of vacuole formation

(Pham et al., 2009).

Jin et al. (2009) have demonstrated that activity of protein

phosphatase Ptc1 is required for maintaining steady state

levels of Vac17. In wild type S. cerevisiae Vac17 is concentrated

on the vacuole membrane at the leading edge of the vacuole.

In a ptc1 mutant Vac17 was distributed throughout the

vacuole membrane, suggesting that Ptc1 is required for the

association of Myo2 with Vac17.

During inheritance of the Golgi in mammalian cells, Golgi

function ceases temporarily (Shorter andWarren, 2002). Yeast

cells have the potential to divide continuously and so shut

down of organelle function during inheritance may be detri-

mental to normal functioning of the cell (Weisman, 2006). In

addition to its role in movement of vacuole vesicles during

vacuole inheritance (Tang et al., 2003), Vac8 is also involved

Fig. 3 e Vacuole inheritance in budding yeast Saccharomyces cerevisiae. The Myo2eVac17eVac8 complex transports vacuole

vesicles into the emerging bud where Vac17 is degraded and vacuole vesicles released. Vac17 levels are affected by cell cycle

dependent phosphorylation by cyclin dependent kinase Cdc28. Protein phosphatase Ptc1 is required for maintaining steady

state levels of Vac17 and may be required for proper association of Myo2 with Vac17. p21 activated kinases Cla4 and Ste20

are involved in resolving the segregation structure and degrading Vac17 in the bud.

Fungal vacuoles 97

in formation of the nuclearevacuole junction (Pan et al., 2000),

formation of vesicles in the cytoplasm-to-vacuole protein tar-

geting pathway (Scott et al., 2000), homotypic vacuole fusion

(Veit et al., 2001; Wang et al., 2001), and caffeine resistance

(Tang et al., 2006). For each of these functions Vac8 associates

with a different binding partner. Vac8 contains eleven arma-

dillo (ARM) repeats thatmediate proteineprotein interactions.

Each binding partner requires overlapping subsets of Vac8

ARM repeats and can compete for access, thus coordinating

vacuole inheritance with other vacuole functions (Tang

et al., 2006).

In the fission yeast S. pombe the regulation of vacuolar

morphology shares similarities with mammalian cells. Under

normal growth conditions cells contain approximately eighty

numerous small vacuoles occupying 3.4 % of a cell (Fig. 1F;

Bone et al., 1998). This differs from the vacuole patterns

observed in the budding yeast or filamentous fungi

(Fig. 1AeE; Veses et al., 2008; Weisman, 2006). The molecular

mechanism of vacuolar inheritance in fission yeast is not yet

clear, although the presence of many small organelles and

the process of cell division by fission would be in favour of

a stochastic inheritance process by simple partitioning

(Warren and Wickner, 1996). If so this would be significantly

different to the mechanism and machinery required for

movement of vacuoles during budding of S. cerevisiae and

other yeast.

Many proteins containing so-called PX protein domains are

involved in vesicle trafficking, protein sorting and lipid modi-

fication. Several of these contain PXA (PX-associated) domains

of which the function is unknown. In S. pombe a protein has

been identified, Pxa1, which contains a PXA but not a PX

domain and which has a function in maintaining normal

vacuole morphology. There is no apparent homologue in

S. cerevisiae (Hosomi et al., 2008) further highlighting the differ-

ences in vacuole dynamics between budding and fission yeast.

5. Mechanism of homotypic vacuole fusion

The ability to purify vacuoles from S. cerevisiae and observe

vacuole fusion using an in vitro assay (Conradt et al., 1992;

Haas et al., 1994; Jun and Wickner, 2007) and to perform

genetic screens to identify components of fusion machinery

(Seeley et al., 2002), has contributed greatly to the under-

standing of vacuole homotypic fusion. This process occurs

in distinct stages: priming, tethering, docking and fusion/

bilayer mixing (Fig. 4; Conradt et al., 1994; Wickner, 2002),

and requires variousmolecular components including soluble

N-ethylmaleimide-sensitive factor attachment protein recep-

tors (SNAREs), the homotypic fusion and vacuole protein sort-

ing (HOPS) complex, the vacuole transporter chaperone (Vtc)

complex, Rab/Ypt GTPases, lipids ergosterol, diacylglycerol,

Fig. 4 e Stages of vacuole homotypic fusion. (A) Priming

is a series of reactions which prepares vacuole membranes

for the next stage. (B) Tethering brings membranes into

close contact, and is reversible. (C) Docking. As vacuoles

are drawn together fusion components become enriched

at the vertex ring, a tightly apposed disc shaped area of

membrane. Vacuoles are firmly docked together by forma-

tion of SNARE complexes. (D) Fusion. Outer cytoplasmic

leaflet lipids mix before luminal contents mixing. Adapted

from Wickner (2002).

98 A. Richards et al.

and phosphoinositides (reviewed in Ostrowicz et al., 2008;

Wickner, 2002; Wickner and Haas, 2000).

Priming is a series of reactions involving changes in the

interaction of SNARE complexes. Disassembly of cis-SNARE

complexes (located on the same membrane) prepares the

individual SNAREs so that they are in an active form and

prepared for interactions in the next stage. No interaction of

vesicles occurs in this stage (Nichols et al., 1997; Price et al.,

2000; Ungermann et al., 1998a). Once primed, vacuole vesicles

are reversibly tethered together. This involves small Rab

GTPase Ypt7 and its effector, the HOPS complex (Mayer and

Wickner, 1997; Price et al., 2000; Seals et al., 2000). Ypt7 func-

tions to binds HOPS to the vacuole membrane (Hickey et al.,

2009). The vacuoles are drawn together so that a tightly

apposed disc-shaped area of membrane exists between

them. Fusion components become enriched at the perimeter

of this disc of membrane, the vertex ring (Wang et al., 2002).

Vesicles are then firmly docked together by interaction of

v-SNAREs (vesicle membrane) and t-SNARES (target

membrane) on opposite membranes to form trans-SNARE

complexes (Collins and Wickner, 2007; Nichols et al., 1997;

Ungermann et al., 1998b). HOPS is an SNARE chaperone, facil-

itating assembly and function of SNARE complexes (Collins

et al., 2005; Stroupe et al., 2006) and proof-reads conformation

of trans-SNARE complexes and reduces the capacity of

mismatched complexes to undergo fusion (Starai et al.,

2008). The Ccz1eMon1 complex binds to the vacuole

membrane as part of the cis-SNARE complex. Association of

Ccz1eMon1 with the vacuole is regulated by the HOPS

complex. The Ccz1eMon1 complex appears to have a role in

SNARE pairing at the tethering and docking stage in associa-

tion with Ypt7 (Hoffman-Sommer et al., 2005; Wang et al.,

2003). During the last stage of vacuole homotypic fusion the

opposing membranes and contents of vacuole vesicles fuse

together. This stage is not well understood but it has been

observed that lipid mixing occurs long before the luminal

contents are combined, suggesting that vacuoles fuse via

a hemifusion intermediate, whereby the outer cytoplasmic

leaflet lipids intermix but the inner luminal leaflets remain

distinct (Chernomordik and Kozlov, 2008; Jun and Wickner,

2007). The extent of trans-SNARE zippering controls the onset

of membrane lipid mixing but does not control the transition

from hemifusion to full fusion and consequent contents

mixing (Schwartz and Merz, 2009).

The complete set of SNARE proteins has been character-

ised in S. cerevisiae (Burri and Lithgow, 2004). A recent study

of the phylogeny of SNARE proteins revealed that S. cerevisiae

and all the other fungi in the study have a simple set of

SNAREs which are relatively consistent and homologous,

and that Vam7 is a fungus-specific soluble SNARE (Kienle

et al., 2009).

6. Vacuole biogenesis: balancing fusion andfission

Vacuoles undergo a continuous balance of antagonistic

membrane fusion and fission reactions to allow changes in

size and shape in response to internal and external stimuli.

How vacuoles maintain the balance between fission and

fusion is not fully understood. Although vacuole fusion has

been well characterised, the mechanism of vacuole fission is

only starting to emerge. The dynamin-like protein Vps1 and

V-ATPase have both been implicated in vacuole fusion and

fission (Baars et al., 2007; Peters et al., 2004; Rothlisberger

et al., 2009), in addition to several other factors (Dove et al.,

2009).

The high osmolarity glycerol (HOG) response pathway

mediates long term response to osmotic shock (Hohmann,

2002). Vacuoles are thought to be involved in the immediate

response to osmotic shock. The numerous small vacuoles of

fission yeast S. pombe fuse quickly in response to hypotonic

stress (Fig. 5). This response is beneficial for adaptation of

fungi to rapid onset of hypotonic stresses in environmental

situations, such as in the response to rainfall. Conversely,

Fig. 5 e Vacuole fusion and fission occur in response to

hypotonic and hypertonic stress respectively. Fission is

accompanied by a rapid rise in phosphatidylinositol-

3,5-bisphosphate (PI[3,5]P2) levels. The HOPS complex

involved in vacuole homotypic fusion can interconvert, by

exchange of subunits, with the CORVET complex involved

in fission. Both fusion and fission require V-ATPase. Fission

also requires acidification of the vacuole. Dynamin-like

protein Vps1 is involved in both fusion and fission

processes. Adapted from Dove et al. (2009).

Fungal vacuoles 99

vacuolar fission takes place in response to high concentra-

tions of salt (hypertonic stress) but is a slower process than

vacuolar fusion (Bone et al., 1998). The same response is

observed in vacuoles of budding yeast S. cerevisiae. Hypotonic

stress increases the rate and extent of vacuole fusion (Brett

and Merz, 2008; Wang et al., 2001). In hypertonic conditions

vacuoles become fragmented and their volume reduced

(Brett and Merz, 2008; Bonangelino et al., 2002). Brett and

Merz (2008) demonstrated that osmolytes affect early teth-

ering/docking events within vacuole fusion. Osmolytes inhibit

Rab-regulated reactions by functionally and biochemically

destabilising Ytp7 and the HOPS complex.

Vacuole fission in response to osmotic shock is accompa-

nied by a rapid rise in phosphatidylinositol-3,5-bisphosphate

(PI[3,5]P2) levels (Fig. 5; Bonangelino et al., 2002; Dove et al.,

1997), whose synthesis and turnover involve phosphotidylino-

sitol 3-phosphate 5-kinase Fab1 and PI(3,5)P2 5-phosphatase

Fig4 (Gary et al., 2002). Fab1 is regulated by Vac7 and Vac14,

whereas Fig4 is regulated by Vac14 alone (Duex et al., 2006;

Rudge et al., 2004). Mutants unable to synthesis PI(3,5)P2 have

a single enlarged vacuole which is unable to undergo

membrane fission whereas over-production of PI(3,5)P2 leads

to more numerous vacuoles of smaller size. This suggests

that PI(3,5)P2 levels regulate surface area to volume ratio of

the vacuole allowing uptake or release of water to restore

the osmotic balance of the cell (Bonangelino et al., 1997, 2002).

Whereas budding yeast S. cerevisiae contains only a single

homologue of the mammalian Rab7, Ypt7 (Haas et al., 1995),

two homologues have been identified in fission yeast S. pombe.

Ypt7 and Ypt71 have been shown to have antagonistic roles in

the regulation of vacuolar fusion and fission. Rab proteins are

required for vesicle fusion with the appropriate target

membrane. The null mutant ypt7 contains large vacuoles,

whereas the ypt71 mutation leads to the presence of small

fragmented vacuoles (Kashiwazaki et al., 2009). This fact that

S. cerevisiae contains only a single Rab7 homologue whereas

S. pombe has two, and the antagonistic roles of Ypt7 and

Ypt71 may explain in part why fission yeast have such small

and numerous vacuoles in comparison to budding yeast,

which have fewer, larger vacuoles (Kashiwazaki et al., 2009).

There is emerging evidence that vacuole fusion and fission

are linked through the use of common molecular compo-

nents. The HOPS complex is a well known component of the

vacuole homotypic fusionmachinery. It shares some common

components with the CORVET-tethering complex and can

interconvert with CORVET by exchange of subunits (Fig. 5).

Whereas HOPS is an effector of Ypt7, CORVET is an effector

of Rab GTPase Vps21, a homologue of mammalian Rab5. COR-

VET catalyses either vacuole fission, vacuoleeendosome

fusion, or both. Links have been suggested between HOPS-

CORVET and the Fab1 pathway (Dove et al., 2009; Peplowska

et al., 2007).

The internal lumen of vacuoles is acidic compared to the

cytoplasm. The gradient is generated and maintained by

the V-ATPase protein complex which pumps protons into

the vacuole (Kane, 2006). The physical presence of the

membrane bound portion of the V-ATPase (V0), but not the

proton translocating activity, is known to be required for

vacuole fusion (Fig. 5; Bayer et al., 2003; Peters et al., 2001). In

contrast, vacuole fission requires acidification of the vacuole

by V-ATPase activity (Fig. 5). In mutant cells which lacked

subunits of the V-ATPase or in which V-ATPase activity was

blocked, vacuoles were unable to undergo normal fission in

response to hyperosmotic stress (Baars et al., 2007).

A dynamin-like protein in S. cerevisiae, Vps1, has been

shown to be involved in both fusion and fission processes.

Although the mechanism is not known, it is demonstrated

that Vps1 interacts with vacuolar SNARE Vam3 which is

involved with vacuole fusion (Peters et al., 2004). A new study

in S. pombe highlights further the tandem role of Vps1 in

vacuole fusion and fission. Mutant vps1 cells exhibited both

fusion and fission defects when placed in water and high

salt solutions respectively. Vps1 acts as a vacuole membrane

tubuliser as over expression results in extensive vacuole tubu-

lation. Scission of vacuole tubules is probably mediated by

another dynamin-related protein Dnm1 (Rothlisberger et al.,

2009), which acts with Vps1 in peroxisome biogenesis

(Jourdain et al., 2008; Kuravi et al., 2006). The mammalian

homologue of Vsp1, DLP1, also tubulates membranes (Yoon

et al., 2001). In hyphae of P. tinctorius a GTP-binding protein,

possibly a dynamin-like GTPase, is involved in vacuolar tubule

production (Hyde et al., 2002).

In addition to the numerous studies on vacuole biogenesis

in the yeast-like fungi, there is also evidence for similar

vacuole machinery in filamentous fungi. Work in the Kita-

moto group has highlighted the participation of GTPases in

vacuolar biogenesis in filamentous fungi (Ohsumi et al., 2002;

Oka et al., 2004; Tarutani et al., 2001). Aspergillus nidulans

AvaA is the homologue of mammalian Rab7 and S. cerevisiae

Ypt7 which plays a role in homotypic vacuole fusion

(Ohsumi et al., 2002). A. nidulans AvaB is similar to S. cerevisiae

Vam6/Vps39, a component of the HOPS complex, which is

100 A. Richards et al.

involved in vacuole membrane tethering during membrane

fusion. AvaB has been shown to interact with AvaA by yeast

two-hybrid analysis and has a critical role in vacuole morpho-

genesis (Oka et al., 2004). A third gene, vpsA, is a homologue of

S. cerevisiae VPS1 (Tarutani et al., 2001). The disruption of any of

these three genes in A. nidulans results in vacuolar fragmenta-

tion suggesting a role in vacuole fusion (Ohsumi et al., 2002;

Oka et al., 2004; Tarutani et al., 2001).

A homologue of S. cerevisiae Vps24 has been identified in

A. oryzae which, unlike in S. cerevisiae, exhibits vacuolar

defects upon gene disruption. Vps24 is a component of the

ESCRT III (endosomal sorting complex required for transport)

complex involved in formation of vesicles within the late

endosome (also called mutivesicular body or prevacuolar

compartment) which is required for transport to the vacuole.

In an A. oryzae vps24 mutant aggregation of structures which

have properties of both vacuoles and the late endosome

suggests that function of the late endosome is required for

vacuole biogenesis (Tatsumi et al., 2006, 2007).

7. Vacuoles, autophagy and apoptosis

Some fungi are able to grow and explore their environments

under highly oligotrophic conditions. For example some plant

and animal fungal pathogens occupy nutrient-limited niches

such as leaf and skin surfaces, yet they are well able to grow

over these surfaces before they invade the host. Magnaporthe,

Puccinia, Ustilago species and C. albicans can grow under

nutrient limited conditions by recycling their internal nutrient

supplies and minimising the biosynthetic costs associated

with increasing their cytoplasmic volume by forming exten-

sively vacuolated hyphae (see above). Autophagy operates

whenamassivenon-specificdegradationof cytoplasmicmate-

rial is required, for recycling cellular nutrients, which can

contribute towards fungal survival through periods of nutrient

scarcity. For example inM. grisea appressorium induction and

growth under low nutrient conditions has been linked to

important features of vacuole biology and autophagy

(Veneault-Fourrey et al., 2006) as has pseudohyphal growth of

S. cerevisiae (Ma et al., 2007). These studies suggest that autoph-

agy may be an important mechanism by which the fungi can

survive,andundergodifferentiation inhostilenutrient-limited

conditions (Palmer et al, 2008; Veses et al., 2008).

Eukaryotes have two major pathways for degradation of

proteins and organelles, the proteosome, which mediates

degradation of ubiquitin-tagged cytosolic and nuclear protein

(Hanna and Finley, 2007; Voges et al., 1999), and the vacuole-

mediated degradation pathway. The major trafficking

pathway that delivers material to be degraded to the yeast

vacuole is autophagy (Reggiori and Klionsky, 2002). This

cellular pathway includes several cellular degradation

processes: macroautophagy, microautophagy, pexophagy

and chaperone-mediated autophagy. All of these converge at

the vacuole. Macroautophagy is the most commonly studied

of the previous pathways, and therefore is itself often referred

to as autophagy. This is a non-selective degradation process

by which eukaryotic cells degrade cytoplasmic material and

organelles. Microautophagy involves direct invagination of

cytosolic material into the vacuole, whereas pexophagy is

the pathway for degradation of targeted peroxisomes. An

additional autophagy process is assisted by chaperone mole-

cules which associate with cytosolic proteins that need to be

degraded (Yorimitsu and Klionsky, 2005). Although macroau-

tophagy and microautophagy are morphologically distinct,

research in S. cerevisiae suggests that they may be regulated

by some of the same molecular machinery (Kissova et al.,

2007). In filamentous fungi, autophagy is typically accompa-

nied by vacuolar enlargement (Zustiak et al., 2008). However,

in mutants defective in autophagy, like atg1D or atg8D in Podo-

spora anserina vacuolation is not suppressed, which suggest

that enlargement of the vacuole is not always autophagy-

dependant (Pinan-Lucarre et al., 2005).

Autophagy is a well studied pathway in S. cerevisiae and in

mammalian cells, because of its involvement in multiple bio-

logical phenomena and its role in human disease, including

ageing, cancer, neurodegeneration and microbial infection

(Klionsky, 2005; Mizushima, 2005; Mizushima et al., 2008). To

date thirty-one autophagy-related (ATG) genes have been iden-

tified in S. cerevisiae (Rubinsztein et al., 2007). Of these, seven-

teen encode gene products constituting the core autophagy

machinery, which can be classified in three major groups

(Xie and Klionsky, 2007): (i) ATG9 and its cycling system

(ATG1, ATG13, ATG2 and ATG18); (ii) the phosphatidylinositol

3-OH kinase complex (VPS34, VPS15, VPS30/ATG6 and ATG14)

and (iii) the ubiquitin-like protein system (ATG8, ATG12,

ATG7, ATG10, ATG3, ATG4, ATG5 and ATG16). ATG9 is the

only integral membrane component gene in the core

machinery that is conserved between species (Noda et al.,

2000). Therefore this gene has been studied thoroughly across

different fungi. Deletion of ATG9 in S. cerevisiae and C. albicans

blocks autophagy (Lang et al., 2000; Palmer et al., 2007, 2008).

However atg9D mutants display additional phenotypes in

cellular development that vary between species. In S. cerevi-

siae, the atg9D mutant is defective in sporulation (Enyenihi

and Saunders, 2003), whereas in C. albicans deletion of ATG9

had no obvious effect on the yeast to hypha transition

(Palmer et al., 2007). Deletion of ATG8 caused block of autoph-

agy in both M. grisea and A. oryzae and, again, these mutants

displayed differential phenotypes in cellular development.

However, the atg8D mutant of the rice blast fungus could not

complete the process of appressorium formation and was

non-pathogenic (Veneault-Fourrey et al., 2006) while the

atg8Dmutation inA. oryzae prevented the formation of conidia

and aerial hyphae (Kikuma et al., 2006). Thus, autophagy plays

a range of ubiquitous and organism-specific roles in fungal

differentiation processes.

8. Concluding remarks

Much light has been shed recently on the workings of the

vacuole and its various roles in fungal physiology. Continuing

advancements and improvements in technologies such as live

cell imaging will no doubt aid in this work in the future. For

example, recent developments in molecular genetics coupled

with advances in image processing capable of multidimen-

sional quantification of subcellular organelles such as the

vacuole (Negishi et al., 2009) will continue to provide valuable

information about the dynamic changes in vacuole

Fungal vacuoles 101

morphology during fungal growth. Fungal vacuoles, more so

than other membrane-bound organelles, are finely tuned to

the requirement and ecology of the fungus. While the molec-

ular details about the processes of fission and fusion of vacu-

oles will no doubt continue to be dissected in model yeast

species, it is clear that the importance of vacuoles in filamen-

tous fungi is now extending to new aspects of cell physiology

in which vacuoles function as nutrient pipelines and media-

tors of cellular morphogenesis and cell cycle control. Collec-

tively, these studies have established the credentials of the

fungal vacuole as a vital organelle at the heart of fungal

physiology.

Acknowledgements

Our work in this area is supported by grants from the BBSRC

and a studentship to AR from the MRC.

r e f e r e n c e s

Baars, T.L., Petri, S., Peters, C., Mayer, A., 2007. Role of the V-AT-Pase in regulation of the vacuolar fissionefusion equilibrium.Molecular Biology of the Cell 18, 3873e3882.

Bagar, T., Altenbach, K., Read, N.D., Bencina, M., 2009. Live-cellimaging and measurement of intracellular pH in filamentousfungi using a genetically encoded ratiometric probe. Eukary-otic Cell 8, 703e712.

Banta, L.M., Robinson, J.S., Klionsky, D.J., Emr, S.D., 1988. Organ-elle assembly in yeast: characterization of yeast mutantsdefective in vacuolar biogenesis and protein sorting. TheJournal of Cell Biology 107, 1369e1383.

Bankaitis, V.A., Johnson, L.M., Emr, S.D., 1986. Isolation of yeastmutants defective in protein targeting to the vacuole.Proceedings of the National Academy of Sciences of theUnited States of America 83, 9075e9079.

Barelle, C.J., Bohula, E.A., Kron, S.J., Wessels, D., Soll, D.R.,Schafer, A., Brown, A.J., Gow, N.A.R., 2003. Asynchronous cellcycle and asymmetric vacuolar inheritance in true hyphae ofCandida albicans. Eukaryotic Cell 2, 398e410.

Barelle, C.J., Richard, M.L., Gaillardin, C., Gow, N.A.R.,Brown, A.J.P., 2006. Candida albicans VAC8 is required forvacuolar inheritance and normal hyphal branching. Eukary-otic Cell 5, 359e367.

Bartholomew, C.R., Hardy, C.F., 2009. p21-activated kinases Cla4and Ste20 regulate vacuole inheritance in Saccharomyces cere-visiae. Eukaryotic Cell 8, 560e572.

Bayer, M.J., Reese, C., Buhler, S., Peters, C., Mayer, A., 2003.Vacuole membrane fusion: V0 functions after trans-SNAREpairing and is coupled to the Ca2þ-releasing channel. TheJournal of Cell Biology 162, 211e222.

Berman, J., 2006. Morphogenesis and cell cycle progression inCandida albicans. Current Opinion in Microbiology 9, 595e601.

Berman, J., Gow, N.A.R., 2004. Cell cycle of fungal pathogens. In:San-Blas, G., Calderone, R.A. (Eds), Pathogenic Fungi: Struc-tural Biology and Taxonomy. Caster Academic Press, Norfolk,pp. 101e125.

Bonangelino, C.J., Catlett, N.L., Weisman, L.S., 1997. Vac7p,a novel vacuolar protein, is required for normal vacuoleinheritance and morphology. Molecular and Cellular Biology17, 6847e6858.

Bonangelino, C.J., Nau, J.J., Duex, J.E., Brinkman, M.,Wurmser, A.E., Gary, J.D., Emr, S.D., Weisman, L.S., 2002.

Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinaseFab1p. The Journal of Cell Biology 156, 1015e1028.

Bone, N., Millar, J.B., Toda, T., Armstrong, J., 1998. Regulatedvacuole fusion and fission in Schizosaccharomyces pombe: anosmotic response dependent on MAP kinases. Current Biology8, 135e144.

Bowman, B.J., Draskovic, M., Freitag, M., Bowman, E.J., 2009.Structure and distribution of organelles and cellular locationof calcium transporters in Neurospora crassa. Eukaryotic Cell 8,1845e1855.

Bretscher, A., 2003. Polarized growth and organelle segregation inyeast: the tracks, motors, and receptors. The Journal of CellBiology 160, 811e816.

Brett, C.L., Merz, A.J., 2008. Osmotic regulation of Rab-mediatedorganelle docking. Current Biology 18, 1072e1077.

Burri, L., Lithgow, T., 2004. A complete set of SNAREs in yeast.Traffic 5, 45e52.

Cakar, Z.P., Sauer, U., Bailey, J.E., Muller, M., Stolz, M.,Wallimann, T., Schlattner, U., 2000. Vacuolar morphology andcell cycle distribution are modified by leucine limitation inauxotrophic Saccharomyces cerevisiae. Biology of the Cell 92,629e637.

Catlett, N.L., Weisman, L.S., 2000. Divide and multiply: organellepartitioning in yeast. Current Opinion in Cell Biology 12,509e516.

Chernomordik, L.V., Kozlov, M.M., 2008. Mechanics of membranefusion. Nature Structural and Molecular Biology 15, 675e683.

Collins, K.M., Thorngren, N.L., Fratti, R.A., Wickner, W.T., 2005.Sec17p and HOPS, in distinct SNARE complexes, mediateSNARE complex disruption or assembly for fusion. The EMBOJournal 24, 1775e1786.

Collins, K.M., Wickner, W.T., 2007. Trans-SNARE complexassembly and yeast vacuole membrane fusion. Proceedings ofthe National Academy of Sciences of the United States ofAmerica 104, 8755e8760.

Conradt, B., Haas, A., Wickner, W., 1994. Determination of fourbiochemically distinct, sequential stages during vacuoleinheritance in vitro. The Journal of Cell Biology 126, 99e110.

Conradt, B., Shaw, J., Vida, T., Emr, S., Wickner, W., 1992. In vitroreactions of vacuole inheritance in Saccharomyces cerevisiae.The Journal of Cell Biology 119, 1469e1479.

Darrah, P.R., Tlalka, M., Ashford, A., Watkinson, S.C.,Fricker, M.D., 2006. The vacuole system is a significant intra-cellular pathway for longitudinal solute transport in basidio-mycete fungi. Eukaryotic Cell 5, 1111e1125.

de Jong, J.C., McCormack, B.J., Smirnoff, N., Talbot, N.J., 1997.Glycerol generates turgor in rice blast. Nature 389, 244e245.

Dove, S.K., Cooke, F.T., Douglas, M.R., Sayers, L.G., Parker, P.J.,Michell, R.H., 1997. Osmotic stress activates phosphatidylino-sitol-3,5-bisphosphate synthesis. Nature 390, 187e192.

Dove, S.K., Dong, K., Kobayashi, T., Williams, F.K., Michell, R.H.,2009. Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIK-fyve underPPIn endo-lysosome function. The BiochemicalJournal 419, 1e13.

Duex, J.E., Nau, J.J., Kauffman, E.J., Weisman, L.S., 2006. Phos-phoinositide 5-phosphatase Fig4p is required for both acuterise and subsequent fall in stress-induced phosphatidylinosi-tol 3,5-bisphosphate levels. Eukaryotic Cell 5, 723e731.

Enyenihi, A.H., Saunders, W.S., 2003. Large-scale functionalgenomic analysis of sporulation and meiosis in Saccharomycescerevisiae. Genetics 163, 47e54.

Fagarasanu, A., Rachubinski, R.A., 2007. Orchestrating organelleinheritance in Saccharomyces cerevisiae. Current Opinion inMicrobiology 10, 528e538.

Gary, J.D., Sato, T.K., Stefan, C.J., Bonangelino, C.J., Weisman, L.S.,Emr, S.D., 2002. Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4,

102 A. Richards et al.

a polyphosphoinositide phosphatase family member. Molec-ular Biology of the Cell 13, 1238e1251.

Gomes de Mesquita, D.S., ten Hoopen, R., Woldringh, C.L., 1991.Vacuolar segregation to the bud of Saccharomyces cerevisiae: ananalysis of morphology and timing in the cell cycle. Journal ofGeneral Microbiology 137, 2447e2454.

Gow, N.A.R., Gooday, G.W., 1982a. Growth kinetics andmorphology of colonies of the filamentous form of Candidaalbicans. Journal of General Microbiology 128, 2187e2194.

Gow, N.A.R., Gooday, G.W., 1982b. Vacuolation, branch produc-tion and linear growth of germ tubes of Candida albicans.Journal of General Microbiology 128, 2195e2198.

Gow, N.A.R., Gooday, G.W., 1984. A model for the germ tubeformation and mycelial growth form of Candida albicans. Sab-ouraudia 22, 137e144.

Gow, N.A.R., Henderson, G., Gooday, G.W., 1986. Cytologicalinterrelationships between the cell cycle and duplication cycleof Candida albicans. Microbios 47, 97e105.

Haas, A., Conradt, B., Wickner, W., 1994. G-protein ligands inhibitin vitro reactions of vacuole inheritance. The Journal of CellBiology 126, 87e97.

Haas, A., Scheglmann, D., Lazar, T., Gallwitz, D., Wickner, W.,1995. The GTPase Ypt7p of Saccharomyces cerevisiae is requiredon both partner vacuoles for the homotypic fusion step ofvacuole inheritance. The EMBO Journal 14, 5258e5270.

Han, B.K., Bogomolnaya, L.M., Totten, J.M., Blank, H.M.,Dangott, L.J., Polymenis, M., 2005. Bem1p, a scaffold signalingprotein, mediates cyclin-dependent control of vacuolarhomeostasis in Saccharomyces cerevisiae. Genes and Develop-ment 19, 2606e2618.

Hanna, J., Finley, D., 2007. A proteasome for all occasions. FEBSLetters 581, 2854e2861.

Herr, F.B., Heath, M.C., 1982. The effects of antimicrotubuleagents on organelle positioning in the cowpea rust fungus,Uromyces phaseoli var vignae. Experimental Mycology 6, 15e24.

Heuser, J., 1989. Changes in lysosome shape and distributioncorrelated with changes in cytoplasmic pH. The Journal of CellBiology 108, 855e864.

Hickey, C.M., Stroupe, C., Wickner, W., 2009. The major role of theRab Ypt7p in vacuole fusion is supporting HOPS membraneassociation. The Journal of Biological Chemistry 284,16118e16125.

Hill, K.L., Catlett, N.L., Weisman, L.S., 1996. Actin and myosinfunction in directed vacuole movement during cell division inSaccharomyces cerevisiae. The Journal of Cell Biology 135,1535e1549.

Hoffman-Sommer, M., Migdalski, A., Rytka, J., Kucharczyk, R.,2005. Multiple functions of the vacuolar sorting protein Ccz1pin Saccharomyces cerevisiae. Biochemical and BiophysicalResearch Communications 329, 197e204.

Hohmann, S., 2002. Osmotic stress signaling and osmoadaptationin yeasts. Microbiology and Molecular Biology Reviews 66,300e372.

Hosomi, A., Kawanishi, Y.Y., Tanaka, N., Takegawa, K., 2008. PXAdomain-containing protein Pxa1 is required for normalvacuole function and morphology in Schizosaccharomycespombe. Bioscience, Biotechnology, and Biochemistry 72,548e556.

Howard, R.J., Ferrari, M.A., Roach, D.H., Money, N.P., 1991. Pene-tration of hard substrates by a fungus employing enormousturgor pressures. Proceedings of the National Academy ofSciences of the United States of America 88, 11281e11284.

Hyde, G.J., Davies, D., Cole, L., Ashford, A.E., 2002. Regulators ofGTP-binding proteins cause morphological changes in thevacuoles system of the filamentous fungus, Pisolithus tinctori-ous. Cell Motility and the Cytoskeleton 51, 133e146.

Hyde, G.J., Davies, D., Perasso, L., Cole, L., Ashford, A.E., 1999.Microtubules, but not actin microfilaments, regulate vacuole

motility and morphology in hyphae of Pisolithus tinctorius. CellMotility and the Cytoskeleton 42, 114e124.

Inselman, A.L., Gathman, A.C., Lilly, W.W., 1999. Two fluorescentmarkers identify the vacuolar system of Schizophyllumcommune. Current Microbiology 38, 295e299.

Ishikawa, K., Catlett, N.L., Novak, J.L., Tang, F., Nau, J.J.,Weisman, L.S., 2003. Identification of an organelle-specificmyosin V receptor. The Journal of Cell Biology 160, 887e897.

Jin, Y., Taylor Eves, P., Tang, F., Weisman, L.S., 2009. PTC1 isrequired for vacuole inheritance and promotes the associationof the myosin-V vacuole-specific receptor complex. MolecularBiology of the Cell 20, 1312e1323.

Jourdain, I., Sontam, D., Johnson, C., Dillies, C., Hyams, J.S., 2008.Dynamin-dependent biogenesis, cell cycle regulation andmitochondrial association of peroxisomes in fission yeast.Traffic 9, 353e365.

Jun, Y., Wickner, W., 2007. Assays of vacuole fusion resolve thestages of docking, lipid mixing, and content mixing. Proceed-ings of the National Academy of Sciences of the United Statesof America 104, 13010e13015.

Kane, P.M., 2006. The where, when, and how of organelle acidi-fication by the yeast vacuolar Hþ-ATPase. Microbiology andMolecular Biology Reviews 70, 177e191.

Kashiwazaki, J., Iwaki, T., Takegawa, K., Shimoda, C.,Nakamura, T., 2009. Two fission yeast rab7 homologs, ypt7and ypt71, play antagonistic roles in the regulation of vacuolarmorphology. Traffic 10, 912e924.

Kienle, N., Kloepper, T.H., Fasshauer, D., 2009. Phylogeny of theSNARE vesicle fusion machinery yields insights into theconservation of the secretory pathway in fungi. BMCEvolutionary Biology 9, 19.

Kikuma, T., Ohneda, M., Arioka, M., Kitamoto, K., 2006. Functionalanalysis of the ATG8 homologue Aoatg8 and role of autophagyin differentiation and germination in Aspergillus oryzae.Eukaryotic Cell 5, 1328e1336.

Kissova, I., Salin, B., Schaeffer, J., Bhatia, S., Manon, S.,Camougrand, N., 2007. Selective and non-selective autophagicdegradation of mitochondria in yeast. Autophagy 3, 329e336.

Klionsky, D.J., 2005. The molecular machinery of autophagy:unanswered questions. Journal of Cell Science 118, 7e18.

Klionsky, D.J., Herman, P.K., Emr, S.D., 1990. The fungal vacuole:composition, function, and biogenesis. MicrobiologicalReviews 54, 266e292.

Kuravi, K., Nagotu, S., Krikken, A.M., Sjollema, K., Deckers, M.,Erdmann, R., Veenhuis, M., van der Klei, I.J., 2006. Dynamin-related proteins Vps1p and Dnm1p control peroxisomeabundance in Saccharomyces cerevisiae. Journal of Cell Science119, 3994e4001.

Lang, T., Reiche, S., Straub, M., Bredschneider, M., Thumm, M.,2000. Autophagy and the cvt pathway both depend on AUT9.Journal of Bacteriology 182, 2125e2133.

Lee, I.H., Kumar, S., Plamann, M., 2001. Null mutants of theNeurospora actin-related protein 1 pointed-end complexshow distinct phenotypes. Molecular Biology of the Cell 12,2195e2206.

Li, S.C., Kane, P.M., 2009. The yeast lysosome-like vacuole:endpoint and crossroads. Biochimica et Biophysica Acta 1793,650e663.

Ma, J., Jin, R., Jia, X., Dobry, C.J., Wang, L., Reggiori, F., Zhu, J.,Kumar, A., 2007. An interrelationship between autophagyand filamentous growth in budding yeast. Genetics 177,205e214.

Mayer, A., Wickner, W., 1997. Docking of yeast vacuoles is cata-lyzed by the Ras-like GTPase Ypt7p after symmetric primingby Sec18p (NSF). The Journal of Cell Biology 136, 307e317.

Mizushima, N., 2005. The pleiotropic role of autophagy: fromprotein metabolism to bactericide. Cell Death and Differenti-ation 12, 1535e1541.

Fungal vacuoles 103

Mizushima, N., Levine, B., Cuervo, A.M., Klionsky, D.J., 2008. Au-tophagy fights disease through cellular self-digestion. Nature451, 1069e1075.

Negishi, T., Nogami, S., Ohya, Y., 2009. Multidimensional quan-tification of subcellular morphology of Saccharomyces cerevisiaeusing CalMorph, the high-throughput image-processingprogram. Journal of Biotechnology 141, 109e117.

Nichols, B.J., Ungermann, C., Pelham, H.R., Wickner, W.T.,Haas, A., 1997. Homotypic vacuolar fusion mediated by t- andv-SNAREs. Nature 387, 199e202.

Noda, T., Kim, J., Huang, W.P., Baba, M., Tokunaga, C.,Ohsumi, Y., Klionsky, D.J., 2000. Apg9p/Cvt7p is an integralmembrane protein required for transport vesicle formation inthe Cvt and autophagy pathways. The Journal of Cell Biology148, 465e480.

Ohsumi, K., Arioka, M., Nakajima, H., Kitamoto, K., 2002. Cloningand characterization of a gene (avaA) from Aspergillus nidulansencoding a small GTPase involved in vacuolar biogenesis.Gene 291, 77e84.

Oka, M., Maruyama, J., Arioka, M., Nakajima, H., Kitamoto, K.,2004. Molecular cloning and functional characterization ofavaB, a gene encoding Vam6p/Vps39p-like protein in Asper-gillus nidulans. FEMS Microbiology Letters 232, 113e121.

Ostrowicz, C.W., Meiringer, C.T., Ungermann, C., 2008. Yeastvacuole fusion: a model system for eukaryotic endomembranedynamics. Autophagy 4, 5e19.

Palmer, G.E., Askew, D.S., Williamson, P.R., 2008. The diverseroles of autophagy in medically important fungi. Autophagy 4,982e988.

Palmer, G.E., Kelly, M.N., Sturtevant, J.E., 2007. Autophagy in thepathogen Candida albicans. Microbiology 153, 51e58.

Pan, X., Roberts, P., Chen, Y., Kvam, E., Shulga, N., Huang, K.,Lemmon, S., Goldfarb, D.S., 2000. Nucleus-vacuole junctions inSaccharomyces cerevisiae are formed through the direct inter-action of Vac8p with Nvj1p. Molecular Biology of the Cell 11,2445e2457.

Penalva, M.A., 2005. Tracing the endocytic pathway of Aspergillusnidulans with FM4-64. Fungal Genetics and Biology 42,963e975.

Peng, Y., Weisman, L.S., 2008. The cyclin-dependant kinase Cdk1directly regulates vacuole inheritance. Developmental Cell 15,478e485.

Peplowska, K., Markgraf, D.F., Ostrowicz, C.W., Bange, G.,Ungermann, C., 2007. The CORVET tethering complex inter-acts with the yeast Rab5 homolog Vps21 and is involved inendo-lysosomal biogenesis. Developmental Cell 12, 739e750.

Peters, C., Baars, T.L., Buhler, S., Mayer, A., 2004. Mutual control ofmembrane fission and fusion proteins. Cell 119, 667e678.

Peters, C., Bayer, M.J., Buhler, S., Andersen, J.S., Mann, M.,Mayer, A., 2001. Trans-complex formation by proteolipidchannels in the terminal phase of membrane fusion. Nature409, 581e588.

Pham, C.D., Yu, Z., Sandrock, B., Bolker, M., Gold, S.E., Perlin, M.H.,2009. Ustilago maydis Rho1 and 14-3-3 homologues participatein pathways controlling cell separation and cell polarity.Eukaryotic Cell 8, 977e989.

Pinan-Lucarre, B., Balguerie, A., Clave, C., 2005. Accelerated celldeath in Podospora autophagy mutants. Eukaryotic Cell 4,1765e1774.

Price, A., Seals, D., Wickner, W., Ungermann, C., 2000. The dock-ing stage of yeast vacuole fusion requires the transfer ofproteins from a cis-SNARE complex to a Rab/Ypt protein. TheJournal of Cell Biology 148, 1231e1238.

Prosser, J.I., Tough, A.J., 1991. Growth mechanisms and growthkinetics of filamentous microorganisms. Critical Reviews inBiotechnology 10, 253e274.

Prosser, J.I., Trinci, A.P., 1979. A model for hyphal growth andbranching. Journal of General Microbiology 111, 153e164.

Raymond, C.K., Howald-Stevenson, I., Vater, C.A., Stevens, T.H.,1992. Morphological classification of the yeast vacuolarprotein sorting mutants: evidence for a prevacuolarcompartment in class E vps mutants. Molecular Biology of theCell 3, 1389e1402.

Rees, B., Shepherd, V.A., Ashford, A.E., 1994. Presence of a motiletubular vacuolar system in different phyla of fungi. Mycolog-ical Research 98, 985e992.

Reggiori, F., Klionsky, D.J., 2002. Autophagy in the eukaryotic cell.Eukaryotic Cell 1, 11e21.

Robinow, C.F., 1963. Observations on cell growth, mitosis, anddivision in the fungus Basidiobolus ranarum. The Journal of CellBiology 17, 123e152.

Robinson, J.S., Klionsky, D.J., Banta, L.M., Emr, S.D., 1988. Proteinsorting in Saccharomyces cerevisiae: isolation of mutantsdefective in the delivery and processing of multiple vacuolarhydrolases. Molecular and Cellular Biology 8, 4936e4948.

Rothlisberger, S., Jourdain, I., Johnson, C., Takegawa, K.,Hyams, J.S., 2009. The dynamin-related protein Vps1 regulatesvacuole fission, fusion and tubulation in the fission yeast,Schizosaccharomyces pombe. Fungal Genetics and Biology. doi:10.1016/j.fgb.2009.07.008.

Rothman, J.H., Howald, I., Stevens, T.H., 1989. Characterization ofgenes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae. The EMBO Journal 8,2057e2065.

Rothman, J.H., Stevens, T.H., 1986. Protein sorting in yeast:mutants defective in vacuole biogenesis mislocalize vacuolarproteins into the late secretory pathway. Cell 47, 1041e1051.

Rubinsztein, D.C., Gestwicki, J.E., Murphy, L.O., Klionsky, D.J.,2007. Potential therapeutic applications of autophagy. NatureReviews Drug Discovery 6, 304e312.

Rudge, S.A., Anderson, D.M., Emr, S.D., 2004. Vacuole size control:regulation of PtdIns(3,5)P2 levels by the vacuole-associatedVac14-Fig4 complex, a PtdIns(3,5)P2-specific phosphatase.Molecular Biology of the Cell 15, 24e36.

Schwartz, M.L., Merz, A.J., 2009. Capture and release of partiallyzipped trans-SNARE complexes on intact organelles. TheJournal of Cell Biology 185, 535e549.

Scott, S.V., Nice 3rd, D.C., Nau, J.J., Weisman, L.S., Kamada, Y., Ke-izer-Gunnink, I., Funakoshi, T., Veenhuis, M., Ohsumi, Y.,Klionsky, D.J., 2000. Apg13p and Vac8p are part of a complex ofphosphoproteins that are required for cytoplasm to vacuoletargeting. The Journal of Biological Chemistry 275, 25840e25849.

Seals, D.F., Eitzen, G., Margolis, N., Wickner, W.T., Price, A., 2000.A Ypt/Rab effector complex containing the Sec1 homologVps33p is required for homotypic vacuole fusion. Proceedingsof the National Academy of Sciences of the United States ofAmerica 97, 9402e9407.

Seeley, E.S., Kato, M., Margolis, N., Wickner, W., Eitzen, G., 2002.Genomic analysis of homotypic vacuole fusion. MolecularBiology of the Cell 13, 782e794.

Seiler, S., Plamann, M., Schliwa, M., 1999. Kinesin and dyneinmutants provide novel insights into the roles of vesicle trafficduring cell morphogenesis in Neurospora. Current Biology 9,779e785.

Shepherd, V.A., Orlovich, D.A., Ashford, A.E., 1993. Cell-to-celltransport via motile tubules in growing hyphae of a fungus.Journal of Cell Science 105, 1173e1178.

Shoji, J.Y., Arioka, M., Kitamoto, K., 2006a. Possible involvementof pleiomorphic vacuolar networks in nutrient recycling infilamentous fungi. Autophagy 2, 226e227.

Shoji, J.Y., Arioka, M., Kitamoto, K., 2006b. Vacuolar membranedynamics in the filamentous fungus Aspergillus oryzae.Eukaryotic Cell 5, 411e421.

Shorter, J., Warren, G., 2002. Golgi architecture and inheritance.Annual Review of Cell and Developmental Biology 18,379e420.

104 A. Richards et al.

Sipiczki, M., Takeo, K., Yamaguchi, M., Yoshida, S., Miklos, I.,1998. Environmentally controlled dimorphic cycle in a fissionyeast. Microbiology 144, 1319e1330.

Starai, V.J., Hickey, C.M., Wickner, W., 2008. HOPS proofreads thetrans-SNARE complex for yeast vacuole fusion. MolecularBiology of the Cell 19, 2500e2508.

Steinberg, G., 2000. The cellular roles of molecular motors infungi. Trends in Microbiology 8, 162e168.

Steinberg, G., 2007a. Preparing the way: fungal motors in micro-tubule organization. Trends in Microbiology 15, 14e21.

Steinberg, G., 2007b. On the move: endosomes in fungal growthand pathogenicity. Nature Reviews Microbiology 5, 309e316.

Steinberg, G., Schliwa, M., 1993. Organelle movements in the wildtype and wall-less fz;sg;os-1 mutants of Neurospora crassa aremediated by cytoplasmic microtubules. Journal of Cell Science106, 555e564.

Steinberg, G., Schliwa, M., Lehmler, C., Bolker, M., Kahmann, R.,McIntosh, J.R., 1998. Kinesin from the plant pathogenicfungus Ustilago maydis is involved in vacuole formation andcytoplasmic migration. Journal of Cell Science 111,2235e2246.

Stroupe, C., Collins, K.M., Fratti, R.A., Wickner, W., 2006. Purifi-cation of active HOPS complex reveals its affinities for phos-phoinositides and the SNARE Vam7p. The EMBO Journal 25,1579e1589.

Sudbery, P.E., Gow, N.A.R., Berman, J., 2004. The distinctmorphogenic states of Candida albicans. Trends in Microbi-ology 12, 317e324.

Swanson, J., Bushnell, A., Silverstein, S.C., 1987. Tubular lysosomemorphology and distribution within macrophages depend onthe integrity of cytoplasmic microtubules. Proceedings of theNational Academy of Sciences of the United States of America84, 1921e1925.

Tang, F., Kauffman, E.J., Novak, J.L., Nau, J.J., Catlett, N.L.,Weisman, L.S., 2003. Regulated degradation of a class Vmyosin receptor directs movement of the yeast vacuole.Nature 422, 87e92.

Tang, F., Peng, Y., Nau, J.J., Kauffman, E.J., Weisman, L.S., 2006.Vac8p, an armadillo repeat protein, coordinates vacuoleinheritance with multiple vacuolar processes. Traffic 7,1368e1377.

Tarutani, Y., Ohsumi, K., Arioka, M., Nakajima, H., Kitamoto, K.,2001. Cloning and characterization of Aspergillus nidulans vpsAgene which is involved in vacuolar biogenesis. Gene 268,23e30.

Tatsumi, A., Kikuma, T., Arioka, M., Kitamoto, K., 2006. Aovps24,a homologue of VPS24, is required for vacuolar formationwhich could maintain proper growth and development in thefilamentous fungus Aspergillus oryzae. Biochemical andBiophysical Research Communications 347, 970e978.

Tatsumi, A., Shoji, J.Y., Kikuma, T., Arioka, M., Kitamoto, K., 2007.Aggregation of endosomal-vacuolar compartments in theAovps24-deleted strain in the filamentous fungus Aspergillusoryzae. Biochemical and Biophysical Research Communica-tions 362, 474e479.

Trinci, A.P.J., 1969. A kinetic study of the growth of Aspergillusnidulans and other fungi. Journal of General Microbiology 57,11e24.

Trinci, A.P.J., 1974. A study of the kinetics of hyphal extension andbranch initiation of fungal mycelia. Journal of GeneralMicrobiology 81, 225e236.

Tuszynska, S., 2006. Ni2þ induces changes in the morphology ofvacuoles, mitochondria and microtubules in Paxillus involutuscells. The New Phytologist 169, 819e828.

Ungermann, C., Nichols, B.J., Pelham, H.R., Wickner, W., 1998a. Avacuolar v-t-SNARE complex, the predominant form in vivoand on isolated vacuoles, is disassembled and activated fordocking and fusion. The Journal of Cell Biology 140, 61e69.

Ungermann, C., Sato, K., Wickner, W., 1998b. Defining the func-tions of trans-SNARE pairs. Nature 396, 543e548.

Uetake, Y., Kojima, T., Ezawa, T., Saito, M., 2002. Extensivetubular vacuole system in an arbuscular mycorrhizal fungus,Gigaspora margarita. New Phytologist 154, 761e768.

Vaughn, L.E., Davis, R.H., 1981. Purification of vacuoles fromNeurospora crassa. Molecular Biology of the Cell 1, 797e806.

Veit, M., Laage, R., Dietrich, L., Wang, L., Ungermann, C., 2001.Vac8p release from the SNARE complex and its palmitoylationare coupled and essential for vacuole fusion. The EMBO Jour-nal 20, 3145e3155.

Veneault-Fourrey, C., Barooah, M., Egan, M., Wakley, G.,Talbot, N.J., 2006. Autophagic fungal cell death is necessary forinfection by the rice blast fungus. Science 312, 580e583.

Veses, V., Gow, N.A.R., 2008. Vacuolar dynamics during themorphogenetic transition in Candida albicans. FEMS YeastResearch 8, 1339e1348.

Veses, V., Richards, A., Gow, N.A.R., 2008. Vacuoles and fungalbiology. Current Opinion in Microbiology 11, 1e8.

Veses, V., Richards, A., Gow, N.A.R., 2009. Vacuole inheritanceregulates cell size and branching frequency of Candida albicanshyphae. Molecular Microbiology 71, 505e519.

Voges, D., Zwickl, P., Baumeister, W., 1999. The 26S proteasome:a molecular machine designed for controlled proteolysis.Annual Review of Biochemistry 68, 1015e1068.

Wada, Y., Ohsumi, Y., Anraku, Y., 1992. Genes for directingvacuolar morphogenesis in Saccharomyces cerevisiae. I.Isolation and characterization of two classes of vammutants. The Journal of Biological Chemistry 267,18665e18670.

Wang, C.W., Stromhaug, P.E., Kauffman, E.J., Weisman, L.S.,Klionsky, D.J., 2003. Yeast homotypic vacuole fusion requiresthe Ccz1-Mon1 complex during the tethering/docking stage.The Journal of Cell Biology 163, 973e985.

Wang, L., Seeley, E.S., Wickner, W., Merz, A.J., 2002. Vacuolefusion at a ring of vertex docking sites leaves membranefragments within the organelle. Cell 108, 357e369.

Wang, Y.X., Kauffman, E.J., Duex, J.E., Weisman, L.S., 2001.Fusion of docked membranes requires the armadillo repeatprotein Vac8p. The Journal of Biological Chemistry 276,35133e35140.

Wang, Y.X., Zhao, H., Harding, T.M., Gomes de Mesquita, D.S.,Woldringh, C.L., Klionsky, D.J., Munn, A.L., Weisman, L.S.,1996. Multiple classes of yeast mutants are defective invacuole partitioning yet target vacuole proteins correctly.Molecular Biology of the Cell 7, 1375e1389.

Warren, G., Wickner, W., 1996. Organelle inheritance. Cell 84,395e400.

Weber, R.W.S., 2002. Vacuoles and the fungal lifestyle. Mycologist16, 10e20.

Weber, R.W.S., Wakley, G.E., Thines, E., Talbot, N.J., 2001. Thevacuole as central element of the lytic system and sink forlipid droplets in maturing appressoria of Magnaporthe grisea.Protoplasma 216, 101e112.

Weisman, L.S., 2003. Yeast vacuole inheritance and dynamics.Annual Review of Genetics 37, 435e460.

Weisman, L.S., 2006. Organelles on the move: insights from yeastvacuole inheritance. Nature Reviews. Molecular Cell Biology 4,243e252.

Weisman, L.S., Bacallao, R., Wickner, W., 1987. Multiple methodsof visualizing the yeast vacuole permit evaluation of itsmorphology and inheritance during the cell cycle. The Journalof Cell Biology 105, 1539e1547.

Weisman, L.S., Emr, S.D., Wickner, W.T., 1990. Mutants ofSaccharomyces cerevisiae that block intervacuole vesiculartraffic and vacuole division and segregation. Proceedings ofthe National Academy of Sciences of the United States ofAmerica 87, 1076e1080.

Fungal vacuoles 105

Weisman, L.S., Wickner, W., 1988. Intervacuole exchange in theyeast zygote: a new pathway in organelle communication.Science 241, 589e591.

Weisman, L.S., Wickner, W., 1992. Molecular characterization ofVAC1, a gene required for vacuole inheritance and vacuoleprotein sorting. The Journal of Biological Chemistry 267,618e623.

Wickner, W., 2002. Yeast vacuoles and membrane fusion path-ways. The EMBO Journal 21, 1241e1247.

Wickner, W., Haas, A., 2000. Yeast homotypic vacuole fusion:a window on organelle trafficking mechanisms. AnnualReview of Biochemistry 69, 247e275.

Xie, Z., Klionsky, D.J., 2007. Autophagosome formation: coremachinery and adaptations. Nature Cell Biology 9, 1102e1109.

Yoon, Y., Pitts, K.R., McNiven, M.A., 2001. Mammalian dynamin-like protein DLP1 tubulates membranes. Molecular Biology ofthe Cell 12, 2894e2905.

Yorimitsu, T., Klionsky, D.J., 2005. Autophagy: molecularmachinery for self-eating. Cell Death and Differentiation 12,1542e1552.

Zhuang, X., Tlalka, M., Davies, D.S., Allaway, W.G.,Watkinson, S.C., Ashford, A.E., 2009. Spitzenkorper, vacuoles,ring-like structures, and mitochondria of Phanerochaete velu-tina hyphal tips visualized with carboxy-DFFDA, CMAC andDiOC6(3). Mycological Research 113, 417e431.

Zustiak, M.P., Pollack, J.K., Marten, M.R., Betenbaugh, M.J., 2008.Feast or famine: autophagy control and engineering in eukary-otic cell culture. Current Opinion in Biotechnology 19, 518e526.