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Volume 23 May 15, 2012 1889
M BoC | ARTICLE
Endosomal maturation by Rab conversion
in Aspergillus nidulans is coupled to dynein-
mediated basipetal movement
Juan F. Abenza*, Antonio Galindo, Mario Pinar, Areti Pantazopoulou, Vivian de los Ríos,
and Miguel A. Peñalva
Departamento de Medicina Molecular y Celular, Centro de Investigaciones Biológicas del Consejo Superior
de Investigaciones Cientificas, 28040 Madrid, Spain
ABSTRACT We exploit the ease with which highly motile early endosomes are distinguished
from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabSRab7
mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole
protein sorting/Vps41–dependent manner. Progression across the endocytic pathway in-
volves endosomal maturation because the end products of the pathway in the absence of
RabSRab7 are minivacuoles that are competent in multivesicular body sorting and cargo deg-
radation but retain early endosomal features, such as the ability to undergo long-distance
movement and propensity to accumulate in the tip region if dynein function is impaired. With-
out RabSRab7, early endosomal Rab5s—RabA and RabB—reach minivacuoles, in agreement
with the view that Rab7 homologues facilitate the release of Rab5 homologues from endo-
somes. RabSRab7 is recruited to membranes already at the stage of late endosomes still lack-
ing vacuolar morphology, but the transition between early and late endosomes is sharp, as
only in a minor proportion of examples are RabA/RabB and RabSRab7 detectable in the same—
frequently the less motile—structures. This early-to-late endosome/vacuole transition is cou-
pled to dynein-dependent movement away from the tip, resembling the periphery-to-center
traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies
establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.
INTRODUCTION
The mechanisms by which membrane and associated proteins traf-
fic across the endocytic pathway toward degradative organelles
(metazoan lysosomes and fungal vacuoles) have been intensively
investigated (Huotari and Helenius, 2011). Rather than being vesicle
mediated, traffic between early endosomes (EEs) and late endo-
somes (LEs) occurs by maturation. According to this view, EEs, which
receive biosynthetic traffic from the Golgi, progressively undergo
changes in lumenal pH and composition as they fuse homotypically
to give rise to gradually larger organelles. Simultaneously, portions
of endosomal membranes that are sorted into the multivesicular
body pathway bud inward, thereby delivering lipids and their asso-
ciated proteins into the lumen of the organelle. Thus maturation
results in organelles that are larger than EEs and display a character-
istic multivesicular appearance. These multivesicular, “late” endo-
somes undergo further fusion between themselves and with the
vacuoles/lysosomes, thus making their cargo accessible to digestion
by the vacuolar/lysosomal hydrolases.
Rabs are small GTPases that, when switched to their GTP confor-
mation, associate with intracellular membranes, recruiting to them
“effector” interacting proteins from the cytosol (Behnia and Munro,
2005). These effectors include tethers, soluble N-ethylmaleimide–
sensitive factor attachment protein receptor (SNARE) regulators,
and lipid-modifying enzymes that are major determinants of mem-
brane identity (Zerial and McBride, 2001; Pfeffer and Aivazian, 2004).
Monitoring Editor
Patrick J. Brennwald
University of North Carolina
Received: Nov 16, 2011
Revised: Mar 1, 2012
Accepted: Mar 20, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-11-0925) on March 28, 2012.
*Present address: The Wellcome Trust/Cancer Research UK Gurdon Institute,
University of Cambridge, Cambridge CB2 1QN, United Kingdom.
Address correspondence to: Miguel A. Peñalva (penalva@cib.csic.es).
© 2012 Abenza et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unport-
ed Creative Commons License (http://creativecommons.org/licenses/by-nc-
sa/3.0).
“ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
Abbreviations used: CMAC, 7-amino-4-chloromethyl-coumarin; CORVET, class C
core vacuole/endosome tethering; EE, early endosome; GDI, GDP dissociation
inhibitor; GEF, guanine nucleotide exchange factor; GST, glutathione S-trans-
ferase; HOPS, homotypic fusion and vacuole protein sorting; MT, microtubule.
1890 | J. F. Abenza et al. Molecular Biology of the Cell
brane/shape of the smaller CMAC-positive structures cannot be re-
solved by optical microscopy. Thus we will use the generic term “LE/
vacuole” to collectively refer to all “mature” structures that are
CMAC positive, irrespective of their size.
Here we characterize in detail RabSRab7 and demonstrate that it
localizes to vacuoles and to smaller structures, that HOPS is a
RabSRab7 effector, and that deletion of rabS leads to the formation of
small yet proteolytically competent CMAC-positive structures that,
unlike normal vacuoles, undergo long-distance movement and con-
tain early endosomal Rabs in their membranes. Time-lapse analyses
showed that the transition between RabA/B-containing EEs and
RabSRab7 structures is sharp, although colocalization can be detected
on large (and thus less motile) endosomal aggregates. Dynein im-
pairment results in the accumulation of vacuoles in a region near the
tip that is normally devoid of them. Taken together, our data sup-
port a model in which endosomal maturation progresses as endo-
somes move away from the tip, losing motility as they enlarge
through homotypic fusion.
RESULTS
RabSRab7 localizes to the vacuolar membranes
We studied the localization of green fluorescent protein (GFP)–
tagged or mCherry-tagged versions of RabSRab7 with constructs
containing the rabS promoter or the regulatable promoter alcAp. All
constructs were single-copy integrated into the genome by targeted
recombination (Calcagno-Pizarelli et al., 2007). Anti-GFP Western
analysis, used to determine the levels of GFP-RabSRab7 expression
obtained with the alcAp driver, showed that these were below de-
tection on glucose (alcAp-repressing conditions), very high on etha-
nol (alcAp-inducing conditions), and low on 0.1% fructose (nonin-
duced, nonrepressed alcAp conditions; Figure 1A). alcAp-driven
fructose levels were similar or slightly below those driven by the
rabSp (Figure 1B). Whether GFP or mCherry tagged, overexpressed
(ethanol; Figure 1C) or expressed to approximately physiological
levels (fructose; Figure 1D), RabSRab7 robustly localizes to the mem-
brane or spherical vacuoles whose lumen is stainable with the vacu-
olar tracer CMAC.
Rabs associate with membranes in their GTP conformation
(Behnia and Munro, 2005). On GTP hydrolysis, GDP-Rabs are ex-
tracted to the cytosol by the GDP dissociation inhibitor (GDI; en-
coded by A. nidulans gdiA). Figure 1F shows that the vacuolar mem-
brane localization of RabSRab7 is strictly dependent on the nucleotide
switch of the GTPase, as mutant Thr22Asn GFP-RabSRab7, locked in
the GDP conformation, is cytosolic (Figure 1F), even though the mu-
tation does not affect the steady-state protein levels (Figure 1E).
This shows that GFP-RabSRab7 reflects the physiological localization
of RabS.
The size and relative abundance of vacuoles increase
with the distance to the tip
We exploited the fact that RabSRab7 specifically labels vacuoles to
measure their distribution along hyphae. We determined the per-
centage of cell projection surface occupied by vacuoles in 30- to
60-μm-long germlings (n = 33), which were segmented into five
zones (Figure 2A). Vacuoles occupied, on average, 40% of the coni-
diospore projection but only 9% of the region nearest to the tip. The
regions between the tip and the base showed intermediate scores,
the overall tendency being that vacuole-occupied surface increases
with the distance to the tip. This increase correlates with a parallel
increase in vacuolar diameter (Figure 1C; Peñalva, 2005; Findon
et al., 2010). Thus the biogenesis of vacuoles is connected to the
relative position within hyphae, such that tip-proximal regions are
Owing to the high specificity of any given Rab for a certain mem-
brane, Rabs have been extensively used in membrane traffic stud-
ies, including endosomal maturation itself, as the transition between
EEs and LEs appears to be mediated by Rab conversion (Rink et al.,
2005; Segev, 2011). In its simplest version, Rab conversion implies
that early endosomal membrane domains containing Rab5 are con-
verted into late endosomal domains containing Rab7. As a result,
Rab7 effectors substitute for Rab5 effectors, leading to a shift in
compartmental identity toward LEs. Replacement of Rab5 by Rab7
on endosomes requires that the positive feedback loops that stabi-
lize Rab5 domains (Zerial and McBride, 2001) need to be inacti-
vated, and the mechanisms facilitating Rab7 recruitment need to be
activated coordinately (Rink et al., 2005; Nordmann et al., 2010;
Segev, 2011).
Major insight into the mechanisms of endosomal maturation
came from work with Saccharomyces cerevisiae (Price et al., 2000;
Peplowska et al., 2007; Markgraf et al., 2009; Brocker et al., 2010;
Cabrera et al., 2010; Nordmann et al., 2010; Ostrowicz et al., 2010).
Endosomal Rab conversion results in substitution of the Vps21p (i.e.,
Rab5) effector class C core vacuole/endosome tethering (CORVET)
complex (Peplowska et al., 2007) by the Ypt7p (i.e., Rab7) effector
homotypic fusion and vacuole protein sorting (HOPS) complex
(Seals et al., 2000). However, the actual mechanism by which HOPS
substitutes for CORVET has not been established. Both HOPS and
CORVET are multiprotein complexes that share four class C pro-
teins—Vps11p, Vps16p, Vps18p, and Vps33p (Rieder and Emr,
1997)—and only differ in two specific components each—Vps8p
and Vps3p for CORVET and Vps41 and Vps39 for HOPS.
A hurdle hindering correlation between subcellular and mecha-
nistic studies of endosomes is implicit in the maturation concept: by
definition, the distinction between EEs and LEs is unavoidably
blurred (Huotari and Helenius, 2011). The basidiomycete Ustilago
maydis and the ascomycete Aspergillus nidulans are ideally suited
for studying endosomal maturation because their EEs can be un-
equivocally identified by their characteristic long-distance bidirec-
tional movement on microtubule-dependent motors (Wedlich-
Soldner et al., 2002; Lenz et al., 2006; Steinberg, 2007; Zekert and
Fischer, 2008; Abenza et al., 2009, 2010; Hervás-Aguilar et al., 2010;
Peñalva, 2010; Zhang et al., 2010, 2011; Schuster et al., 2011).
A. nidulans has two Rab5 paralogues—RabA and RabB—localiz-
ing to EEs (Abenza et al., 2009, 2010) and a single Rab7 homologue
(Ohsumi et al., 2002; Sánchez-Ferrero and Peñalva, 2006), which we
denoted RabS (Rab seven, RabSRab7). The CORVET complex is an
effector of RabB and, to a lesser extent, of RabA (Abenza et al.,
2010). Whether HOPS is a RabSRab7 effector was unknown. Because
maturation involves the CORVET-mediated coalescence of EEs into
progressively larger endosomes (Markgraf et al., 2009), it seems
plausible that the resulting increase in size inevitably results in de-
creased motility. Such decreased motility would be a bona fide fea-
ture of LEs. Indeed, overexpression of the CORVET recruiters RabA/
RabB leads to formation of endosomal aggregates with reduced
motility and multivesicular appearance (a landmark of LEs; Abenza
et al., 2009, 2010; Griffith et al., 2011). However, as discussed by
Huotari and Helenius (2011), the presence of internal vesicles can-
not be the sole criterion to define LEs. Indeed ESCRT-III appears to
operate in A. nidulans EEs (Galindo et al., 2007; Hervás-Aguilar
et al., 2010). LEs and vacuoles can be visualized with 7-amino-4-
chloromethyl-coumarin (CMAC), a fluorescent compound that labels
the lumen of hydrolase-containing acidic organelles, including vacu-
oles and smaller structures. Here we use the term “vacuole” to refer
to spherical CMAC-positive organelles whose limiting membrane is
stainable with FM4-64 (Peñalva, 2005). However, the limiting mem-
Volume 23 May 15, 2012 Endosomal maturation in Aspergillus | 1891
Partial overlap of the target-SNARE Pep12 with RabSRab7
The A. nidulans orthologue of S. cerevisiae Pep12p is the only syn-
taxin across the endovacuolar system (Sánchez-Ferrero and Peñalva,
relatively devoid of these organelles. Vacuoles rarely underwent
long-distance movements (Figure 3; see later discussion for a de-
tailed consideration), making unlikely that the mechanism underly-
ing their asymmetrical distribution involves basipetal transport. The
strict and robust GFP-RabSRab7 localization to vacuolar membranes
facilitated time-lapse studies permitting visualization of vacuoles un-
dergoing fusion (Supplemental Figure S1).
FIGURE 1: Subcellular localization of RabSRab7. (A) Anti-GFP Western
blot analysis showing the different steady-state levels of GFP-RabSRab7
achieved under repressing (1% glucose, Glc), noninducing,
nonrepressing (0.1% fructose, Frc) and inducing (1% ethanol, EtOH)
conditions for the alcAp driver. Actin was used as loading control.
(B) GFP-RabSRab7 levels driven by the physiological promoter
compared with those obtained with alcAp. (C) Vacuolar localization
of GFP-RabSRab7 (alcAp driver, ethanol conditions). (D) Vacuolar
localization of GFP-RabSRab7 (alcAp driver, fructose conditions).
(E) Western blot demonstrating that Thr22Asn substitution does not
affect the steady-state level of GFP-RabSRab7 (T22N). (F) GFP-RabSRab7
(T22N) locked in the GDP conformation is cytosolic. Bars, 5 μm.
FIGURE 2: A. nidulans vacuolar distribution. Pep12 is present in EEs
and LEs/vacuoles. (A) Vacuolar distribution: 30- to 60-μm germlings were
arbitrarily divided into the “conidium” and four “rectangular” sections
of approximately equivalent length, as indicated. The percentage of the
total projection area occupied by vacuoles in each section was plotted in
n = 33 germlings. (B) mCherry-RabSRab7 colocalizes with GFP-Pep12 on
vacuoles. However, GFP-Pep12 additionally localizes to punctate
structures that are not labeled by mCherry-RabSRab7. Regions of interest
are shown at double magnification in the indicated red, green, and
merge channels. (C) Kymograph of a 4-frame/s time-lapse sequence of a
cell coexpressing GFP-Pep12 and the EE marker mCherry-RabA. Pep12
and RabA colocalize in the population of rapidly moving EEs and in less
abundant larger and static RabA-containing structures but not on
vacuoles, which do not contain RabA.
1892 | J. F. Abenza et al. Molecular Biology of the Cell
rabSΔ strictly cosegregates with the growth/
conidiation defects in the progeny of crosses
established that these phenotypic features
are solely attributable to the rabSΔ allele.
rabSΔ results in proteolytically
competent minivacuoles capable of
undergoing movement
rabS disruption results in small vacuoles
(Ohsumi et al., 2002). Strains carrying rabSΔ
indeed showed minute vacuoles (CMAC
staining; Figure 3B) whose small lumen is
hardly resolvable by optical microscopy
(using GFP-Pep12 to label their membranes;
Figure 3C). Thus, in agreement with previ-
ous data and with work in S. cerevisiae
Ypt7p, RabSRab7 is involved in the biogene-
sis of “normal-sized” vacuoles. rabSΔ “mini-
vacuoles” show two noteworthy features: 1)
They are evenly distributed across hyphae,
unlike wild-type vacuoles (Figure 3, D and
E), and 2) a proportion of mutant minivacu-
oles undergoes long-range movements
similar to those of EEs (reflected by diago-
nal lines in kymographs), contrasting with
the essentially immotile wild-type vacuoles
(vertical lines in kymographs; Figure 3, D and E, and Supplemental
Movies S2 [wild-type] and S3 [rabSΔ]). Given that the motility of EEs
is RabB dependent (Abenza et al., 2010), we hypothesized that the
inability of endosomes to exchange RabSRab7 for RabB might result
in RabB persisting on minivacuoles (see later discussion).
We next determined whether rabSΔ minivacuoles are competent
in proteolysis of an endocytic cargo, the plasma membrane amino
acid permease AgtA. AgtA is expressed on glutamate medium, lo-
calizing to the plasma membrane and vacuoles (Figure 4A). If gluta-
mate-cultured cells are shifted to ammonium, AgtA synthesis is
strongly repressed, and the plasma membrane-resident pool of the
transporter is delivered to the vacuole via endocytosis (Apostolaki
et al., 2009). In rabSΔ cells, AgtA-GFP is also delivered to the lumen
of (mini-) vacuoles (Figure 4A). Western blots revealed similar kinet-
ics of AgtA-GFP degradation in rabSΔ and rabS+ cells (Figure 4B).
Thus rabSΔ does not affect the sorting of cargoes into the MVB
pathway, and minivacuoles are proteolytically competent.
RabSRab7 recruits HOPS
We next used glutathione S-transferase (GST)–RabSRab7, loaded with
GDP or GTP-γ-S, and cell extracts containing endogenously 3× he-
magglutinin (HA)-tagged interactors in pull-down experiments
(Figure 5A). GdiA was specifically and efficiently pulled down by GDP-
loaded RabSRab7, indicating that GTP-γ-S shifts RabSRab7 toward a con-
formation that prevents its interaction with the Rab-GDP effector
GdiA (Figure 5A). The fusogenic activity of Ypt7p involves HOPS.
Vps41p is the Ypt7p effector subunit in HOPS linking the complex to
the GTP-loaded Rab (Cabrera et al., 2010; Ostrowicz et al., 2010). In
agreement, A. nidulans Vps41 was specifically pulled down by GTP-γ-S
RabSRab7 but not at all by RabB, which instead pulled down Vps8 ef-
ficiently (Vps8 is the CORVET equivalent of Vps41; Peplowska et al.,
2007; Markgraf et al., 2009). In contrast, Vps39, which also showed
strict RabSRab7 specificity, was preferentially retained by GTP-γ-S Rab-
SRab7 but interacted substantially with GDP-RabSRab7 (Figure 5A).
Given that we used unfractionated “prey extracts,” this finding is con-
sistent with the finding that whereas the Vps39-containing HOPS
2006; Findon et al., 2010; S. cerevisiae has two—Pep12p for pre-
vacuolar endosomes and Vam3p for vacuoles). mCherry-RabSRab7
strictly colocalizes with GFP-Pep12, but the reverse is not true, as
GFP-Pep12 labels punctate structures that do not contain RabSRab7
(Figure 2B). These punctate structures undergo long-distance bidi-
rectional movements, suggesting that they are EEs. Thus we con-
structed a strain coexpressing GFP-Pep12 and the EE marker
mCherry-RabA and recorded time-lapse sequences (∼4 frames/s)
simultaneously in the GFP and mCherry channels. These sequences
were analyzed with kymographs in which the y-axis represents the
time scale. Thus static and short-range moving structures appear as
vertical lines, whereas fast-moving structures appear as diagonal
lines whose slope reflects the speed/direction and length reflects
the range of movement of any given particle. Figure 2C shows that
GFP-Pep12 localizes to static vacuoles (bright vertical lines) that do
not contain RabA, to static structures containing RabA (LEs; see later
discussion), and, in addition, to a population of small, bidirectionally
motile structures (thus EEs; Figure 2C diagonal lines; Supplemental
Movie S1) in which it colocalizes with RabA. This important finding
indicates that Pep12 plays roles at the levels of the A. nidulans EEs,
LEs, and vacuoles.
rabSΔ slightly impairs vegetative growth and results in a
temperature-dependent defect in conidiation
We constructed a null rabSΔ allele, which results in a minor vegeta-
tive growth defect at any of the three temperatures tested (30, 37,
and 42°C), more noticeable on synthetic than on rich medium
(Figure 3A). Its most conspicuous phenotype was the nearly com-
plete absence of conidiation at 42°C (Figure 3A). These phenotypic
aspects of rabSΔ somewhat resemble those of rabBΔ (RabB is the
major Rab5), but they are markedly less pronounced (Figure 3A;
compare middle and right columns on SC). This indicates that
RabSRab7 plays a less important physiological role than RabB. The
findings that rabSp::gfp-rabS and alcAp::gfp-rabS transgenes comple-
ment the 42°C rabSΔ growth and conidiation defects (demonstrating
that GFP-RabSRab7 is functional; Supplemental Figure S2) and that
FIGURE 3: rabSΔ results in abnormally motile minivacuoles. (A) Growth phenotypes of the
indicated strains at the indicated media and temperatures. MCA, complete medium. SC,
synthetic complete medium. (B) CMAC and (C) GFP-Pep12 visualization of rabSΔ minivacuoles.
All images are depicted at the same magnification. Bar, 2 μm. (D) Still frames (blue) and
kymographs (inverted contrast, shown at the same scale as still images) correspond to 10-s time-
lapse series of CMAC-stained vacuoles. Note the multiple diagonal lines seen in the rabSΔ
example. Both images are at the same magnification. Bar, 5 μm.
Volume 23 May 15, 2012 Endosomal maturation in Aspergillus | 1893
contained all six components of HOPS—
four components shared with CORVET
(Vps11, Vps18, Vps16, and Vps33) and the
HOPS-specific components Vps39 and
Vps41 (Figure 5B and Supplemental Table
S1). HOPS components were only slightly
less efficiently retained by the GDP column,
suggesting that GST-RabSRab7 immobilized
in the Sepharose support does not com-
pletely undergo the nucleotide-induced
conformational changes mediating RabSRab7
function in vivo. (Work with S. cerevisiae
Ypt7p showed that purified HOPS shows
selectivity for the GTP-γ-S–loaded Ypt7p
form; Ostrowicz et al., 2010.) In summary,
these experiments demonstrate that HOPS
is an effector of RabSRab7.
Endosomal maturation is essential but
vacuolar fusion is not
The two Rab5s (RabA and RabB) and their
effectors mediate the coalescence of EEs
and their maturation into LEs (Abenza et al.,
2010). Vps45 and its binding partner
Vac1Vps19 are specific RabB effectors,
whereas the CORVET complex (containing
Vps8) is a RabB and RabA effector (Abenza
et al., 2010). A double rabAΔ rabBΔ dele-
tion is nearly lethal, and single vps45Δ and
vps8Δ mutations are severely debilitating
(Abenza et al., 2010), as are deletions involving ESCRT-0, -I, -II, and
-III components mediating the biogenesis of multivesicular endo-
somes (Calcagno-Pizarelli et al., 2011). These data indicate that
maturation and coalescence of EEs into larger structures (LEs) is es-
sential. In contrast, rabSΔ mutants are perfectly viable, and thus the
RabSRab7-mediated fusion of LEs with vacu-
oles, of LEs with LEs to form vacuoles, and
the homotypic fusion of vacuoles is largely
dispensable (Figure 3A).
These conclusions led to three predic-
tions: 1) vac1Δ should be as severely debili-
tating as vps45Δ; 2) deletion of any one
gene encoding a HOPS class C component
shared with CORVET should lead to near
lethality due to its role in CORVET; and 3)
deletion of the gene encoding the HOPS-
specific subunit Vps41 should be viable,
phenocopying rabSΔ mutations. We de-
leted vac1 and vps18 (Vps18 is a class C
CORVET and HOPS subunit). Figure 6A
shows the corresponding growth pheno-
types. vac1Δ colonies show severely re-
duced growth, similar to that of vps45Δ
colonies. The growth defect of vps18Δ colo-
nies is even more marked, indicating that
this class C protein is virtually essential.
Vps41 is the HOPS equivalent of COR-
VET Vps8 (Peplowska et al., 2007). In marked
contrast with vps8Δ or vps18Δ, vps41Δ
strains are relatively healthy and indistin-
guishable from a rabSΔ strains in growth
and conidiation (note the slight reduction in
complex is a Ypt7p (GTP) effector, “free” Vps39p binds Ypt7p irre-
spective of the nucleotide status (Ostrowicz et al., 2010).
We passed cell-free extracts through GDP- or GTP-γ-S–loaded
GST-RabSRab7 affinity columns (Figure 5B). SDS–PAGE and tandem
mass spectrometry (MS/MS) analyses showed that GTP-γ-S eluates
FIGURE 4: rabSΔ minivacuoles are proteolytically competent. (A) Normal traffic to the vacuoles
of the 12-TMD protein AgtA, which mediates the uptake of dicarboxylic amino acids. In these
assays, AgtA is endogenously tagged with GFP in its (cytosolic) C-terminus. Wild-type and rabSΔ
cells expressing AgtA-GFP were initially cultured on glutamate. Under these conditions, AgtA
localizes to the plasma membrane and, to a lesser extent, to the vacuoles. These cells were then
shifted to ammonium, which shuts the agtA promoter off and promotes the endocytic delivery
of plasma membrane–localizing AgtA-GFP to the vacuolar lumen. (B) Western blots showing the
proteolytic vacuolar degradation of AgtA-GFP following transfer of cells to ammonium and
further incubation for the indicated times. GFP is recalcitrant to degradation by vacuolar
proteases but more so in the rabSΔ mutant.
FIGURE 5: RabSRab7 recruits HOPS. (A) Glutathione–Sepharose beads containing GST-RabB or
GST-RabSRab7 loaded with the indicated nucleotides were used as bait in pull-downs with
whole-cell protein extracts, which were obtained from strains expressing, at physiological levels,
GdiA (the only A. nidulans guanine nucleotide dissociation inhibitor), the CORVET component
Vps8, or the HOPS components Vps39 and Vps41, endogenously tagged with 3× HA. The 3×
HA baits were detected by Western blotting. (B) A. nidulans whole-cell extracts were passed
through GST-RabSRab7 affinity columns loaded with the indicated nucleotides, and the bound
proteins were eluted, separated in an SDS–polyacrylamide gel, and stained. The identity of the
bands was determined by MS/MS (Supplemental Table S1). Vps41* and Vps39** indicate that
these bands are degradation products. Full-length Vps39 is also detectable in the gel.
1894 | J. F. Abenza et al. Molecular Biology of the Cell
alcAp-driven transgenes. Using this regulatable (by the carbon
source) promoter allowed us exploit three different levels of expres-
sion: low (fructose), intermediate (glycerol), and high (ethanol). None
of these carbon source conditions led to any detectable effect on
colony growth (Supplemental Figure S4), strongly indicating that ex-
pression of the fusion proteins does not interfere with the physiol-
ogy of the endosomal system.
We acquired high-speed time-lapse sequences (10–20 frames/s)
of these strains in the red and green channels simultaneously using
a Dual-Viewer. RabSRab7 and RabA/RabB showed little colocalization
(Figure 7A and Supplemental Movies S4 and S5). RabSRab7 localized
to the vacuolar membranes, to relatively static structures, and, oc-
casionally, to moving structures (see later discussion). By contrast,
RabA or RabB did not label the vacuolar membranes at all, localizing
to moving EEs and to less abundant static structures. As in U. may-
dis (Schuster et al., 2011), moving RabA/RabB EEs usually traveled
for several seconds in one direction before reversing movement in
the opposite direction, with (U-shaped tracing in the kymograph;
Figure 7B) or without (V-shaped tracings; Figure 8) arresting for
some time in between. This behavior reflects the tug-of-war action
of dynein and kinesins on the same endosome (Schuster et al.,
2011). Time-lapse sequences revealed very unusual examples of
vacuolar movement, like the ∼6-μm journey of the small vacuole in
Figure 7C to dock at a large vacuole (Supplemental Movie S6). This
movement was short ranged and slower than that of EEs (compare
slopes of green- and red-channel kymographs).
The transition between RabA/RabB and RabSRab7 is sharp
RabB is the major determinant of endosomal maturation (Abenza
et al., 2010). Fructose kymographs (Figure 8, A1 and A2) revealed
occasional yet rare examples of moving GFP-RabB EEs faintly
stained with mCherry-RabSRab7 (Figure 8A2, arrows). In contrast,
static structures containing RabB and RabSRab7 were clearly more
abundant (Figure 8A1, arrows). Similar overall absence of colocaliza-
tion in moving structures was seen on glycerol, despite the increased
colony size and lack of conidiation at 42°C; Figure 6B). CMAC stain-
ing (Supplemental Figure S3) showed that vps41Δ mutants also re-
semble rabSΔ mutants in the “minute vacuole” phenotype and in
the loss of the tip-to-base gradient of vacuolar distribution. Thus the
rabSΔ minute vacuole phenotype results from inability to recruit
HOPS. Vps41, able to bind membranes of LEs via its ALPS domain
(Cabrera et al., 2010), might conceivably contribute to the mem-
brane recruitment of its Rab. However, GFP-RabSRab7 is recruited to
the membranes of CMAC-stained vps41Δ minivacuoles in the ab-
sence of Vps41 (Figure 6D).
The motility of the rabSΔ minivacuoles suggests that they retain
endosome features, even though they have the characteristic spheri-
cal shape of vacuoles and their lumen is large enough to be optically
resolvable. However, in the absence of RabSRab7 (and therefore of
HOPS), endosomes can only fuse into minivacuoles by using COR-
VET (i.e., in a RabA/RabB-dependent manner; Abenza et al., 2010).
rabBΔ and rabAΔ are synthetically sick with rabSΔ, the double
mutations leading to a stronger impairment of growth and conidia-
tion than either single deletion (Figure 6C). Given that vps41Δ and
rabSΔ cause similar synthetic growth defects in combination with
rabBΔ (Figure 6C), the debilitating effect of the double rabSΔ rabBΔ
mutation involves the inability to recruit HOPS and not another
RabSRab7-mediated process. Two non–mutually exclusive interpreta-
tions can account for these observations. One is that in the absence
of RabSRab7/HOPS, impairing the recruitment of CORVET to endo-
somes critically compromises the formation of functional rabSΔ
minivacuoles. A second is that RabSRab7/HOPS has RabA/RabB-in-
dependent functions. For example, rabSΔ could prevent, in addition
to LE–vacuole and vacuole–vacuole fusion, the entry of Vps41-
dependent traffic into the vacuoles through a pathway akin to the
S. cerevisiae AP-3 pathway (Cabrera et al., 2010).
Subcellular distribution of Rab5s and RabSRab7
To study endosomal Rab transitions, we constructed strains express-
ing mCherry-RabSRab7 and GFP-RabA or GFP-RabB from single-copy
FIGURE 6: Genetic studies of endosomal maturation. (A) Growth phenotypes of vps18Δ (encoding a class C subunit
shared by the CORVET and HOPS complexes) and vac1Δ (Vac1, also denoted Vps19, is a specific RabB effector). vps18Δ
and vac1Δ are very severely debilitating. Note that vps18 was previously called digA. A digA1 early-truncating mutation
removing the Vps18 C-terminal RING finger leads to a temperature-dependent growth defect (Geissenhoner et al., 2001).
(B) Growth phenotypes at the indicated temperatures of vps41Δ compared with rabSΔ. The two mutations are
phenotypically indistinguishable. (C) Both rabAΔ and rabBΔ result in a synthetic growth defect in double mutants with
rabSΔ. rabBΔ also results in a similar synthetic growth defect in double mutants with vps41Δ (rabAΔ was not tested in
combination with vps41Δ). (D) Like rabSΔ, vps41Δ results in minivacuoles. In the absence of Vps41, the limiting membranes
of CMAC-containing minivacuoles are labeled with GFP-RabSRab7. Images of a z-series are shown, with numbers indicating
relative z-position in micrometers. For comparison, GFP-RabSRab7 wild-type vacuoles are shown on the right.
Volume 23 May 15, 2012 Endosomal maturation in Aspergillus | 1895
motile, generally moved for shorter dis-
tances (Figure 8, B1 and B2). RabSRab7 was
clearly present in a proportion of RabB-con-
taining structures, generally corresponding
to these large aggregates (Figure 8, B1 and
B2, double arrowed; one “control” RabSRab7-
negative EE is single arrowed). However,
RabB was absent from vacuolar membranes,
even though these were strongly positive for
RabSRab7. Therefore, although these strongly
overexpressing conditions are not physio-
logical, these experiments establish that
RabSRab7 can be recruited to endosomal
membranes containing high levels of RabB.
In contrast, RabB is excluded from vacuolar
membranes, where RabSRab7 is abundant.
These data are consistent with a maturation
model in which endosomes acquire RabSRab7
in their late steps of maturation, losing RabB
as they become vacuoles.
Similar experiments using GFP-RabA re-
vealed a previously unnoticed feature of
RabA endosomes: even under fructose con-
ditions leading to GFP-RabA levels similar
to those attained with the endogenous pro-
moter (Abenza et al., 2009), we detected a
background of very abundant small, moving
endosomes that we did not see with GFP-
RabB; their kymograph tracings formed a
net of diagonal lines against which vertical
lines of more static aggregates were promi-
nent (Figure 8, C and D, Supplemental Fig-
ure S5, and Supplemental Movie S9). As
with RabB, we only very occasionally de-
tected EEs labeled with RabSRab7 and RabA.
The tracings arrowed in Figure 8D and Sup-
plemental Figure S5B are the only examples
among the numerous moving RabA EEs in
which RabSRab7 was detectable. We thus
conclude that the transition between RabA/
RabB and RabSRab7 domains is relatively
sharp.
RabB and RabA reach the vacuoles
in rabSΔ
rabSΔ markedly increases the abundance of
RabA/RabB structures. In 30-μm-long pro-
jections of wild-type tip regions (n = 8 hy-
phae), GFP-RabB structures accounted for
4.4 ± 0.6% of the area. This figure was markedly higher (sixfold; 23.3
± 2.5%) in the mutant. This hypertrophy of the rabSΔ endosomal
compartment might reflect the inability of LEs to undergo HOPS-
mediated fusion or the ability of EE Rabs to invade membranes oth-
erwise restricted to RabSRab7 (or both). One observation lent cre-
dence to the “Rab5 invasion” possibility. In the wild type, GFP-RabA/
RabB eventually label aggregates of endosomes that are not vacu-
oles (they are multivesicular, irregularly shaped, and CMAC negative;
Abenza et al., 2009, 2010; Griffith et al., 2011). In contrast, in rabSΔ
cells, both RabA (Supplemental Movie S10) and RabB (Figure 9A)
clearly label the membranes of minivacuoles. To further demonstrate
this, cells expressing GFP-RabB were pretreated with benomyl (to
abolish the microtubule [MT]-dependent motility of minivacuoles)
fluorescence signal. Again, colocalization predominated in the more
static structures or in those motile structures that moved for shorter
distances and at slower speeds than EEs (Supplemental Figure S5A).
Figure 7D and Supplemental Movies S7 and S8 show structures
containing RabB and RabSRab7 moving at 1–1.8 μm/s, more slowly
than EEs (∼2.5 μm/s; Abenza et al., 2009). We speculate that these
double-labeled structures, whose size would impede/retard move-
ment, represent late or mature endosomes where Rab conversion is
taking place.
To confirm that RabB and RabSRab7 can actually coincide on the
less motile endosomes, we induced the transgenes with ethanol,
which results in strong overexpression of the Rabs. RabB still local-
ized to fast-moving EEs. It also localized to aggregates that, albeit
FIGURE 7: Coimaging of early endosomal Rabs and RabSRab7. (A) Still frames showing that
RabSRab7 and RabB or RabA show little colocalization. (B) Examples of static and moving
endosomes: RabB (green) and RabSRab7 (mCherry). A kymograph was plotted across the
indicated line. Three static RabB endosomes, one moving EE, and two small vacuoles strongly
labeled with RabSRab7are shown. The tip-distal static RabB endosome is docked at the most
anterior RabSRab7 vacuole (yellow in the channel merge). The movement of a fourth RabB
endosome is schematically represented. (C) One example (arrows) of a basipetally moving small
RabSRab7 vacuole (smV) that docks against a large vacuole (LgV). Kymographs on the right depict
how the speed (diagonal slope, 1.34 μm/s) of this moving vacuole is markedly lower than that of
RabA endosomes. See Supplemental Movie S6. (D) RabB and RabSRab7 can coexist on LEs: two
LEs containing RabB and RabSRab7, located between two vacuoles. One of the endosomes
moves in one direction before shifting toward the opposite direction, but its speed (1.8 μm/s) is
lower than the average speed of EEs. Frames correspond to supplemental Movie S7.
1896 | J. F. Abenza et al. Molecular Biology of the Cell
and loaded with CMAC. Whereas hardly any overlap of RabB with
CMAC was detectable in the wild type, most RabB-containing struc-
tures in the rabSΔ mutant were CMAC positive (Figure 9B). Because
vacuolar membrane labeling by RabA/RabB is never seen in the wild
type, these data suggest that displacement of RabA/RabB from EEs
once they reach the LE stage requires RabSRab7.
We next tested whether GFP-RabSRab7 localization to vacuoles ne-
cessitates RabB. GFP-RabSRab7 is recruited to the membrane of rabBΔ
vacuoles, although clearly less efficiently than in the wild type (Figure
9C). This finding is consistent with the interpretation that the vacuolar
RabSRab7 localization involves CORVET, as RabA is also able to recruit
CORVET to endosomes, albeit less efficiently than RabB (note that
lethality-causing rabAΔ rabBΔ double deletion cannot be tested).
The role of dynein-mediated basipetal movement in
endosomal maturation
Given that the amount and size of vacuoles increase with the dis-
tance to the tip, we hypothesized that maturation of endosomes
into vacuoles might be coupled to the dynein-mediated movement
of endosomes away from the tip (MTs are oriented with their plus
ends toward the apex). Thus, if dynein function is compromised, EEs
would be expected to mature in the proximity of the tips. Indeed
the dynein (heavy chain) nudA1ts mutation caused a pronounced
effect on the apicobasal distribution of GFP-RabSRab7–labeled vacu-
oles. In nudA1ts germlings (Figure 10A) and hyphae (Figure 10B)
there was a marked accumulation of vacuoles near the tip that was
never seen in the wild type (wild-type tip-proximal regions are actu-
ally devoid of vacuoles). Clusters of nudA1ts vacuoles did not invade
the actual tip of hyphae (Figure 10B). These findings strongly sup-
port a model according to which endosomes mature to LEs that
become engaged in homotypic fusion events to coalesce into larger
vacuoles as they move away from the tip (Figure 11). We also
constructed nudA1 rabBΔ double-mutant strains expressing GFP-
RabSRab7. This double mutation does not prevent the vacuolar local-
ization of RabSRab7 (as noted, RabA is able to recruit CORVET), even
though these strains display the characteristic nudA1 accumulation
of vacuoles near the tip (Supplemental Figure S6B).
We conducted additional experiments using CMAC to visualize
vacuoles in cells that do not express any fluorescent Rab. nudA1
hyphal tip cells indeed accumulate clusters of small vacuoles within
the apicalmost 10- to 20-μm region. These clusters were absent in
the wild type (Figure 10, D and E), in agreement with data presented
earlier. We also noticed that in rabSΔ cells, the otherwise ubiquitous
minivacuoles are absent from the tip (Figure 10, D and E), suggest-
ing that they are excluded from this region, predictably by dynein-
mediated transport. If this were true, in nudA1ts cells minivacuoles
should invade the tip, forming a minivacuolar analogue of the EE
cluster characteristic of dynein-deficient hyphal tips (Lenz et al.,
FIGURE 8: Kymographs of “stream” time-lapse series of GFP-labeled
Rab5s and mCherry-RabSRab7. Images in the GFP (Rab5s) and mCherry
(RabSRab7) channels were simultaneously acquired using a Dual-View
beam splitter with “stream” acquisition, a routine by which images are
directly discharged from the camera chip to the computer RAM
memory at maximal speed. The resulting time series were used to
draw kymographs. All kymographs, whose time and length
dimensions are indicated, are displayed to the same scale and are
thus directly comparable. (A, B) GFP-RabB and (C, D) GFP-RabA, as
indicated. Expression of all three fluorescent fusion proteins was
governed by the regulatable alcAp driver and thus can be manipulated
by the carbon source in the medium. All transgenes were integrated
in single copy into known chromosomal locations. For GFP-RabB: (A1)
Arrows show two static endosomes containing RabB and mCherry-
RabSRab7. (A2) Arrows show two rare examples of moving endosomes
containing RabB and RabSRab7. (B1, B2) Strongly overexpressing
conditions. Examples of moving endosomes containing RabB and
mCherry-RabSRab7 are indicated with red and green arrows. (B2) One
RabB endosome that does not contain mCherry-RabSRab7 is indicated
with a single green arrow. GFP-RabA: (C) Virtually no colocalization of
GFP-RabA and mCherry-RabSRab7 is seen in the numerous examples of
moving endosomes detected across this 60-μm-long region. (D) The
endosome on the left (red and green arrows of equal size), containing
GFP-RabA and mCherry-RabSRab7, moves toward the tip until it docks
at a LE/vacuole. Two examples of GFP-RabA moving endosomes
faintly stained with mCherry-RabSRab7 are indicated with green (large)
and red (small) arrows. Note that moving RabA structures (diagonals
in C and D) are clearly more numerous than moving RabB structures
(A and B).
Volume 23 May 15, 2012 Endosomal maturation in Aspergillus | 1897
in A. nidulans strains deficient in the NudA
dynein, in the dynein activator NudF, in the
KinA kinesin-1 transporting dynein to MT
plus ends, or in the dynactin p25 subunit
linking EEs to dynein, EEs aggregate in the
tips (Zekert and Fischer, 2008; Abenza et al.,
2009; Zhang et al., 2010, 2011).
In time-course experiments, the endo-
cytic tracer FM4-64 arrives first to motile
EEs and, in a posterior stage, to a popula-
tion of larger, static LEs (Peñalva, 2005;
Hervás-Aguilar et al., 2010). Thus the transi-
tion from EE to LEs takes place with simulta-
neous loss of motility. In our model, matura-
tion of EEs is coupled to their movement
away from the tip (Figure 11), resembling
the situation in mammalian cells, in which
Rab5 EEs that form in the cell periphery (the
hyphal tip equivalent) move centripetally
(i.e., basipetally in fungal hyphae) on dynein
while they augment their size (Rink et al.,
2005). However, in mammalian cells, LEs/
lysosomes also undergo dynein-mediated
centripetal transport toward the minus ends
of MTs. Recruitment of dynein-dynactin to
LEs/lysosomes is mediated by the Rab7 ef-
fector RILP (Jordens et al., 2001). RILP ho-
mologues are absent from fungi. Thus inef-
ficient dynein recruitment might contribute
to the reduced motility of A. nidulans LEs
enriched in RabSRab7.
Our model in Figure 11 accounts for the
observations that tip-proximal regions con-
tain very few vacuoles (the end product of endosomal maturation)
and that vacuolar size increases with the distance to the tip. It is
strongly supported by three findings: 1) Vacuoles accumulate near
the tip region of nudA1ts cells; 2) unlike wild-type vacuoles, rabSΔ
minivacuoles, which are abnormally motile, are uniformly dispersed
across the cytosol; and 3) In nudA1ts cells, overlap of Rab5 and
RabSRab7 membranes is detectable in the tip, suggesting that if ret-
rograde EE movement is prevented, endosomes mature on the
spot.
The absence of RabSRab7 prevents the formation of normal-
sized vacuoles. The resulting minivacuoles have features of EEs:
1) They are able to eventually undergo long-distance movement;
2) they invade the apicalmost regions of nudA1ts tips, resembling
EEs; and 3) they contain EE Rabs on their membranes. The finding
that Rab5s invade LE/vacuolar membranes only if RabSRab7 is ab-
sent is consistent with the models in which maturation of EEs into
LEs/vacuoles occurs by Rab conversion (Rink et al., 2005; Peplowska
et al., 2007; Brocker et al., 2010). In a simplified model, this im-
plies that Rab5/CORVET membrane domains are converted into
Rab7/HOPS domains (Rink et al., 2005). A key regulator of this
conversion is a complex of two proteins formed by Mon1p/Ccz1p
in S. cerevisiae (Nordmann et al., 2010) and SAND-1/CCZ-1 in
C. elegans (Kinchen and Ravichandran, 2010). The findings that
SAND-1/CCZ-1 is itself a Rab5 effector (Kinchen and Ravichandran,
2010), that the localization to endosomes of Mon1p/Ccz1p is
Vps21p (Rab5) and CORVET dependent (Nordmann et al., 2010),
that the complex interacts with HOPS components (Nordmann
et al., 2010; Poteryaev et al., 2010), and, of importance, that
Mon1p/Ccz1p is the guanine nucleotide exchange factor (GEF) of
2006; Zekert and Fischer, 2008; Abenza et al., 2009). This is indeed
the case (Figure 10, D and E). Double-mutant nudA1ts rabSΔ cells
accumulate most of their minivacuoles at and near the tip, with the
rest of the cell largely devoid of them. Invasion of nudA1ts tip re-
gions by minivacuoles is very conspicuous in young branches (Sup-
plemental Figure S6A).
Finally, we hypothesized that by impairing EE movement, nudA1
would confine maturation of EEs into LEs/vacuoles within the tip.
Thus we examined nudA1 cells coexpressing fluorescent RabB and
RabSRab7 that had been shifted to the restrictive temperature for 4 h.
These cells contained the characteristic nudA1 aggregate of RabB-
containing endosomes unable to depart from the tip (Figure 10C)
and the also characteristic tip-proximal clusters of RabSRab7 vacuolar
structures. RabB aggregates and RabSRab7 structures were very
closely associated and showed partial colocalization (Figure 10C
and Supplemental Figure S7). However, in every example analyzed
(n = 47 tips examined with Dual-Viewer z-stacks), the cluster of LE/
vacuolar structures was less apical or, at most, overlapped with the
EE aggregate (Supplemental Figure S7), strongly supporting the
view that conversion of RabB EEs into RabSRab7 LEs/vacuoles is cou-
pled to basipetal movement of the former.
DISCUSSION
In U. maydis and A. nidulans kinesin-3 and dynein mediate the long-
distance movement of EEs (Wedlich-Soldner et al., 2002; Lenz et al.,
2006; Zekert and Fischer, 2008; Abenza et al., 2009, 2010). Endocy-
tosis predominates in the tip region (Araujo-Bazán et al., 2008;
Taheri-Talesh et al., 2008), which is the major source of EEs (Figure
11). These endosomes move away from the tip using dynein. Thus,
FIGURE 9: In rabSΔ strains, RabB invades vacuolar membrane domains normally occupied by
RabSRab7. (A) GFP-RabB in rabSΔ. Inverted contrast images correspond (at double magnification)
to the indicated regions of interest in the Nomarski panel. They show how the early endosomal
RabB reaches the membrane of rabSΔ minivacuoles. Similar data obtained for GFP-RabA are
displayed in Supplemental Movie S10. (B) Microtubules were depolymerized with benomyl.
Under such conditions, endosomes form aggregates. In the wild type, endosomal aggregates
show little colocalization with CMAC-stained LEs/vacuoles (one colocalizing particle is shown by
the arrow). In contrast, colocalization is very conspicuous in the rabSΔ mutant, strongly
indicating that CMAC-stained rabSΔ minivacuoles retain early endosomal identity. (C) In rabBΔ
cells, GFP-RabSRab7 localizes to the vacuolar membranes but less efficiently than in the wild type.
The relative difference of fluorescence is illustrated by the line scans on the right. The inset
below the rabBΔ cell shows the faint yet clear labeling of vacuolar membranes.
1898 | J. F. Abenza et al. Molecular Biology of the Cell
LEs whose mobility is constrained by their
size. Thus RabSRab7 is recruited to mem-
branes already at the level of LEs and it is
not confined to vacuoles, in agreement with
data in S. cerevisiae (Balderhaar et al., 2010).
In contrast, RabA or RabB, both of which re-
side in EEs, can reach LEs but are normally
absent from vacuoles.
Fusion of A. nidulans LEs with vacuoles
and among vacuoles themselves is dispens-
able, as demonstrated by the relatively mild
growth defects resulting from rabSΔ, vps41Δ
(this work) and vps39Δ (Oka et al., 2004). In
contrast, double rabAΔ rabBΔ deletion is
lethal (Abenza et al., 2010), and single dele-
tion of each tested gene involved in matu-
ration of EEs into LEs is very severely debili-
tating. Tested mutations include vps45Δ,
vps8Δ (CORVET; Abenza et al., 2010),
vps18Δ and vac1Δ (this work), ESCRTΔ al-
leles (single vps27Δ, vps23Δ, vps20Δ,
vps24Δ, and vps32Δ mutations), and a con-
ditional expression allele of Vps4 (Rodríguez-
Galán et al., 2009; Calcagno-Pizarelli et al.,
2011; Galindo et al., 2012). Of note, the se-
verely debilitating effect of ESCRTΔ alleles
is suppressible by loss-of-function muta-
tions in the filamentous fungal–specific sltA
gene regulating cation homeostasis (Findon
et al., 2010; Calcagno-Pizarelli et al., 2011),
suggesting that the essential role of endo-
somal maturation might be related to cation
homeostasis
rabSΔ minivacuoles are proteolytically
competent and contain Pep12. Thus resi-
dent proteases or Pep12 must be able to
reach vacuoles through endosomes, be-
cause the direct AP-3 pathway to the vacu-
ole requires Ypt7p/HOPS/Vps41p (Angers
and Merz, 2009; Cabrera et al., 2010). In
the absence of RabSRab7, the formation of
minivacuoles possibly occurs through the
CORVET-mediated homotypic fusion of
endosomes (Markgraf et al., 2009), but
CORVET alone appears to be insufficient to sustain further en-
largement of minivacuoles. Thus an attractive and as yet untested
possibility is that CORVET and HOPS complexes mediate homo-
typic fusion events but that these events involve partners with dif-
ferent degrees of membrane curvature, depending on the Rab/
tether combination.
MATERIALS AND METHODS
Aspergillus media and molecular biology
Synthetic complete medium (SC; Cove, 1966) contained 1% glu-
cose and 5 mM ammonium tartrate unless otherwise indicated.
Complete medium for Aspergillus (MCA) was used for strain main-
tenance. Strains are listed in Supplemental Table S2 (vps18Δ, Vac1Δ,
and the double mutants rabAΔ-rabSΔ, rabBΔ-rabSΔ, and vps41Δ-
rabBΔ strains were impossible to keep beyond the described ex-
periments due to their severely debilitated phenotype). For growth
tests involving different carbon sources, we cultivated the strains
on SC containing 5 mM ammonium tartrate and 3% glucose,
Ypt7p (yeast Rab7) provide the mechanistic bases for the Rab5-
to-Rab7 conversion: Rab5 recruits Mon1/Ccz1, then Mon1/Ccz1
activates Rab7, and the GEF is further stabilized by Rab7 effectors.
Mon1/Ccz1 influences Rab5 localization (Nordmann et al., 2010).
Given that C. elegans SAND-1 displaces the Rab5 GEF from mem-
branes (Poteryaev et al., 2010), fungal Mon1/Ccz1 could plausibly
act in a similar manner. The relatively sharp Rab transition that we
observe is indeed consistent with this possibility. Moreover, the
finding that, in the absence of RabSRab7, Rab5s (RabA/RabB) reach
the vacuolar membranes suggests that RabSRab7 contributes to de-
stabilizing the association of Rab5s with membranes (Figure 11).
Our data strongly indicate that the transition between Rab5s and
RabSRab7 is sharp, as examples of rapidly moving RabA/RabB EEs
that also contain RabSRab7 are very rare. Inspection of a large number
of movies taken under a variety of conditions revealed that colocal-
ization of RabA/B and RabSRab7 is relatively frequent in static or short-
range moving particles or in large particles that move at speeds con-
siderably below the average speed of EEs, most likely representing
FIGURE 10: nudA1ts results in the accumulation of vacuoles near the tip. (A) nudA1ts and control
wild-type germlings and (B) a nudA1 hyphal tip expressing GFP-RabSRab7 under the control of
the alcAp driver. In both cases cells were cultured at 37°C. (C) GFP-RabB and mCherry-RabSRab7
colocalize in tip aggregates of nudA1ts cells cultured on fructose. Shown are x-z orthogonal
views of z-stacks in the positions of aggregates containing mCherry-RabSRab7 only (red, bottom),
GFP-RabB only (green, middle), or both (top). In the latter, the overlap across several z-planes of
red and green signals shows that RabB and RabSRab7 membranes overlap. (D) Accumulation of
CMAC-stained vacuoles near the tip of nudA1ts and nudA1ts rabSΔ cells. Note that, in the
nudA1ts rabSΔ cell, CMAC minivacuoles invade the hyphal tip. (E) Quantification of the surface
occupied by CMAC-stained structures in the different mutant conditions shown in D. Hyphal tip
cells were segmented into four 6-μm-long regions as indicated.
Volume 23 May 15, 2012 Endosomal maturation in Aspergillus | 1899
Western blotting
To determine levels of GFP-RabSRab7 by
Western blotting, we extracted proteins as
described (Hervás-Aguilar et al., 2007). GFP
fusion proteins were detected using mouse
anti α-GFP (monoclonal cocktail, 1:5000;
Roche, Indianapolis, IN). Actin (detected
with mouse anti α-actin from ICN Biomedi-
cals (Irvine, CA) using 1:80,000 dilution of
the antibody) was used as loading control.
For the extraction of AgtA-GFP we used a
reported procedure (Hervás-Aguilar and
Peñalva, 2010) involving solubilization of
proteins from lyophilized mycelial biomass
with NaOH, followed by their precipitation
with trichloroacetic acid.
Microscopy
Cells were grown in submerged cultures at
25°C in watch minimal medium (WMM), us-
ing Lab-Tek chambers (Nalge Nunc Interna-
tional, Rochester, NY) for 14–16 h before
proceeding to microscopy (Pantazopoulou
and Peñalva, 2009). To modulate the ex-
pression levels of the fluorescent chimeras
expressed under the control of alcAp, we
used different carbon sources (Abenza
et al., 2009): low levels were attained with
0.1% (wt/vol) fructose, and high levels were
obtained using 1% ethanol (vol/vol) or by
preculturing cells on 0.02–0.05% (wt/vol)
glucose and shifting them to 1% (vol/vol)
ethanol for 3–4 h. In RabA-RabSRab7 and in
RabB-RabSRab7 colocalization experiments,
intermediate expression levels were
achieved by culturing cells in WMM con-
taining 1% (vol/vol) glycerol as carbon
source. Mature endosomes/vacuoles were
detected with CMAC as described (Abenza
et al., 2009; Pantazopoulou and Peñalva,
2009). To determine the effects of the
nudA1ts mutation, cells were cultivated at
37°C, which is a semirestrictive tempera-
ture, or overnight at 25°C (permissive tem-
perature) and then shifted to 37°C for a few
hours before image acquisition. To impede
movement of rabSΔ minivacuoles, benomyl
was added to WMM at a final concentration of 4.8 μg/ml as de-
scribed (Abenza et al., 2009). For experiments involving delivery of
AgtA-GFP to the vacuole, cells were cultured at 25°C for 14–16 h in
WMM containing 5 mM l-glutamate as nitrogen source and then
shifted to the same medium in which 5 mM ammonium tartrate sub-
stituted for l-glutamate. AgtA-GFP images were taken at the 0-min
time point and 80 min after the shift (Abenza et al., 2009, 2010).
Images were acquired using a Hamamatsu ORCA ER-II camera
(Hamamatsu, Hamamatsu, Japan) coupled to a Leica DMI6000B
microscope (Leica, Wetzlar, Germany) driven by MetaMorph soft-
ware (Molecular Dynamics, Sunnyvale, CA) and equipped with an
EL6000 external light source for epifluorescence excitation. The mi-
croscope was equipped with HCX 63×, 1.4 numerical aperture
(NA), and 100×, 1.4 NA, objectives and BrightLine GFP-3035B
(Semrock, Rochester, NY), TXRED-4040B (mCherry), and standard
0.1% fructose, or a combination of 1% ethanol and 0.02% glucose
during 72 h at the indicated temperatures.
A. nidulans vps18 (digA), Vac1, vps41, and gdiA were identified
as AN2266, AN3144, AN4876, and AN5895, respectively, in the
A. nidulans genomic database. Deletion cassettes were constructed
by PCR (Szewczyk et al., 2006), using primers listed in Supplemental
Table S3 and Aspergillus fumigatus pyrGAf (for vps18Δ, rabSΔ,
vac1Δ, and vps41Δ) or pyroAAf (for a second rabSΔ deletion allele) as
selection markers. Recipient strains carried nkuAΔ, preventing non-
homologous recombination (Nayak et al., 2006). Constructs for inte-
grative transformation were confirmed by sequencing. They were
targeted to the pyroA or argB locus after transformation, using pub-
lished methodology (Calcagno-Pizarelli et al., 2007; Pantazopoulou
and Peñalva, 2009). Single-copy integration was verified by South-
ern blots.
FIGURE 11: A model for endosomal maturation in A. nidulans. This model for endosomal
maturation incorporates the conclusions of this work into the interpretation of the endocytic
pathway derived from previous reports (Ohsumi et al., 2002; Peñalva, 2005; Abenza et al., 2009,
2010; Hervás-Aguilar and Peñalva, 2010; Hervás-Aguilar et al., 2010; Zhang et al., 2010, 2011;
Griffith et al., 2011; Pantazopoulou and Peñalva, 2011). Endocytosis predominates in the tip.
Endocytic vesicles reach a hypothetical endosomal compartment that would act as a sorting
endosome, organized as a mosaic of domains (magenta). We speculate that this mosaic includes
domains from which membrane and cargo can recycle to the plasma membrane, segregating
from other domains in which the two Rab5s—RabA and RabB—determine “degradative” EE
identity (i.e., specify the identity of membranes destined to the vacuole). It is generally accepted
that efficient endocytic recycling is required for hyphal morphogenesis. Thus the hypothetical
existence of a sorting endosome acting upstream of RabB is supported by the fact that rabBΔ
has a relatively minor effect on hyphal morphogenesis (Abenza et al., 2010). Degradative EEs
become loaded on dynein. RabB is the major player in establishing this “degradative identity,”
as it is required for EE movement and mediates recruitment of Vps45 and Vps34 to endosomes
(not depicted; Abenza et al., 2010). Vps45 enables endosomes to accept Golgi traffic required
for maturation, whereas Vps34 synthesizes phosphatidyl inositol-3-phosphate, the landmark of
degradative endosome identity, initiating the MVB pathway at the stage of EEs (Abenza et al.,
2010; Hervás-Aguilar et al., 2010). RabB and, less efficiently, RabA recruit CORVET, mediating
homotypic fusion between EEs, with key involvement of Vps8 (Markgraf et al., 2009). As
endosomes increase their size, they decrease their motility and acquire their final composition
(i.e. become LEs). Then RabSRab7 substitutes Rab5s. Finally, LEs undergo further fusion between
them and with vacuoles in a HOPS-dependent manner. The negative feedback loop that may
help to release Rab5s from endosomes is depicted.
1900 | J. F. Abenza et al. Molecular Biology of the Cell
4′,6-diamidino-2-phenylindole (CMAC) filter sets. For colocaliza-
tion experiments, a strictly simultaneous imaging of GFP and
mCherry was carried out using a Dual-View imaging system (Photo-
metrics, Tucson, AZ), using the recommended filter set (Pantazo-
poulou and Peñalva, 2011). MetaMorph software was used for con-
trast adjustment, Dual-View channel alignments, color combining,
z-stack maximal-intensity projections (contrasted, when indicated,
with the MetaMorph “unsharp mask” filter) and for the assembly of
kymographs from time-lapse series. Images were converted to 8-bit
grayscale (and usually shown in inverted contrast) or to 24-bit RGB
and annotated with Corel Draw (Corel, Ottawa, Canada). For some
z-stacks we improved image quality by using a blind deconvolution
procedure (AutoDeblur software; Media Cybernetics, Bethesda,
MD). Time-lapse sequences were converted to QuickTime using
ImageJ 1.37 (National Institutes of Health, Bethesda, MD). Statisti-
cal analyses were performed using GraphPad Prism 5.00 for Win-
dows (GraphPad Software, La Jolla, CA).
Affinity purification of RabSRab7 effectors
Affinity purification of effectors using GDP- or GTP-γ-S GST-RabSRab7
glutathione-Sepharose beads was made as described for GST-
RabB. Glutathione-Sepharose 4B beads containing GST-RabSRab7
were incubated with A. nidulans protein extracts in 50-ml Falcon
tubes before eluting the interacting proteins, which were resolved
by SDS–PAGE cells, excised, and analyzed by matrix-assisted laser
desorption/ionization peptide mass fingerprinting and tandem
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ACKNOWLEDGMENTS
We thank E. Reoyo for technical assistance. This work was supported
by the Spanish government through Dirección General de Investi-
gación Científica y Técnica Grant BIO2009-7281 and by Comunidad
de Madrid Networking Grant SAL/0246/2006 to M.A.P. J.F.A. was a
Consejo Superior de Investigaciones Cientificas I3P predoctoral
fellow.
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