Post on 24-Apr-2023
Cyclase-Associated Protein is Essential for theFunctioning of the Endo-Lysosomal System andProvides a Link to the Actin Cytoskeleton
Hameeda Sultana1, Francisco Rivero1,Rosemarie Blau-Wasser1, Stephan Schwager2,Alessandra Balbo3, Salvatore Bozzaro3, MichaelSchleicher2 and Angelika A. Noegel1,*
1Center for Biochemistry and Center for MolecularMedicine Cologne, Medical Faculty, University of Cologne,50931 Koln, Germany2Institute of Cell Biology, Ludwig-Maximilians-Universitat,80336 Munchen, Germany3Dipartimento di Scienze Cliniche e Biologiche, OspedaleS. Luigi, 10043 Orbassano, Italy*Corresponding author: Dr Angelika A. Noegel,noegel@uni-koeln.de
Data from mutant analysis in yeast and Dictyosteliumindicate a role for the cyclase-associated protein (CAP)in endocytosis and vesicle transport. We have usedgenetic and biochemical approaches to identify novelinteracting partners of Dictyostelium CAP to help explainits molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunitsof the highly conserved vacuolar H+-ATPase (V-ATPase)and co-localizes to some extent with the V-ATPase.Furthermore, CAP is essential for maintaining the struc-tural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology ofV-ATPase- and Nramp1-decorated membranes were dis-turbed in a CAP mutant (CAP bsr) accompanied by anincreased endosomal pH. Moreover, concanamycin A(CMA), a specific inhibitor of the V-ATPase, had a moresevere effect on CAP bsr than on wild-type cells, and themutant did not show adaptation to the drug. Also, thedistribution of green fluorescent protein-CAP wasaffected upon CMA treatment in the wildtype and recov-ered after adaptation. Distribution of the V-ATPase inCAP bsr was drastically altered upon hypo-osmoticshock, and growth was slower and reached lower satura-tion densities in the mutant under hyper-osmotic condi-tions. Taken together, our data unravel a link of CAP withthe actin cytoskeleton and endocytosis and suggest thatCAP is an essential component of the endo-lysosomalsystem in Dictyostelium.
Key words: CAP, contractile vacuole system, F-actin-depolymerizing drugs, Nramp1, V-ATPase, V-ATPaseinhibitor
Received 14 January 2005, revised and accepted for pub-lication 8 July 2005, published on-line 8 August 2005
It has been increasingly realized that rearrangement of the
actin cytoskeleton is essential for the process of endocy-
tosis. Localized recruitment and polymerization of actin
are observed at the sites of endocytosis, and cytoskeletal
components are shown to assemble and help in localizing
the endocytic machinery to domains of the plasma mem-
brane (1). Genetic studies in yeast have revealed many
genes required for receptor-mediated endocytosis such as
Sla1p/end3, Sla2p/end4/Mop2, Abp1p, Rvs167p, Srv2p/
CAP (cyclase-associated protein), Pan1, Arc15, Act1p and
Aip1p, which regulate actin dynamics and act as bridges or
adapters between the actin cytoskeleton and the endocy-
tic machinery (2–4). These proteins assemble into modular
complexes that can induce actin polymerization at the
sites of endocytosis. In elegant studies, Kaksonen et al.
(5) have unraveled a finely choreographed pathway of the
assembly of cortical patches of differing protein composi-
tion at the plasma membrane during endocytic internaliza-
tion and have shown that actin and Sla2p are directly
involved in the internalization and are required for patch
motility. Sla2p is a protein functioning at the interface
between the actin cytoskeleton and the endocytic machin-
ery and is suggested to link actin polymerization and endo-
cytic internalization based on findings that in sla2D cells
patch motility is blocked and actin remains at the cell
cortex. Furthermore, Sla2p is a candidate for negatively
regulating the Arp2/3 complex-mediated actin nucleation
as endocytic sites in sla2D cells had more cortical F-actin
than the wildtype.
The mammalian homologs of the yeast patch proteins
Sla2p and Pan1p and Hip1R and Eps15, localize to
clathrin-coated pits (CCPs), the sites of endocytic interna-
lization.Hip1R isbothacomponentofCCPsandclathrin-coated
vesicles (CCVs) and functions in the receptor-mediated endo-
cytosis similar to Sla2p. Hip1R is associated with clathrin
during CCVs’ formation and frequently localizes to the
edges of the forming coated pits, similar to cortactin,
and is suggested to connect clathrin to F-actin at the
cortex. Furthermore, Hip1R binds to cortactin and can
physically connect actin and clathrin in vitro (6). The deple-
tion of Hip1R did not disrupt the transient association
between endocytic and cytoskeletal proteins but rather
stabilized it, whereas RNAi double-depletion experiments
for Hip1R and cortactin demonstrated that accumulation
of the cortical actin–endocytic complexes depended on
cortactin (7).
Traffic 2005; 6: 930–946Copyright # Blackwell Munksgaard 2005
Blackwell Munksgaard doi: 10.1111/j.1600-0854.2005.00330.x
930
The other candidates that may link the actin cytoskeleton
to endocytosis are Abp1p, Rvs167p and Srv2p. They co-
localize with the cortical actin cytoskeleton and relocalize
to sites of cytoskeletal rearrangements. More recent stu-
dies of the mammalian Abp1p have strengthened this role
by demonstrating an interaction with dynamin, a large
GTPase controlling the fission reaction during endocytosis
(8). Furthermore, amphiphysins, the homologs of yeast
Rvs167p, also interact with dynamin to regulate endocy-
tosis of synaptic vesicles (9).
Srv2p/cyclase-associated protein, a further component
of the endocytic machinery and an evolutionarily
conserved regulator of the G-actin/F-actin ratio, is sug-
gested to provide a link to the dynamin–Abp1p– amphy-
physin complex, because the yeast protein interacts
directly or indirectly with many key components of the
actin cytoskeleton and endocytosis like Abp1p, Sla2p
and Rvs167p (10). Recent papers provided evidence
for an active role of Srv2/CAP in controlling actin fila-
ment dynamics and showed that Srv2/CAP exists as a
high molecular weight structure (approximately
600 kDa) composed of actin and Srv2 (1:1 M ratio),
which is linked to actin filaments via the SH3 domain
of Abp1 (11). This Srv2–actin complex functions as a
monomer-processing intermediate, which catalytically
accelerates cofilin-dependent actin turnover by releasing
cofilin from ADP-G-actin monomers allowing recycling
of cofilin for new rounds of filament depolymerization.
The nucleotide exchange on ADP-G-actin is enhanced,
and due to the lower affinity of Srv2/CAP for ATP-G-
actin, other cellular factors, such as profilin or WASP,
may take over ATP-G-actin and facilitate actin assembly
(12–15). Profilin and Srv2 were also reported to physi-
cally interact (14,16), which might further facilitate a
monomer handoff. The cofilin-induced acceleration of
actin turnover has been shown to be achieved through
the integration of the activities of CAP, which involves a
coordinated interplay between its N- and C-terminal
domains. These findings provide evidence on the phy-
siological significance of the reported interaction
between the N- and C-terminal domains within CAP
molecules (10).
Data from Dictyostelium also indicate an involvement of
CAP in areas of high actin rearrangements during cell
motility, pinocytosis and phagocytosis and in the genera-
tion of cell polarity (17–19). Here, we have made an
attempt to define and strengthen the role of CAP in linking
the actin cytoskeleton to endocytosis by identifying its
interacting partners and by further analysis of the
Dictyostelium CAP bsr mutant. In this mutant, the CAP
gene has been altered by homologous recombination in
such a way that the mutant had less than 5% of the
protein concentration in wild-type cells. The mutant had
a severe growth defect reaching saturation densities dur-
ing growth in suspension at 6 � 106 cells/mL in contrast
to the parent strain with 1.2 � 107 cells/mL, which
resulted from an endocytosis defect. When analyzing the
developmental properties of the mutant, we noted
defects in cAMP signaling, cell polarization and chemotac-
tic motility, which suggested an interaction of CAP with
adenylyl cyclase and an influence of the protein on signal-
ing pathways directly and through its function as a regula-
tory component of the cytoskeleton. All defects observed
in the mutant could be rescued by expression of a green
fluorescent protein (GFP)-tagged CAP (18,19).
We report here that CAP interacts with components of the
vacuolar Hþ-ATPase (V-ATPase). Vacuolar Hþ-ATPases are
highly conserved multisubunit enzymes composed of an
integral (transmembrane) V0 domain that consists of five
subunits (subunits a–d) serving as proton channel and a
cytosolic catalytic sector (peripheral V1 domain) composed
of eight subunits (subunits A–H) that contains the ATP-
binding site involved in ATPase activity. Vacuolar Hþ-
ATPases are present in the endomembranes of all and in
the plasma membrane of many eukaryotic cells. They are
responsible for the acidification of lysosomes, endo-
somes, the Golgi complex, and secretory vesicles, and
function in processes such as receptor-mediated endocy-
tosis, intracellular targeting of lysosomal enzymes, protein
processing and degradation, and the transport of small
molecules across the plasma membrane of various cell
types (20). In Dictyostelium discoideum, the V-ATPase is
also present in membranes of the contractile vacuole (CV),
an osmoregulatory organelle of freshwater and soil proto-
zoa which pumps water out of the cell (21).
Our data suggest that loss of CAP affects the structural
organization and integrity of V-ATPase and natural resis-
tance-associated macrophage protein (Nramp1)-decorated
endo-lysosomal membranes and alters the distribution and
morphology of the vesicular network. This occurs in a
manner probably similar to the action of actin-depolymer-
izing drugs. The endosomal pH in the mutant is altered
and is highly sensitive to concanamycin A (CMA), a spe-
cific inhibitor of the V-ATPase. Together, our studies pro-
vide an insight into the role and functioning of CAP as a
general regulator of the actin cytoskeleton and
endocytosis.
Results
Cyclase-associated protein interacts directly with the
V-ATPase in Dictyostelium
In a search for components that interact with CAP, we
performed a series of immunoprecipitation assays using
cell extracts of wild-type AX2, AX2 cells expressing GFP-
CAP and the CAP mutant CAP bsr for control and antibo-
dies specific to GFP and CAP. In the immunoprecipitates,
we repeatedly detected the B subunit (vatB, approxi-
mately 31 kDa) of the V1 peripheral complex and the d
subunit (41 kDa) of the integral membrane V0 complex of
the V-ATPase with highest mass spectrometric scores,
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 931
whereas these proteins were absent in control experi-
ments performed with CAP bsr. Our immunoprecipitation
assays also revealed other elements of the endocytic
machinery associated with CAP and the V-ATPase (our
unpublished data). We have extended these findings by
carrying out immunoprecipitation with the GFP-specific
antibody, mAb K3-184-2, using lysates from AX2 cells
that expressed GFP-CAP. The immunoprecipitate was
probed for the presence of the 69-kDa vatA subunit of
the V-ATPase (Figure 1A) for which a monoclonal antibody
is available (22). The presence of CAP was confirmed with
mAb 223-445-1. In an independent yeast two-hybrid
screen using a Dictyostelium cDNA library, we identified
the H subunit (50 kDa) of the V1 peripheral complex of the
V-ATPase as interaction partner of the N-terminal domain
of CAP. Further studies revealed that V-ATPase H inter-
acted with full-length CAP, the N-domain (aa 1–215) and a
100 amino acids N-terminal stretch of CAP (aa 1–102)
narrowing down the interaction site (Figure 1B).
Subsequently, we also observed a direct interaction of
CAP with the d subunit of the V-ATPase by the yeast
two-hybrid assay. The association of CAP with the
V-ATPase complex and the physical interaction with its
subunits suggested a direct link of CAP with the early
and late endo-lysosomal system, because V-ATPase is a
component of these endo-membrane systems. Together,
our results indicate that CAP interacts directly with the
V-ATPase and may play a crucial role during endocytosis
and vesicular trafficking.
Cyclase-associated protein associates and co-localizes
with vacuolar membranes
The availability of reagents specific for several V-ATPase
components allowed us to investigate this interaction of
CAP by indirect immunofluorescence. AX2 cells expres-
sing GFP-CAP were immunostained for vatA, a marker for
the endo-lysosomal system and the CV (22). Green fluores-
cent protein-cyclase-associated protein is present near the
plasma membrane and is diffusely distributed in the cyto-
sol with enrichments on vesicles and vacuoles, which are
stained by the vatA antibodies as well, although not all
GFP-CAP-decorated vesicles are stained by the vatA anti-
bodies. Conversely, not all vatA positive structures carry
GFP-CAP (Figure 2A). In further studies, we also observed
a co-localization of CAP with the vatB subunit, which was
expressed as GFP fusion protein in AX2 cells, whereas
CAP was detected by a mAb. However, in this case, the
co-localization was less prominent and the CAP antibody
did not stain intracellular vesicles as strongly as is seen
when a GFP-tagged CAP is used (Figure 2B). This could be
due to the specificity of the antibody or to the higher levels
of GFP-CAP as compared with the endogenous protein,
which might allow to observe an otherwise rather transi-
ent interaction. Green fluorescent protein – vatB was pre-
sent on endo-membranes and co-localized with the vatA
subunit (data not shown). Our previous observations that
GFP-CAP rescued all phenotypic defects in the mutant
that we had observed argues that the fusion protein
behaves like the wild-type protein and is most likely prop-
erly targeted. On the basis of our interaction studies, the
distribution and association of CAP with the vesicular
membranes could be due to the direct physical interaction
of CAP with subunits of the V-ATPase.
Cyclase-associated protein is indispensable for
maintaining the integrity and organization of the
V-ATPase-decorated endo-membrane system
CAP bsr mutant cells have a severe growth and endo-
cytosis defect (18), but a link to the endosomal system
has not been tested so far. Here, we investigated the
distribution and integrity of the V-ATPase system in CAP
bsr cells with the vatA-specific mAb. In AX2 cells, vatA is
found at the periphery of large and small vesicles and also
present in the cytosol, whereas in CAP bsr cells the
structure and shape of the vatA labeled membranes
were strongly affected. We observed a reduced staining
of vatA as compared with the wildtype and many
vesicles of smaller sizes, which were present throughout
kDa
67
N-CAP
A B
CAP 300
Pro-C-CAP
fl-CAP
pAS2-143
Figure 1: Cyclase-associated protein interacts directly with
the vacuolar H+-ATPase (V-ATPase). A) Immunoprecipitation
of green fluorescent protein (GFP)-cyclase-associated protein
(CAP) from AX2 cells expressing GFP-CAP was performed with
GFP-specific mAb K3-184-2, the immunoblot containing
the immunocomplexes was probed for the presence of the
V-ATPase A subunit with mAb 221-35-2. The band observed at
67 kDa corresponds to vatA and the band at approximately
50 kDa corresponds to the IgG heavy chain. B) The H-subunit of
the V-ATPase interacts with the N-domain of CAP in a yeast two-
hybrid assay. Polypeptides corresponding to various CAP domains
were linked to the Gal4-activating domain, whereas the sequence
encoding the V-ATPase H subunit (residues 33–443) was fused to
the Gal4 DNA-binding domain. The corresponding plasmids were
transformed into yeast cells, and the interactions were assessed
by the filter lift b-galactosidase assay. Growth on selective media
and blue color development due to b-galactosidase activity are
shown. Cyclase-associated protein domains: N-CAP, aa 1–215;
CAP 300, aa 1–102; Pro-C-CAP, aa 216–464; fl-CAP, 1–464 and
pAS2-1, vector control.
Sultana et al.
932 Traffic 2005; 6: 930–946
the cytosol and enriched at the cell periphery (Figure 3A,
supplementary Figure 1S available online at http://
www.traffic.dk/suppmat/6_10.asp). An analysis of the
distribution of the vesicles in the mutant at different focal
planes using confocal microscopy showed that the vatA-
stained structures do not form a regular network and do not
appear throughout the cell, whereas in AX2 we detected
vatA in all focal planes (videos 1 and 2 available online at
http://www.traffic.dk/suppmat/6_10.asp). We also found
that CAP bsr cells were more flattened (videos 3 and 4
available online at http://www.traffic.dk/suppmat/6_10.asp).
The weaker staining for vatA in the mutant was due to
reduced levels of the protein as revealed by Western blot
analysis (Figure 3B). The reduced vatA levels further sug-
gested that CAP contributes to the integrity of the V-
ATPase and thus acts as a general regulator of endocytosis.
When we introduced a plasmid allowing expression of full-
length GFP-CAP, the vatA staining and distribution of the
vacuolar network were restored to the wild-type pattern
(Figure 3C). We extended this analysis and tested the
individual domains of CAP for their capability to re-estab-
lish an intact vesicular network. In CAP bsr expressing the
GFP-tagged N-terminal domain, the vesicular network was
restored but only to a partial extent. The vesicles were
large, less numerous and strongly stained for vatA. We
also observed a co-localization of GFP-N-CAP-Pro with
vatA at these structures (Figure 3C). Furthermore, expres-
sion of the C-terminal domain as GFP–Pro-C-CAP fusion
had some positive effect on the structure of vatA-contain-
ing vesicles but only the full-length protein efficiently cor-
rected the observed alterations (Figure 3C).
Morphology and functioning of the CV system is
altered in CAP bsr
In Dictyostelium about 10% of the total cellular V-ATPase is
associated with the endo-lysosomal system, whereas the
remainder is associated with the membranes of the tubular
system of the CV (23). The CV system (CVS) consists of
ducts, cisternae and bladders and is transiently connected
to the plasma membrane by pores through which the blad-
der expels water and thus is involved in osmoregulation
(24). The presence of V-ATPase as marker on these sys-
tems suggests that both CVS and endo-lysosomal compart-
ments are connected to each other by fusion of their
membranes, and the interaction of CAP with V-ATPase
suggested their co-ordinated role in the functioning of the
CVS. To understand the osmoregulatory role of the V-
ATPase in the absence of CAP, we performed studies
under hypo-osmotic stress conditions, as the V-ATPase-
rich CVS rapidly collects and expels water. Phase-contrast
microscopy revealed that most of the wild-type cells were
attached to the surface and exhibited an amoeboid shape
and morphology. Under hypo-osmotic conditions, wild-type
CVSs were visualized as phase-lucent compartments
(Figure 4, arrowheads) that gradually filled and swiftly con-
tracted. After successful discharge (shown by arrow), addi-
tional phase-lucent vacuoles were filled to repeat the cycle.
The mutant cells were attached and appeared to be mor-
phologically normal in growth medium; however, in water,
they rounded up and detached from the plastic surface.
CAP bsr cells hardly contained phase-lucent vacuoles and
further showed no size changes, refilling or discharge of
vacuoles for a period of more than 30 mins (Figure 4).
GFP-CAP
GFP-vatB CAP
vatA MergeA
B
Figure 2: Cyclase-associated protein co-localization with vacuolar H+-ATPase (V-ATPase). A) AX2 cells expressing green fluores-
cent protein (GFP)-cyclase-associated protein (CAP) were fixed and immunolabeled with V-ATPase subunit A (vatA)-specific mAb 221-35-2
followed by a Cy3-labeled secondary antibody. Green fluorescent protein-cyclase-associated protein co-localized partially with vatA-
stained membranes (arrowhead). The images were obtained using a confocal laser-scanning microscope. B) AX2 cells expressing GFP-
vatB were labeled with the CAP mAb 230-18-8 to analyze the co-distribution of CAP with GFP-vatB. Cyclase-associated protein was
enriched at the cell cortex and was also present on cytoplasmic structures. A closer analysis revealed its association with vacuolar
membranes and a partial co-localization with GFP-vatB (arrowhead). Bars, 5 mm.
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 933
After 1–2 h, 70% of the cells had lysed, suggesting that
CAP may regulate water homeostasis because the
mutants retain low level of osmoregulation.
To support our findings on the altered CVS in CAP bsr, we
performed experiments using the styryl dye FM 4-64 (25).
By time-lapse video microscopy, we followed the inter-
nalization of FM 4-64 from the plasma membrane into the
CV. The rapid and fast events of dye redistribution, CV
filling and discharge were observed by recording one
frame per second in wild-type cells. Cyclase-associated
protein bsr also exhibited these events; however, they
appeared much slower and fewer as compared with
AX2. Mostly, the mutants swelled and appeared round
and lost their typical morphology. Occasionally, the
appearance of enlarged and filled CVs at the plasma mem-
brane was observed, which appeared to slowly expel fluid
suggesting an impaired functioning of the CV (videos
A B
C
AX2 CAP bsr
GF
P-C
AP
GF
P-N
-CA
P-P
roG
FP
-Pro
-C-C
AP
vatA Merge
vatA
CAP bsr
AX2
Comitin
Figure 3: The vacuolar H+-ATPase (V-ATPase)-labeled membrane system is disturbed in CAP bsr. A) VatA-stained vacuoles and
vesicles of varying sizes are present throughout the AX2 cells. In CAP bsr, the vesicles are smaller and less numerous. Detection was
with mAb 221-35-2 followed by secondary Cy3-labeled antibody (supplementary videos 1–4 available online at http://www.traffic.dk/
suppmat/6_10.asp) correspond to this figure). B) VatA protein levels are lower in CAP bsr. Total homogenates from 2 � 105 cells of AX2
and CAP bsr were subjected to SDS – PAGE (10% acrylamide). The blot was probed with mAb 221-35-2 for detection of vatA and mAb
190-68 for labeling of comitin as loading control. C) Restoration of vacuolar organization in CAP bsr cells expressing green fluorescent
protein (GFP) fusions of CAP. Expression of GFP-CAP completely restored the vacuolar network. The disturbed vacuolar organization and
distribution was partially complemented by the expression of GFP-N-CAP-Pro and GFP-Pro-C-CAP in CAP bsr. mAb 221-35-2 labeling
revealed the presence of vesicles and a co-localization of vatA with GFP-CAP and GFP-N-CAP-Pro. Green fluorescent protein-Pro-C-CAP
distributed more diffusely and did not co-localize with vatA. Expression of CAP domains restores mostly large but less numerous vesicles
illustrating that both domains are essential for the restoration of the disturbed vacuolar network in CAP bsr. Arrowheads denote
co-localization of GFP fusions of CAP with vatA-labeled membranes. Bars, 10 mm.
Sultana et al.
934 Traffic 2005; 6: 930–946
5 and 6 available online at http://www.traffic.dk/suppmat/
6_10.asp).
As our data indicated alterations in the functioning of the
V-ATPase in CAP bsr, we analyzed its distribution under
hypo-osmotic conditions by staining the cells for vatA. In
AX2, the morphology and distribution of the V-ATPase
was different from that in CAP bsr. The V-ATPase localized
to one side of the wild-type cells; in CAP bsr, the
V-ATPase structures were present in the cell center in a
large clustered patch with few small vesicles around
(Figure 5A,B) and agreed well with the observations in
videos 5 and 6. The actin staining suggested that in AX2
actin is also enriched in those areas of the cell cortex
where the V-ATPase is found. In CAP bsr, actin is present
in some regions at the cell periphery, and the staining was
much weaker in comparison to AX2 (Figure 5C). The
altered distribution of the V-ATPase in the absence of
CAP supports the importance of CAP for this process.
We also tested the sensitivity of the mutant cells to
hyperosmotic conditions by growing them in media of
different osmotic strength and supplementing the med-
ium either with 30 mM NaCl or 115 mM sorbitol. In the
presence of 30 mM NaCl, AX2 was only marginally
affected in growth whereas the mutant reached saturation
densities, which were about 60% of untreated control
cells. Sorbitol (115 mM) affected both strains. AX2 cells
reached saturation densities that were 60% of control
cells, CAP bsr was strongly affected and reached final
densities that were only 20–30% of untreated mutant
cells. Moreover, the duplication times were significantly
increased.
Loss of CAP results in an increased endo-lysosomal
pH and slower adaptability to CMA
Because the activity of the V-ATPase affects the endo-
lysosomal pH, we measured this parameter in living AX2
and CAP bsr cells using fluorescein isothiocyanate (FITC)-
dextran as a pH probe. Growing Dictyostelium amoebae
contain acidic lumen with a pH value of 5.4–5.8 (26). We
found that the basal pH in CAP bsr was higher (pH,
6.0 � 0.2) in comparison with the wild-type cells (pH,
5.7 � 0.2). The increased basal pH in CAP bsr was statis-
tically significant (p < 0.01, n ¼ 7, Student’s t-test). This
increase suggests an involvement of CAP in maintaining
the acidic nature of the endo-lysosomal system.
To examine whether CAP is also required for the acidifica-
tion and functioning of the endo-lysosomal compartments,
AX2C
ontr
olH
ypo-
osm
otic
CAP bsr
Figure 4: CAP bsr cells are impaired
in osmoregulation. Cells were
allowed to attach to coverslips in
nutrient medium (control) or water
(hypo-osmotic condition), and phase-
contrast images were obtained after
60 min. Wild-type cells appeared fre-
quently swollen and round under
hypo-osmotic load but were still able
to crawl and change shape and
showed fast and rapid filling and dis-
charging of contractile vacuoles
(CVs). Many strong filled (arrow-
heads) and discharging vesicles
(arrow) were observed. In CAP bsr
cells, the appearance of vesicles
was drastically reduced and only a
few vesicles were observed (arrow-
head). The CVs of CAP bsr were not
able to expel water efficiently, and
the cells exhibited enlarged filled
vacuoles (arrow). Furthermore, they
barely remained attached to the cov-
erslip. Bars, 10 mm.
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 935
we investigated the endosomal pH evolution following a
5-min FITC-dextran pulse in CAP bsr cells. In both AX2 and
CAP bsr, endosomal acidification was rapid, reaching a pH
value of approximately 5 during the pulse phase. During
the next 30 min, the pH value steadily rose to stabilize at
values around 6 (Figure 6A). We next investigated the
effects of CMA in CAP bsr cells. Concanamycin A is an
extremely potent inhibitor of V-ATPases and as such can
inhibit endosomal and phagosomal membrane traffic in
Dictyostelium (26). The effects of CMA (10 mM) on the
pH of endosomal and lysosomal compartments of AX2
and CAP bsr were determined by dual excitation ratio
fluorimetry of cells using FITC-dextran. To determine
how rapidly the increase in pH of the endo-lysosomes
occurs after CMA treatment, we exposed FITC-dextran-
loaded cells to CMA, and the pH was measured over the
indicated times. We observed that the rise in pH was rapid
and reached a value of approximately 6.5 at 1 h after drug
addition in both the wild-type and CAP bsr cells with no
significant difference (Figure 6B). Later on, AX2 cells
adapted to CMA and the pH dropped. Such behavior has
been reported for wild-type cells previously (26). In con-
trast, in the mutant cells, the pH increased further to a
value close to 7.0 and remained there for more than 3 h of
CMA treatment indicating that the cells did not adapt. The
increase in basal pH and the low degree of adaptability to
the effects of CMA in CAP bsr indicate that CAP is essen-
tial for the functioning of the V-ATPase.
Furthermore, to understand the complexity, dynamics and
the association of CAP with the endo-lysosomal system,
we studied the effects of CMA in vivo. The distribution of
GFP-CAP in AX2 wildtype was affected upon treatment
with CMA and showed a punctuated pattern in the cyto-
plasm. Macropinocytosis, cell shape changes and the relo-
calization of GFP-CAP to the sites of endocytosis
disappeared upon CMA treatment in comparison with the
control; however, after 60 min, these effects were re-
established upon adaptation to CMA (videos 7 and 8 avail-
able online at http://www.traffic.dk/suppmat/6_10.asp).
During endocytic transit, the V-ATPase accumulates at
phagosomes shortly after internalization occurs and is
retrieved prior to exocytosis (27). We investigated
whether absence of CAP alters the behavior of V-ATPase
during phagocytosis of yeast cells. For this, AX2 and CAP
bsr were pulsed with yeasts for 15 min followed by a
short (15 min) or long (120 min) chase period prior to
fixation (Figure 6C–F). In both strains, vatA was found
around phagosomes devoid of an actin coat. Early phago-
somes (Figure 6C,D: arrowheads) were actin-coated and
lacked vatA. Prior to exocytosis vatA detached from some
phagosomes (Figure 6E,F: asterisks). We conclude that
relocalization of V-ATPase during phagocytosis is not
noticeably impaired in CAP-bsr.
Nramp1-decorated vesicles are disturbed in CAP bsr
Dictyostelium amoebae are professional phagocytes,
which ingest bacteria as their principal food source in
both natural and laboratory conditions. They harbor a set
of proteins required for endocytosis, which are similar to
the one in higher eukaryotes. One of these proteins is the
membrane protein Nramp1 that in mammals causes resist-
ance to microbe infection with intracellular parasites such
as Mycobacteria, Salmonella and Leishmania (28). During
infection, Nramp1 is recruited to phagosomal membranes
from the late endosomal/lysosomal compartment where it
is involved in the acidification of the lumen by promoting
the fusion of the vesicles with membranes carrying
AX2
A
B
C
CAP bsr
Figure 5: The morphology and distribution of the contractile
vacuole system is disturbed in CAP bsr under hypo-osmotic
stress. A) The vatA staining showed that in AX2 cells the
V-ATPase carrying membranes localize to one side of the cells.
In contrast, CAP bsr cells had the V-ATPase-stained structures
distributed in the cytoplasm exhibiting also an altered morphology
(Bars, 5 mm). B) The corresponding phase-contrast images reveal
the cell boundaries. C) AX2 stained for actin shows the actin
cortex mostly at one side of the cells, whereas in CAP bsr, actin
was found in some peripheral regions and the actin staining
appeared reduced under the hypo-osmotic load. Bars, 10 mm.
Sultana et al.
936 Traffic 2005; 6: 930–946
6.5
A
C
D
E
*
*
F
B
6
5.5
pH pH
5
7.5
7
6.5
AX2-CMAAX2-EtOHCAP-CMACAP-EtOH
6
5.5
54.50 10 20 30
Time (min)
vatA Actin Merge Transmission
40 50
AX2CAP
60 0 30 60Time (min)
90 120 150
Figure 6: Behavior of vacuolar H+-ATPase (V-ATPase) in CAP bsr. A) Change of endosomal pH during transit of a fluid phase marker.
Cells were pulsed with fluorescein isothiocyanate (FITC)-dextran (2 mg/mL) for 10 min (dotted line), washed and resuspended in fresh
nutrient medium. Samples were withdrawn at the times indicated, and dual excitation ratio was used to calculate the endosomal pH. B)
Effects of concanamycin A (CMA) on the endosomal pH in CAP bsr. Cells were loaded with FITC-dextran (2 mg/mL) for 3 h; the specific
V-ATPase inhibitor CMA (20 mM) or its diluent ethanol (0.01% v/v) was then added, and samples were withdrawn at the times indicated for
determination of endosomal pH as described in (A). Concanamycin A inhibited acidification and increased the endo-lysosomal pH in a
significant manner. After 60 min of drug treatment, a slight recovery was gained as the pH decreased to a value of 6.0 in the wild-type
cells. In CAP bsr, the endosomal pH was near to neutral and the cells failed to adapt to CMA. Data of A and B are the average �SD of four
independent experiments. For simplicity, error bars are depicted only in one direction. (Also see supplementary videos 5 and 6 available
online at http://www.traffic.dk/suppmat/6_10.asp). C – F) Distribution of V-ATPase during phagocytosis. Cells of AX2 (C, E) and CAP bsr
(D, F) were deposited on coverslips and incubated with yeasts for 15 min, then washed and incubated in Soerensen buffer for additional
15 min (C, D) or 120 min (E, F) and fixed with picric acid/paraformaldehyde. Vacuolar Hþ-ATPase was detected with mAb 221-35-2 for
vatA followed by Cy3-labeled secondary antibody. F-actin was stained with FITC-phalloidin. The images were obtained using a confocal
laser-scanning microscope. VatA localizes around phagosomes devoid of an actin coat. Early phagosomes (arrowheads) are actin-coated
and lack vatA. In the cell shown in D, the early phagosomes are located adjacent to vatA-stained vesicles. Prior to exocytosis, vatA
detaches from the phagosomes (asterisks). V-ATPase behaves similarly in AX2 and CAP bsr. Bar, 10 mm.
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 937
V-ATPase. Mammalian cells have also an Nramp2, which
is much more widely expressed in the body. Dictyostelium
discoideum harbors two homologs of Nramp, NrampA and
NrampB. They are encoded by separate genes giving rise
to 70- and 53-kDa proteins, respectively. NrampA and
NrampB (in the following designated as Nramp1) have
32% identity and 57% similarity amongst each other and
42–45% identity with the mammalian proteins.
We studied the distribution of Nramp1-GFP and found that
it is targeted to internal membranes that resemble the
vatA-stained structures. In CAP bsr, the Nramp1-GFP dis-
tribution was different from wildtype. The Nramp1-GFP-
stained membranes showed a disturbed organization, and
the stained vesicles were smaller (Figure 7A). Upon
co-staining with vatA-specific antibodies, we detected a
considerable amount of overlap; however, not all vatA-labeled
membranes carried Nramp1-GFP suggesting that Nramp1
is present on a subset of vatA-positive membranes
(Figure 7B), which are however, not identical to the CVS
(S. Bozzaro, unpublished). The altered distribution and
difference in the organization of Nramp1-GFP-
decorated membranes in AX2 and CAP bsr cells were
confirmed by live microscopy studies (videos 9 and
10 available online at http://www.traffic.dk/suppmat/
6_10.asp). Live imaging revealed that the Nramp1 vesicles
fail to undergo fast and dynamic events of movement and
fusion in the absence of CAP. The disappearance of larger
and numerous Nramp1-decorated vesicles resembles and
correlates with the situation noted for the V-ATPase. We
conclude that membranes carrying vatA, Nramp1 or both
are grossly disturbed in the mutant.
We also examined the association of CAP with the com-
partments decorated by Nramp1-GFP and stained AX2
cells expressing Nramp1-GFP with CAP-specific mAb
230-18-8 (17). This antibody strongly labels the cell cortex
and in addition localizes to some Nramp1-GFP-positive
membranes in the cytosol (Figure 8A). Furthermore, as
Nramp1 tightly associates with lysosomal-associated
membrane protein 1 (Lamp1)-positive compartments in
mammalian cells (28), we studied such an association in
A
B
AX2
GFP-Nramp1
GF
P-N
ram
p1A
X2
CA
P b
sr
vatA Merge
CAP bsr
Figure 7: Disturbance of the
endo-lysosomal system in CAP
bsr as detected by natural resist-
ance-associated macrophage
protein (Nramp1). A) AX2 and
CAP bsr cells expressing green
fluorescent protein (GFP)-Nramp1
were imaged to analyze the organi-
zation and distribution of Nramp1-
decorated vesicles. In CAP bsr the
vesicles were small, diffused and
appeared as a clustered network.
B) Organization and integrity of the
early and late endo-lysosomal sys-
tem is disturbed in CAP bsr. Early
and late endosomes are detected
using vatA staining and GFP-
Nramp1 labels late endosomes.
Mutant and wild-type cells expres-
sing GFP-Nramp1 were labeled with
mAb 221-35-2 for vatA followed by
Cy3-labeled secondary antibody. In
CAP bsr, the GFP-Nramp1 and vatA-
labeled vesicles were smaller and
vatA staining was reduced (also
see supplementary videos 7 and 8
available online at http://www.
traffic.dk/suppmat/6_10.asp).
Sultana et al.
938 Traffic 2005; 6: 930–946
Dictyostelium and found that CAP co-localizes with the
postlysosomal marker vacuolin A (Figure 8B).
Disruption of the actin cytoskeleton also disturbs the
vesicular network
Our results indicate a direct link between the actin cyto-
skeleton and membranes of the endo-lysosomal system
through CAP and subunits of the vacuolar ATPase. We
therefore tested whether the structure and integrity of the
V-ATPase system is disturbed when the actin cytoskele-
ton is disrupted using cytochalasin A (20 mM) and latruncu-
lin B (1 mM). The action of these drugs is different:
cytochalasin A binds to the barbed ends of actin filaments
inhibiting both the association and dissociation of sub-
units, whereas latrunculin B is an actin monomer seques-
tering toxin inhibiting actin polymerization and disrupting
microfilament organization as well as microfilament-
mediated processes and is 10- to 100-fold more potent
than cytochalasin (29).
We first analyzed the action of both drugs on the actin
cytoskeleton of wildtype and mutant. The treatments led
to an altered actin staining in both strains; however, CAP
bsr cells showed a different response to cytochalasin A.
After a 20 min treatment with cytochalasin A, actin was
no longer present in the cortex in AX2 cells. Instead, it
was distributed in spots all over the cells. At 40 min actin
was concentrated in large clumps inside the cells. In the
mutant, actin was still present at the cortex after 40 min
of incubation and, in addition, we observed rod-like struc-
tures in most cells. Also, the cortical staining remained
after 60 and 80 min of incubation (data not shown). In both
strains the cell shape was altered by cytochalasin A, and
cells were more rounded and did not adhere well to the
coverslips (Figure 9A). When we investigated the distribu-
tion of the vacuolar network in AX2 and CAP bsr cells
upon treatment with cytochalasin A, we observed an
altered vacuolar organization in AX2, whereas CAP bsr
cells did not show much difference in the vacuolar staining
in comparison with the control cells. In AX2 the vesicles
were fewer and small in size, and the vatA staining was
more diffused (Figure 9B).
We also found that the distribution of GFP-CAP expressed
in CAP bsr cells was affected in a way resembling the one
observed for actin upon treatment with cytochalasin A for
60 min, thus suggesting that the actin-depolymerizing
drug also affects the actin regulatory protein CAP
(Figure 10). The effect of latrunculin B was comparable
in AX2 and CAP bsr. At 10 min, the cortical staining was
less prominent and appeared fragmented, and the inten-
sity of actin staining seemed to decrease over the time
course of incubation (30 min) (Figure 11A). The vacuolar
staining was also altered in both strains. The vesicular
structures appeared to be clustered at the edges of the
cells, and the enrichment of vatA on vacuolar membranes
was less prominent upon treatment with latrunculin B
(Figure 11B). Taken together, the treatment of AX2 cells
with actin-affecting drugs had effects on the distribution
and organization of the V-ATPase that were comparable
to the ones in CAP bsr suggesting that CAP contributes
to V-ATPase localization and structural organization prob-
ably through a general role in actin organization and
dynamics.
A GFP-Nramp 1
CAP-GFP Vacuolin A
CAP Merge
MergeB
*
Figure 8: CAP co-localizes with natural resistance-associated macrophage protein (Nramp1) and vacuolin, markers of the
endo-lysosomal system. A) In AX2 cells expressing green fluorescent protein (GFP)-Nramp1, cyclase-associated protein (CAP) associ-
ates to some extent with the Nramp1-decorated vesicular endomembranes. Cyclase-associated protein was detected with mAb 230-18-
8. Arrowheads point to co-stained vesicles and star represents the association of CAP with a subset of early endosomes. B) Presence of
GFP-CAP at the postlysosomal compartment and co-localization with vacuolin A. AX2 cells expressing GFP-CAP were fixed and immuno-
stained with the postlysosomal marker vacuolin A using mAb 221-1-1. Green fluorescent protein-cyclase-associated protein was found on
some of the vacuolin A-positive vesicles (arrowheads) but did not completely overlap. Bar, 5 mm.
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 939
Discussion
Cyclase-associated protein as a key component in
maintaining the organization and functioning of
V-ATPase-decorated endo-lysosomal and CV
membranes
The V-ATPases are ATP-dependent proton pumps and are
universal components of eukaryotic organisms present in
the membranes of many intracellular organelles. They
function to couple the energy of ATP hydrolysis to the
active transport of protons from the cytoplasm to the
lumen, creating a proton gradient and generating the low
intravacuolar pH found in endosomes and lysosomes (30).
The endocytic pathway defines membrane traffic from the
cell surface to the degradative compartments like lyso-
somes (animals) and vacuoles (plants and fungi), and
V-ATPases have a vital role in both endocytosis and vesicle
trafficking (20). Our attempts to identify binding partners
of CAP resulted in the isolation of various subunits of the
V-ATPase. Furthermore, we also found in an independent
yeast two-hybrid screen that the N-terminal domain of
CAP directly interacted with the H subunit of the peri-
pheral V1 complex, thus providing a direct link of CAP
with V-ATPase and establishing CAP as a component of
the endo-lysosomal membranes. The association of CAP
with the V-ATPase complex was further established by
our immunofluorescence studies where GFP-CAP co-loca-
lized with vatA-stained membranes, and endogenous CAP
overlapped with GFP-vatB to some degree (Figure 2). The
subunit B appears to be essential for the assembly of the
V-ATPase probably via its interaction with the actin cyto-
skeleton (31,32).
Several proteins have been identified that bind to subunits
of the V-ATPase and thus provide multiple links of this
enzyme to cellular components. The direct interactions
between Nef, a HIV accessory protein, and the V-ATPase
subunit H have been shown by Lu et al. (33). The binding
of Nef to the subunit H facilitates the internalization of
CD4, a primary receptor for HIV on the surface of infected
cells suggesting an important role for V-ATPase subunit H
in viral infectivity (34). Presumably, if the interaction of
CAP with the V-ATPase subunit H is conserved in humans,
this interaction may hinder the binding of the subunit H to
Nef and in turn may influence the viral infectivity.
However, this is a mere hypothesis that needs to be
thoroughly explored.
In Dictyostelium, unlike yeast, V-ATPase associates with a
variety of organelles including lysosomes and phago-
somes. Additionally, V-ATPase, calmodulin and Rab-
GTPases are present at the osmoregulatory CV complex
(25,35,36). Interestingly, in our immunoprecipitation
experiments, we found CAP in association with Rab-
GTPases (Rab4, Rab11, RabA and RabB), and in immuno-
fluorescence studies CAP co-localized with Rab-GTPases
(our unpublished results). Our findings that the functioning
of endocytic processes and the osmotic resistance are
disturbed and impaired in the CAP-deficient mutant further
provide functional support for the association and inter-
action of CAP with the V-ATPase. The sensitivity to and
lysis of CAP bsr cells in hypo-osmotic medium provided
evidence for a poor functioning of the CV system to expel
water under these stress conditions. The altered distribu-
tion of the V-ATPase under hypo-osmotic stress in the
absence of CAP further suggested the requirement of
the tight interaction of CAP with V-ATPase to achieve
the normal morphology and functioning of the proton
ControlA
X2
CA
P b
srC
AP
bsr
AX
2
A
B
Cytochalasin A
Figure 9: Cytochalasin A alters the distribution of the actin
cytoskeleton and the vacuolar network. Cells were treated
with 20 mM cytochalasin A or DMSO (control) for 40 min and
stained for actin (mAb act-1–7) (A) or vatA (mAb 221-35-2) (B). In
AX2, cytochalasin A leads to an accumulation of actin in spots and
clumps in the cytosol (A), and the vatA staining is weaker (B). In
CAP bsr cells, actin was found in the cell cortex and in rods that
centered in the cytoplasm (A). The vatA staining was less influ-
enced and did not vary much from the control (B). Bars, 10 mm.
Sultana et al.
940 Traffic 2005; 6: 930–946
pumps. Moreover, the observed reduced expression
levels of vatA in CAP bsr suggest an impaired functioning
of the V-ATPase as vatA is the subunit containing the site
of ATP hydrolysis in the enzyme. In yeast and fungi, dis-
ruption of the V-ATPase subunit A (vmaA1) inhibited nor-
mal growth, abolished sporulation and resulted in
morphological changes and reduced growth similar to
those observed after addition of CMA (37). In
Dictyostelium, reduced expression levels of VatM have
effects on cell growth and cytosolic pH regulation (38).
Our rescue analysis revealed that CAP is essential for the
structure and integrity of the endo-lysosomal membrane
network as expression of GFP-CAP led to its complete
restoration in the mutant. A partial restoration was
achieved by the expression of N- and C-domains. The
rather modest effects observed with the C-terminal
domain could be due to its association with actin or its
linkage to other components through the proline rich
region, which has the property to bind to SH3 domains
or a combination of both.
Involvement of CAP in the maintenance of endosomal
pH
The maintenance of an appropriate pH within a mem-
brane-surrounded organelle (both secretory and endo-
cytic) is a challenge from the simplest eukaryote to
complex multicellular organisms, and the balance
between active Hþ pumping and passive Hþ efflux activ-
ity of the V-ATPase achieves maintenance of an optimal
pH. The pH varies in different subcompartments of the
endocytic and secretory pathways, and the pH of each
organelle critically determines the coordinated biochem-
ical reactions. The altered structural organization of the
endo-lysosomal system correlated with the impaired
functional activity of V-ATPase, because the endosomal
pH was significantly higher in CAP bsr (Figure 6A,B).
These findings suggest an impaired function of the pro-
ton pumps to generate the proton gradient and maintain
the acidic pH of the endo-lysosomal system. There are
several lines of evidence supporting the notion that acid-
ification of the endo-lysosomes by V-ATPase is essential
for efficient targeting of molecules through the endo-
lysosomal pathway, and alterations in the normal pH
homeostasis can lead to significant functional changes
(26,39). The increase in the endosomal pH may also
account for the reduced endocytosis in CAP bsr.
Similarly, Dictyostelium mutants in distinct ABC transpor-
ters showed an altered endosomal pH and endocytosis
defects (40).
The proton pumping activity of V-ATPase is inferred
from the effects of pharmacological agents that cause
a rapid alkalinization of the acidic organelles (39). The
macrolide antibiotic bafilomycin and the related CMA
are highly specific inhibitors of V-ATPases that slow
down the receptor-mediated endocytosis and the recy-
cling of the endo-lysosomal system (30). In
Dictyostelium, CMA results in the neutralization of the
lumen of the endo-lysosomal system, inhibition of endo-
cytosis, exocytosis and phagocytosis, delays protein
processing and induces missorting of the lysosomal
enzyme a-mannosidase and causes gross morphological
changes. Concanamycin A inhibited the acidification of
endo-lysosomal vesicles and led to an increase of the
pH; however, after 60 min, an adaptation to CMA was
observed in AX2 that was associated with a decrease in
pH. Addition of fresh CMA had no effect on increasing
GFP-CAPC
ontr
olC
ytoc
hala
sin
AActin Merge
60 min
Figure 10: Cytochalasin A affects
the distribution of green fluores-
cent protein (GFP)-CAP. Cyclase-
associated protein bsr cells expres-
sing GFP-CAP were fixed and
stained to visualize actin. The distri-
bution of GFP-CAP was affected
upon treatment with the drug and
distributed in actin rich patches fail-
ing to reach the cell cortex and
showed a pattern comparable to
the actin distribution, suggesting
that the actin-depolymerizing drug
through altering the actin cytoskele-
ton also affects the CAP distribu-
tion. Bar, 10 mm.
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 941
the pH, and the recovery of the pH did not require
protein synthesis. Adaptation to CMA was therefore
proposed to result from reactivation of V-ATPase func-
tion by cytosolic activators (26). In the CAP bsr mutant,
CMA caused a rapid increase in endosomal pH (nearing
a neutral pH of 7) and a failure to adapt to CMA sug-
gesting a higher sensitivity of the mutant’s V-ATPase.
The altered distribution and dynamics of GFP-CAP upon
CMA treatment further suggested an association of
CAP with the V-ATPase (videos 7 and 8 available online
at http://www.traffic.dk/suppmat/6_10.asp). It might
well be that CAP and the link to the actin cytoskeleton
provided through CAP might be identical to the ‘cyto-
solic activators’ proposed by Temesvari et al. (26).
ControlA
B
AX2
10 m
in30
min
10 m
in30
min
Latrunculin B Latrunculin BControl
CAP bsr
AX2 CAP bsr
Figure 11: Effects of latrunculin B. A) Cytoskeletal rearrangements in response to latrunculin B. AX2 and CAP bsr cells were either
treated with ethanol (control) or latrunculin B (1 mM) in phosphate buffer for the times indicated prior to fixation. Cells were labeled with
actin-specific mAb Act 1–7 followed by labeling with secondary antibody. The confocal images reveal reduction in the actin staining upon
treatment with latrunculin B in wildtype and mutant alike. B) The vacuolar network is affected during cytoskeletal changes upon treatment
with latrunculin B. Cells were treated with ethanol or latrunculin B and fixed as described in (A). The vatA labeling revealed a cytoskeleton
association of actin with the endo-lysosomal and contractile vacuole system marked by mAb 221-35-2 for vatA. In latrunculin B-treated
AX2 cells, the vatA-stained vesicles were small and clustered to one region of the cell thereby altering the vacuolar organization. Also, we
observed a weaker vacuolar staining. CAP bsr showed similar effects upon treatment with latrunculin B. Bars, 10 mm.
Sultana et al.
942 Traffic 2005; 6: 930–946
Previously, CAP has been shown to be involved in endo-
cytic internalization (18) with reduced endocytosis, while
in the current study, we have found that the vatA levels
and the vacuolar organization are affected in the absence
of CAP. Based on the role of CAP in endocytosis, it is also
formally possible that CAP regulates the V-ATPase local-
ization by contributing to the overall internalization of lipids
and proteins at the plasma membrane. In wild-type cells,
the efficient internalization of V-ATPase may lead to nor-
mal endocytosis, whereas in CAP bsr due to inefficient
internalization of the V-ATPase, it may remain at the
plasma membrane due to a general defect in endocytosis.
It is also noteworthy that the treatment of cells with actin
drugs had similar effects on the organization and distribu-
tion of the V-ATPase as the loss of CAP, which highlights a
role of CAP in facilitating and controlling the actin
dynamics that may contribute to V-ATPase localization
and distribution. However, on the basis of the data that
indicate a direct CAP–V-ATPase interaction, we assume
that the altered distribution and functioning of V-ATPase is
directly related to the loss of CAP and the interaction to
the cytoskeleton, which it provided.
Requirement of CAP for the integrity of the Nramp1-
associated membrane system
Nramp1, an integral membrane protein, is localized to the
endosomal/lysosomal compartment in macrophages and
is rapidly recruited to the membranes of the particle/
microbe-containing phagosomes upon phagocytosis (41).
Dictyostelium, a professional phagocyte principally feed-
ing on bacteria, contains two homologs of human Nramp.
Here, we have studied NrampB/Nramp1, a 53-kDa protein,
which is closer to human Nramp1 than Dictyostelium
NrampA. We have found that the Nramp1-decorated
membrane network is disturbed in CAP bsr cells suggest-
ing that CAP is involved in maintaining also this membrane
compartment. In wild-type cells, Nramp1-GFP localizes to
a subset of vatA-positive vesicles and shows some degree
of co-localization with CAP on vesicles (Figures 7 and 8).
Dictyostelium Nramp1 can therefore be classified as a
protein of the endo-lysosomal system; however, it is also
present on membranes of different origin. Our live
imaging showed that the fusion, fission and movement
events of Nramp1-GFP-decorated vesicles were much
slower in the mutant as compared with the wildtype
further supporting the disturbed V-ATPase functioning
associated with high endosomal pH (videos 9 and 10
available online at http://www.traffic.dk/suppmat/
6_10.asp).
Nramp1 tightly associates with Lamp1-positive compart-
ments and Rab7-positive late endosomal structures,
which are clearly distinct from those of the early endo-
somal marker Rab5 (28). In the endocytic pathway of
Dictyostelium, four distinct phases have been distin-
guished: the uptake of particle/fluid by phagocytosis and
macropinocytosis, an acidic phase initiated by the associa-
tion of V-ATPase with the endosomes, targeting of
lysosomal enzymes and members of the Rab family to
the V-ATPase complex and followed by a postlysosomal
period where the pH is close to neutral and vesicles are
devoid of the V-ATPase and finally exocytosis (22). The
indigestible remnants of the postlysosomal compartment
are released by exocytosis, and this compartment is
characterized by two isoforms of vacuolin, A and B,
which are encoded by different genes.
Our finding that GFP-CAP associates to some extent with
membranes carrying the lysosomal marker vacuolin A
further supported the possible connection of CAP with
Nramp1-labeled membranes (Figure 8). Acidification of
late endosomes causes the release of lysosomal enzymes
and permits receptor recycling, uncoupling of receptor–
ligand complexes, degradative processes and biosynth-
esis and sorting of lysosomal hydrolases (20,26).
Therefore, association of CAP with the lysosomal marker
and its involvement in pH homeostasis suggest a consid-
erable role of CAP in the complete endocytic circuit.
Nramp1 is initially located in the late endosomal/lysosomal
compartment and is then recruited to the phagosomes
during the course of its maturation from early plasma
membrane-derived phagosomes to phagolysosomes and
becomes enriched with the membranous compartments
containing the ingested particle. In Dictyostelium, relocal-
ization of CAP upon a stimulus during phagocytosis to the
phagocytic cups and phagosomes (our unpublished
results) and its association with Nramp1 and vacuolin A-
positive membranes may also propose a role for CAP in
phagolysosomes.
Role of CAP in linking endocytosis to the actin
cytoskeleton
Insight into the mechanism and requirement of the actin
cytoskeleton for endocytosis has come through the
identification of the genes affected in fluid phase and
receptor-mediated endocytosis with defects in cytoskeleton
organization. In addition, mutations in proteins that
regulate the integrity and modulation of the actin cytoske-
leton such as fimbrin, calmodulin and type I unconven-
tional myosin blocked endocytosis, suggesting that
mutations that disturb the integrity of the actin cytoskele-
ton affect endocytosis (1). Adessi et al. (42) proposed that
the presence of actin on Dictyostelium endocytic vesicles
reflects tight interactions between the vesicles and the
actin cytoskeleton, postulating that interactions occur via
unknown vesicle-associated actin-binding proteins. Our
data provide a link of endocytosis to the actin cytoskeleton
through CAP.
Several recent studies have suggested an essential link
between the V-ATPase to the actin cytoskeleton where
both the V1 complex and the holoenzyme are shown to
directly interact with F-actin. Interactions between
V-ATPase and microfilaments have been reported during
osteoclast activation, where ruffled membrane V-ATPases
directly bind to F-actin (31). Binding to F-actin was
CAP Links Endocytosis to the Actin Cytoskeleton
Traffic 2005; 6: 930–946 943
attributed to the amino-terminal domain of the subunit B
implicating a role for the actin–V-ATPase complex in
controlling the transport of V-ATPase to the membrane
ruffles (31,32). The interaction of CAP with the B subunit
may also provide a direct link of V-ATPase to the actin
regulatory network. We have found that the actin assem-
bly inhibitors and depolymerization inducers cytochalasin
A and latrunculin B affected the organization of V-ATPase-
decorated vesicles in a similar manner as observed for the
loss of CAP in CAP bsr, suggesting a requirement of the
actin regulatory network and making it likely that the pri-
mary function of CAP in these processes is through its
role in regulation of the actin dynamics.
Materials and Methods
Strains and growth conditionsDictyostelium discoideum wild-type strain AX2, the CAP-deficient mutant
CAP bsr and the transformants were cultured as described (18). For rescue
experiments, CAP bsr cells were transformed with vectors allowing for the
expression of GFP fusions of CAP under the control of the actin15 gene
promoter and the actin8 gene terminator. The construction of the GFP-CAP
fusions was described previously (18,19). The GFP fusion proteins
expressed in the transformants varied in their amounts with levels nearly
identical to wild-type protein level up to twofold higher amounts. Only cells
expressing moderate amounts were chosen for the analysis. A genomic
DNA fragment of 1713 bp coding for NrampB/Nramp1 was cloned into
pDEX27 to produce an NrampB/Nramp1–GFP fusion. The vatB cDNA
was cloned into pDEX79 resulting in the expression of GFP-vatB. AX2 or
CAP bsr transformants expressing GFP fusion proteins of vatB or Nramp1
were analyzed for the presence of the fusion proteins by Western blots
using CAP domain-specific mAbs (17) or GFP-specific mAb K3-184-2 (19)
and by fluorescence microscopy.
Yeast two-hybrid assayThe yeast strain PJ69 4a was a kind gift from Dr Jurgen Dohmen
(University of Cologne). Strain Y190 and plasmids pGBKT7, pGADT7,
pAS2 and pACT2 were obtained from BD Biosciences (Palo Alto, CA).
cDNA fragments comprising the complete Dictyostelium CAP and the
vacuolar ATPase d subunit were amplified by polymerase chain reaction
(PCR) with the primers: 5´-CGGGATCCCGATGTCAGAAGCAACTATTGT-3´
and 5´-CGGGATCCCGTTAAATATGTGAAGTTGATTCA-3´ with a BamH1
linker at either flanking ends, and 5´-GGATCCCGATGGGTTTATTTG
GTGGTAGAAAACATGGTG-3´ and 5´-CTCGAGTTAAAAGATTGGAATTAT
TGATTCTTTTTG-3´ with BamHI and XhoI linker, respectively. Cyclase-
associated protein and N-CAP, a fragment of 663 bp obtained as
described (18), were, respectively, cloned in the GAL4 DNA-binding
domain vectors pGBKT7 and pAS2 using BamHI linker for CAP and
NdeI-BamHI-cloning sites for N-CAP. The V-ATPase d subunit was cloned
into the GAL4 activation domain vector pGADT7 using BamHI and XhoI
linker. A Dictyostelium library of cDNAs pooled from vegetative amoebae
was cloned into the pACT2 vector using the EcoRI-XhoI restriction sites
(43). Yeast transformations, library screening and X-Gal tests were as
described (44).
Immunoprecipitation assayFor analyzing the in vivo interactions, AX2 cells or cells expressing GFP-
CAP were axenically grown, harvested and washed twice with Soerensen
phosphate buffer (pH 6.0) and suspended in twice the volume of immuno-
precipitation (IP) buffer (PB; pH 7.4, 2 mM benzamidine, 4 mM DTT, 2 mM
EDTA and 0.5 mM PMSF). Cells were then lysed by sonication and the
solutions were adjusted to 1 � IP buffer in the presence of 0.5% Triton-X-
100 or 1% NP-40 (Fluka, Munich, Germany). The cell lysate prior to IP was
precleared with protein A Sepharose beads (Amersham Biosciences,
Freiburg, Germany) for 30 min, and the cell debris and proteins bound
nonspecifically were removed by centrifugation (2000 � g for 3 min,
4 �C). The lysate was incubated with GFP mAb K3-184-2 or CAP mAb
230-18-8 bound to protein A Sepharose beads for 2 h at 4 �C, then cen-
trifuged as above and washed several times with IP buffer. The immuno-
complexes were electrophoresed on SDS–PAGE (10–15%) and stained
with Coomassie Blue or processed for immunoblotting and probed either
with vatA or CAP-specific mAbs. The immunoprecipitated bands were
excised and analyzed by MALDI-MS (matrix-associated laser desorption-
ionization mass spectrometry) in the central facilities of the Center of
Molecular Medicine Cologne (CMMC).
Immunofluorescence microscopy and live-cell
imagingAxenically grown cells were harvested and allowed to adhere onto 18 mm
glass coverslips for 30 min, fixed with methanol for 10 min (�20 �C) or
picric acid/paraformaldehyde and processed as described by Weiner et al.
(45). Alternatively, cells on coverslips were quick frozen by floating the
coverslips on liquid N2 for 20 s and then immediately submerging them in
cold MeOH (�20 �C). For studies of cytoskeletal rearrangements, the cells
were incubated with 20 mM cytochalasin A or 1 mM latrunculin B (Sigma-
Aldrich, Munich, Germany) in 17 mM Soerensen phosphate buffer, pH 6.0,
for the desired period of time prior to fixation. For examining the sensitivity
to hypo-osmotic conditions, cells were either treated with medium or
water for 1 h, and images were obtained using a phase-contrast micro-
scope at 40� magnification or alternatively cells were fixed and stained for
vatA. Cyclase-associated protein was detected using mAb 230-18-8, the
V-ATPase was stained with mAb 221-35-2 for the A subunit (22), GFP using
mAb K3-184-2, postlysosomal compartments were detected using vacuo-
lin A-specific mAb 221-1-1 (46) and actin was labeled with mAb act-1–7 (47)
followed by incubation with Cy3-labeled goat anti-mouse IgG secondary
antibody. In paraformaldehyde-fixed cells, actin was stained with FITC-
phallodin (Sigma). Confocal microscopy was done as described (43). The
confocal images obtained at different focal planes were assembled at
equal and optimized averaging and a sectioning of 200 nm. The three
dimensional reconstitution of the images was done using the 3D option
in the Leica confocal software.
To monitor the alterations in the distribution of the CVS system, we labeled
cells with the styryl dye FM 4–64 (Molecular Probes, Karlsruhe, Germany)
(1 mg/mL) in Soerensen phosphate buffer, and time-lapse video micro-
scopy was performed according to Heuser et al. (25). To analyze the live
dynamics of GFP-CAP and Nramp1-GFP, 2–3 � 106 cells/mL were washed
and resuspended in Soerensen phosphate buffer, transferred onto a cover-
slip and allowed to adhere for 15 min. Live dynamics of GFP-CAP were
performed either with ethanol (control) or CMA (10 mM). Images were
obtained every 5 s and were processed using the accompanying Leica
software.
Determination of the endo-lysosomal pHThe endosomal pH was measured by a dual excitation ratio method (exci-
tation at 450 and 495 nm and emission at 520 nm) using FITC-dextran as a
pH probe as described by Temesvari et al. (26). Briefly, cells were grown to
2–5 � 105 cells/mL, harvested and resuspended at a concentration of
3 � 106 cells/mL in fresh axenic medium and loaded with FITC-dextran
(2 mg/mL) (70 000 Mr, Sigma-Aldrich). To monitor changes of endosomal
pH during transit of the fluid phase marker, we pulsed cells for 10 min,
washed and resuspended them in fresh nutrient medium. Basal endoso-
mal pH was measured after loading for 3 h, the time period shown to be
sufficient for the complete loading of all the endo-lysosomal compartments
with a fluid phase marker (23). The cells were then either treated with
CMA (10 mM) (Fluka) or ethanol, the CMA diluent, as control. At various
time points, cells were collected by centrifugation, washed and resus-
pended in 50 mM MES buffer, pH 6.5, and the fluorescence intensity
was measured using a PTI fluorimeter.
Sultana et al.
944 Traffic 2005; 6: 930–946
Miscellaneous methodsStandard molecular biology methods were done as described by Sambrook
et al. (48). SDS–PAGE was performed according to the method of
Laemmli (49) and immunoblotting as described in Towbin et al. (50). The
V-ATPase subunit vatA was detected with mAb 221-35-2 and comitin
(loading control) with mAb 190-68 (22,45) and the osmoregulation assay
was done as described in Gerald et al. (51).
Acknowledgments
We thank Dr S. Muller (CMMC) for performing MALDI-MS analysis,
Dr M. Maniak and B. Gassen for providing monoclonal antibodies and
R. Muller for help with IP experiments. This work was supported by grants
from the DFG (NO 113/7-3, RI 1034/2 and SFB 413), the FCI (Fonds der
Chemischen Industrie) and Koln Fortune.
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