We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

Apodaca
G.
,
Enrich
C.
,
Mostov
K. E.
(
1994
).
The calmodulin antagonist, W-13, alters transcytosis, recycling and morphology of the endocytic pathway in Madin-Darby Canine Kidney Cells.
J. Biol. Chem
269
,
19005
19013
Aubry
L.
,
Klein
G.
,
Martiel
J.-I.
,
Satre
M.
(
1993
).
Kinetics ofendosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy.
J. Cell Sci
105
,
861
866
Aubry
L.
,
Klein
G.
,
Satre
M.
(
1993
).
Endosomal acidification in Dictyostelium discoideum amoebae. Effects of two endocytosis inhibitors: caffeine and cycloheximide. Eur.
J. Cell Biol
61
,
225
228
Bauerfeind
R.
,
Huttner
W. B.
(
1993
).
Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles.
Curr. Opin. Cell Biol
5
,
628
635
Bidani
A.
,
Heming
T. A.
(
1995
).
Effects of bafilomycin A1 on functional capabilities of LPS-activated macrophages.
J. Leuk. Biol
57
,
275
281
Bof
M.
,
Brenot
F.
,
Gonzalez
C.
,
Klein
G.
,
Martin
J.-B.
,
Satre
M.
(
1992
).
Dictyostelium discoideum mutants resistant to the toxic action of methylene diphosphonate are defective in endocytosis.
J. Cell Sci
101
,
139
144
Bowman
E. J.
,
Siebers
A.
,
Altendorf
K.
(
1988
).
Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells.
Proc. Nat. Acad. Sci. USA
85
,
7972
7976
Brenot
F.
,
Aubry
L.
,
Martin
J.-B.
,
Satre
M.
,
Klein
G.
(
1992
).
Kinetics of endosomal acidification in Dictyostelium discoideum amoebae.31P NMR evidence for a very acidic early endosomal compartment.
Biochimie
74
,
883
895
Bucci
C.
,
Parton
R. G.
,
Mather
I. H.
,
Stunnenberg
H.
,
Simons
K.
,
Hoflack
B.
,
Zerial
M.
(
1992
).
The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.
Cell
70
,
715
728
Bush
J.
,
Nolta
K.
,
Rodriguez-Paris
J.
,
Kaufman
N.
,
O'Halloran
T.
,
Ruscetti
T.
,
Temesvari
L.
,
Steck
T.
,
Cardelli
J.
(
1994
).
A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H+-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes.
J. Cell Sci
107
,
2801
2812
Cardelli
J. A.
,
Golumbeski
G. S.
,
Dimond
R. L.
(
1986
).
Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates.
J. Cell Biol
102
,
1264
1270
Cardelli
J. A.
,
Richardson
J.
,
Miears
D.
(
1989
).
Role of acidic intracellular compartments in the biosynthesis of lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting.
J. Biol. Chem
264
,
3454
3463
Clague
M. J.
,
Urbe
S.
,
Aniento
F.
,
Gruenberg
J.
(
1994
).
Vacuolar ATPase activity is required for endosomal carrier vesicle formation.
J. Biol. Chem
269
,
21
24
Crider
B. P.
,
Xie
X.-S.
,
Stone
D. K.
(
1994
).
Bafilomycin inhibits proton flow through the H+ channel of vacuolar proton pumps.
J. Biol. Chem
269
,
17379
17381
Fok
A. K.
,
Clarke
M.
,
Ma
L.
,
Allen
R. D.
(
1993
).
Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study.
J. Cell Sci
106
,
1103
1113
Griffiths
G.
,
Simons
K.
(
1986
).
The trans Golgi Network: sorting at the exit site of the Golgi complex.
Science
234
,
438
443
Gonzales
C.
,
Satre
M.
(
1991
).
Endocytosis in Dictyostelium discoideum amoebae. Inhibition by cycloheximide and other inhibitors of protein synthesis.
J. Cell Sci
99
,
535
543
Henomatsu
N.
,
Yoshimori
T.
,
Yamamoto
A.
,
Moriyama
Y.
,
Tashiro
Y.
(
1993
).
Inhibition of intracellular transport of newly synthesized prolactin by bafilomycin A1 in a pituitary tumor cell line, GH3 cells.
Eur. J. Cell Biol
62
,
127
139
Heuser
J.
,
Zhu
Q.
,
Clarke
M.
(
1993
).
Proton pumps populate the contractile vacuoles of Dictyostelium amoebae.
J. Cell Biol
121
,
1311
1327
Kataoka
T.
,
Takaku
K.
,
Magae
J.
,
Shinohara
N.
,
Takayama
N. H.
,
Kondo
S.
,
Nagai
K.
(
1994
).
Acidification is essential for maintaining the structure and function of lytic granules of CTL.
J. Immunol
153
,
3938
3947
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lenhard
J. M.
,
Kahn
R. A.
,
Stahl
P. D.
(
1992
).
Evidence for ADP-ribosylation factor (ARF) as a regulator of in vitro endosome-endosome fusion.
J. Biol. Chem
267
,
13047
13052
Lenhard
J. M.
,
Mayorga
L.
,
Stahl
P. D.
(
1992
).
Characterization of endosome-endosome fusion in a cell free system using Dictyostelium discoideum.
J. Biol. Chem
267
,
1896
1903
Mattsson
J. P.
,
Väänänen
K.
,
Wallmark
B.
,
Lorentzon
P.
(
1991
).
Omeprazole and bafilomycin A1: two proton pump inhibitors: differentiation of their effects on gastric, kidney and bone H(+)-translocating ATPases.
Biochim. Biophys. Acta
1065
,
261
268
Mayorga
L. S.
,
Beron
W.
,
Sarrouf
M. N.
,
Colombo
M.
,
Creutz
C.
,
Stahl
P. D.
(
1994
).
Calcium-dependent fusion among endosomes.
J. Biol. Chem
269
,
30927
30934
Mellman
I.
,
Fuchs
R.
,
Helenius
A.
(
1986
).
Acidification of the endocytic and exocytic pathways.
Annu. Rev. Biochem
55
,
663
700
Mellman
I.
,
Simons
K.
(
1992
).
The Golgi complex: in vitro veritas?.
Cell
68
,
829
840
Mierendorf
R. C.
,
Cardelli
J. A.
,
Livi
G.
,
Dimond
R. L.
(
1985
).
Synthesis of related forms of the lysosomal enzyme-mannosidase in Dictyostelium discoideum.
J. Biol. Chem
258
,
5878
5884
Mierendorf
R. C.
,
Dimond
R. L.
(
1983
).
Functional heterogeneity of monoclonal antibodies using different screening assays.
Anal. Biochem
135
,
221
229
Moriyama
Y.
,
Maeda
M.
,
Futai
M.
(
1992
).
Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes.
FEBS Lett
302
,
18
20
Myers
M.
,
Forgac
M.
(
1993
).
The coated vesicle vacuolar (H+)-ATPase associates with and is phosphorylated by the 50-kDa polypeptide of the clathrin assembly protein AP-2.
J. Biol. Chem
268
,
9184
9186
Nolta
K. V.
,
Rodriguez-Paris
J. M.
,
Steck
T. L.
(
1994
).
Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation.
Biochim. Biophys Acta
1224
,
237
246
Nordstrom
T.
,
Rotstein
O. D.
,
Romanek
R.
,
Asotra
S.
,
Heersche
J. N. M.
,
Manolson
M. F.
,
Brisseau
G. F.
,
Grinstein
S.
(
1995
).
Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.
J. Biol. Chem
270
,
2203
2212
O'Halloran
T. J.
,
Anderson
R. G. W.
(
1992
).
Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles and development in Dictyostelium.
J. Cell Biol
118
,
1371
1378
Okuma
S.
,
Poole
B.
(
1978
).
Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.
Proc. Nat. Acad. Sci. USA
75
,
3327
3331
Orci
L.
,
Halban
P.
,
Amherdt
M.
,
Pavazzola
M.
,
Vassali
J. D.
,
Perrelet
A.
(
1984
).
A clathrin-coated, golgi related compartment of the insulin secreting cell accumulates pro-insulin in the presence of monensin.
Cell
39
,
39
47
Palokangas
H.
,
Metsikko
K.
,
Vaananen
K.
(
1994
).
Active vacuolar H+-ATPase is required for both endocytic and exocytic processes during viral infection of BHK-21 cells.
J. Biol. Chem
269
,
17577
17585
Patterson
D. J.
(
1980
).
Contractile vacuoles and associated structures: their organization and function.
Biol. Rev.
55
,
1
46
Poitras
M.
,
Ribeiro-Do-Valle
R.-M.
,
Poirier
S. N.
,
Guillemette
G.
(
1995
).
Bidirectional activity of the endoplasmic reticulum Ca2+-ATPase of bovine adrenal cortex.
Biochemistry
34
,
9755
9761
Reaves
B.
,
Banting
G.
(
1994
).
Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane.
FEBS Lett
345
,
61
66
Reijngoud
D. J.
,
Tager
J. M.
(
1977
).
The permeability properties of the lysosomal membrane.
Biochim. Biophys. Acta
472
,
419
449
Richardson
J. M.
,
Woychik
N. A.
,
Ebert
D. L.
,
Dimond
R.
,
Cardelli
J. A.
(
1988
).
Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum.
J. Cell Biol
107
,
2097
2107
Rodriguez-Paris
J. M.
,
Nolta
K. V.
,
Steck
T. L.
(
1993
).
Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fraction.
J. Biol. Chem
268
,
9110
9116
Ruscetti
T.
,
Cardelli
J. A.
,
Niswonger
M. L.
,
O'Halloran
T. J.
(
1994
).
Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.
J. Cell Biol
126
,
343
352
Sandvig
K.
,
Olsnes
S.
,
Peterson
O. W.
,
van Deurs
B.
(
1987
).
Acidification of the cytosol inhibits endocytosis from coated pits.
J. Cell Biol
105
,
679
689
Schatzle
J.
,
Rathi
A.
,
Clarke
M.
,
Cardelli
J. A.
(
1991
).
Developmentalregulation of the -mannosidase gene in Dictyostelium discoideum: control is at the level of transcription and is affected by cell density.
Mol. Cell Biol
11
,
3339
3347
Spiro
D. J.
,
Taylor
T. C.
,
Melancon
P.
,
Wessling-Resnick
M.
(
1995
).
Cytosolic ADP-ribosylation factors are not required for endosome-endosome fusion but are necessary for GTP gamma S inhibition of fusion.
J. Biol. Chem
270
,
13693
13697
Steinman
R. M.
,
Silver
J. M.
,
Cohn
Z. A.
(
1974
).
Pinocytosis in fibroblasts. Quantitative studies in vitro.
J. Cell Biol
63
,
949
969
Swallow
C. J.
,
Grinstein
S.
,
Sudsbury
P. A.
,
Rotstein
O. D.
(
1993
).
Relative roles of Na+/H+exchange and vacuolar-type H+ ATPases in regulating cytoplasmic pH and function of murine peritoneal macrophages.
J. Cell Physiol
157
,
453
460
Temesvari
L.
,
Rodriguez-Paris
J.
,
Bush
J.
,
Steck
T. L.
,
Cardelli
J.
(
1994
).
Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.
J. Biol. Chem
269
,
25719
25727
Towbin
H.
,
Staehelin
T.
,
Gordon
J.
(
1979
).
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Nat. Acad. Sci. USA
76
,
4350
4354
van Weert
A. W. M.
,
Dunn
K. W.
,
Geuze
H. J.
,
Maxfield
F. R.
,
Stoorvogel
W.
(
1995
).
Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.
J. Cell Biol
130
,
821
834
Woo
J. T.
,
Shirohara
C.
,
Sakai
K.
,
Hasumi
K.
,
Endo
H.
(
1992
).
Inhibition of acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774.
Eur. J. Biochem
207
,
8638
8644
Yamashiro
C. T.
,
Kane
P. M.
,
Wolczyk
D. F.
,
Preston
R. A.
,
Stevens
T. H.
(
1990
).
Role of vacuolar acidification in protein sorting and zymogenactivation; a genetic analysis of the yeast vacuolar proton-translocating ATPase.
Mol. Cell Biol
10
,
3737
3749
Yamashiro
D. J.
,
Maxfield
F. R.
(
1988
).
Kinetics of endosome acidification in mutant and wild type chinese ovary cells.
J. Cell Biol
105
,
2713
2721
Yilla
M.
,
Tan
A.
,
Kouichi
I.
,
Miwa
K.
,
Ploegh
H.
(
1993
).
Involvement of vacuolar H+-ATPases in the secretory pathway of HepG2 cells.
J. Biol. Chem
268
,
19092
19100
Yoshimori
T.
,
Yamamoto
A.
,
Moriyama
Y.
,
Futai
M.
,
Tashiro
Y.
(
1991
).
Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.
J. Biol. Chem
266
,
17707
17712
Zeuzem
S.
,
Feick
P.
,
Zimmermann
P.
,
Haase
W.
,
Kahn
R. A.
,
Schulz
I.
(
1992
).
Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes.
Proc. Nat. Acad. Sci. USA
89
,
6619
6623
Zeuzem
S.
,
Zimmermann
P.
,
Schulz
I.
(
1992
).
Association of a 19-and 21-kDa GTP-binding protein to pancreatic microsomal vesicles is regulated by the intravesicular pH established by a vacuolar-type H+-ATPase.
J. Membr. Biol
125
,
231
241
Zhang
J.
,
Feng
Y.
,
Forgac
M.
(
1994
).
Proton conduction and bafilomycin binding by the Vodomain of the coated vesicle V-ATPase.
J. Biol. Chem
269
,
23518
23523
Zhang
K.
,
Wang
Z.-Q.
,
Gluck
S.
(
1992
).
Identification and partial purification of a cytosolic activator of vacuolar H+-ATPases from mammalian kidney.
J. Biol. Chem
267
,
9701
9705
Zhu
Q.
,
Clarke
M. J.
(
1992
).
Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum.
J. Cell Biol
118
,
347
358
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