Dynamics of the vacuolar H(+)-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells.
2002
The vacuolar H + -ATPase (V-ATPase) is a multi-subunit enzyme that
plays important roles in eukaryotic cells. In Dictyostelium , it is
found primarily in membranes of the contractile vacuole complex, where it
energizes fluid accumulation by this osmoregulatory organelle and also in
membranes of endolysosomes, where it serves to acidify the endosomal lumen. In
the present study, a fusion was created between vatM , the gene
encoding the 100 kDa transmembrane subunit of the V-ATPase, and the gene
encoding Green Fluorescent Protein (GFP). When expressed in
Dictyostelium cells, this fusion protein, VatM-GFP, was correctly
targeted to contractile vacuole and endolysosomal membranes and was competent
to direct assembly of the V-ATPase enzyme complex. Protease treatment of
isolated endosomes indicated that the GFP moiety, located on the C-terminus of
VatM, was exposed to the cytoplasmic side of the endosomal membrane rather
than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex
revealed clearly the dynamics of this pleiomorphic vesiculotubular organelle.
VatM-GFP labeling of endosomes allowed direct visualization of the trafficking
of vacuolar proton pumps in this pathway, which appeared to be entirely
independent from the contractile vacuole membrane system. In cells whose
endosomes were pre-labeled with TRITC-dextran and then fed yeast particles,
VatM-GFP was delivered to newly formed yeast phagosomes with the same time
course as TRITC-dextran, consistent with transfer via a direct fusion of
endosomes with phagosomes. Several minutes were required before the intensity
of the VatM-GFP labeling of new phagosomes reached the level observed in older
phagosomes, suggesting that this fusion process was progressive and
continuous. VatM-GFP was retrieved from the phagosome membrane prior to
exocytosis of the indigestible remnants of the yeast particle. These data
suggest that vacuolar proton pumps are recycled by fusion of advanced with
newly formed endosomes.
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