Abstract
Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM, Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 μM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn2+-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 919-923 |
| Number of pages | 5 |
| Journal | Biochemical Journal |
| Volume | 312 |
| Issue number | 3 |
| DOIs | |
| State | Published - Jan 1 1995 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology
Cite this
Zn2+ depletion blocks endosome fusion. / Aballay, Alejandro; Sarrouf, M. N.; Colombo, M. I.; Stahl, P. D.; Mayorga, L. S.
In: Biochemical Journal, Vol. 312, No. 3, 01.01.1995, p. 919-923.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Zn2+ depletion blocks endosome fusion
AU - Aballay, Alejandro
AU - Sarrouf, M. N.
AU - Colombo, M. I.
AU - Stahl, P. D.
AU - Mayorga, L. S.
PY - 1995/1/1
Y1 - 1995/1/1
N2 - Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM, Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 μM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn2+-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.
AB - Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM, Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 μM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn2+-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.
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U2 - 10.1042/bj3120919
DO - 10.1042/bj3120919
M3 - Article
C2 - 8554539
AN - SCOPUS:0029416968
VL - 312
SP - 919
EP - 923
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 3
ER -