Showing papers by "María Isabel Colombo published in 1995"

A regulatory role for ARF6 in receptor-mediated endocytosis.

Abstract: Adenosine diphosphate-ribosylation factor 6 (ARF6), ARF6 mutants, and ARF1 were transiently expressed in Chinese hamster ovary cells, and the effects on receptor-mediated endocytosis were assessed. Overexpressed ARF6 localized to the cell periphery and led to a redistribution of transferrin receptors to the cell surface and a decrease in the rate of uptake of transferrin. Similar results were obtained when a mutant defective in guanosine triphosphate hydrolysis was expressed. Expression of a dominant negative mutant, ARF6(T27N), resulted in an intracellular distribution of transferrin receptors and an inhibition of transferrin recycling to the cell surface. In contrast, overexpression of ARF1 had little or no effect on these parameters of endocytosis.

Zn2+ depletion blocks endosome fusion.

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 microM 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+. Zn(2+)-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.

GTPases: Key regulatory components of the endocytic pathway

Abstract: GTP-binding proteins or GTPases are versatile cyclic molecular switches (Gilman, A.G. 1987 ; Bourne et al., 1990, 1992). In the past few years there has been an explosion of interest in unraveling the role of GTPases in membrane traffic (Balch et al., 1990; Bourne, H.R., 1988). GTP-binding proteins are classified into two broad families: ras-like monomeric GTP-binding proteins and the heterotrimeric G proteins. Evidences from genetics, immunolocalization, and functional assays has established that two subfamilies of monomeric GTP-binding proteins, the Rab and ARF subfamilies, are required for membrane traffic (Pfeffer, S.R., 1992; Rothman, J.E. and Orci, L., 1992). More recently, attention has turned to the role of heterotrimeric G proteins in membrane traffic (Balch et al., 1992; Barr et al., 1992). This review will focus solely on the recent data that suggest that heterotrimeric G proteins and monomeric GTP-binding proteins are involved in endosome-endosome fusion and in endocytic transport.