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© The Rockefeller University Press, 0021-9525/2000//1053 $5.00
The Journal of Cell Biology, Volume 149, Number 5, , 2000 1053-1062

The Role of Intraorganellar Ca²⁺In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

^a Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2XY, United Kingdom
^b Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2XY, United Kingdom
Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2XY, UK.44-1223-76263044-1223-336780

jpl10{at}cam.ac.uk

We have investigated the requirement for Ca²⁺ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca²⁺. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca²⁺ reduced the amount of fusion. The requirement for Ca²⁺ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca²⁺ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Key Words: calcium • calmodulin • endosome • lysosome • membrane fusion

© 2000 The Rockefeller University Press

Abbreviations used in this paper: Av-ASF, avidin-asialofetuin; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; bpIgA, biotinylated polymeric IgA; EGTA-AM, methyl ester of EGTA; GDI, GDP dissociation inhibitor; GMP-PNP, 5'-guanylylimidodiphosphate; NSF, N-ethylmaleimide–sensitive factor; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; STKM, STM containing 25 mM KCl.

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