The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.
We have investigated the requirement for Ca(2+) 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 Ca(2+). 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 Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) 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 Ca(2+) 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.
Pryor
P R
PR
Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2XY, United Kingdom.
Mullock
B M
BM
Bright
N A
NA
Gray
S R
SR
Luzio
J P
JP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism; D002474:Cell-Free System / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011992:Endosomes / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism; D007070:Immunoglobulin A / Q000378:metabolism / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008247:Lysosomes / Q000502:physiology* / Q000648:ultrastructure; D018942:Macrolides; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008854:Microscopy, Electron; D051381:Rats
D000900:Anti-Bacterial Agents; D002147:Calmodulin; D002614:Chelating Agents; D004791:Enzyme Inhibitors; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D007070:Immunoglobulin A; D007457:Iodine Radioisotopes; D018942:Macrolides; D004533:Egtazic Acid; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; C046770:EGTA acetoxymethyl ester; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium