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      The Role of Intraorganellar Ca 2+In Late Endosome–Lysosome Heterotypic Fusion and in the Reformation of Lysosomes from Hybrid Organelles

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          Abstract

          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.

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          Most cited references57

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          Mechanisms of intracellular protein transport.

          Recent advances have uncovered the general protein apparatus used by all eukaryotes for intracellular transport, including secretion and endocytosis, and for triggered exocytosis of hormones and neurotransmitters. Membranes are shaped into vesicles by cytoplasmic coats which then dissociate upon GTP hydrolysis. Both vesicles and their acceptor membranes carry targeting proteins which interact specifically to initiate docking. A general apparatus then assembles at the docking site and fuses the vesicle with its target.
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            Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants.

            The collection of vacuolar protein sorting mutants (vps mutants) in Saccharomyces cerevisiae comprises of 41 complementation groups. The vacuoles in these mutant strains were examined using immunofluorescence microscopy. Most of the vps mutants were found to possess vacuolar morphologies that differed significantly from wild-type vacuoles. Furthermore, mutants representing independent vps complementation groups were found to share aberrant morphological features. Six distinct classes of vacuolar morphology were observed. Mutants from eight vps complementation groups were defective both for vacuolar segregation from mother cells into developing buds and for acidification of the vacuole. Another group of mutants, represented by 13 complementation groups, accumulated a novel organelle distinct from the vacuole that contained a late-Golgi protein, active vacuolar H(+)-ATPase complex, and soluble vacuolar hydrolases. We suggest that this organelle may represent an exaggerated endosome-like compartment. None of the vps mutants appeared to mislocalize significant amounts of the vacuolar membrane protein alkaline phosphatase. Quantitative immunoprecipitations of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) were performed to determine the extent of the sorting defect in each vps mutant. A good correlation between morphological phenotype and the extent of the CPY sorting defect was observed.
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              The fungal vacuole: composition, function, and biogenesis.

              The fungal vacuole is an extremely complex organelle that is involved in a wide variety of functions. The vacuole not only carries out degradative processes, the role most often ascribed to it, but also is the primary storage site for certain small molecules and biosynthetic precursors such as basic amino acids and polyphosphate, plays a role in osmoregulation, and is involved in the precise homeostatic regulation of cytosolic ion and basic amino acid concentration and intracellular pH. These many functions necessitate an intricate interaction between the vacuole and the rest of the cell; the vacuole is part of both the secretory and endocytic pathways and is also directly accessible from the cytosol. Because of the various roles and properties of the vacuole, it has been possible to isolate mutants which are defective in various vacuolar functions including the storage and uptake of metabolites, regulation of pH, sorting and processing of vacuolar proteins, and vacuole biogenesis. These mutants show a remarkable degree of genetic overlap, suggesting that these functions are not individual, discrete properties of the vacuole but, rather, are closely interrelated.
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                Author and article information

                Contributors
                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                29 May 2000
                : 149
                : 5
                : 1053-1062
                Affiliations
                [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
                Article
                9911059
                10.1083/jcb.149.5.1053
                2174832
                10831609
                84d0e268-2f75-4f34-a8f8-bba3e172c868
                © 2000 The Rockefeller University Press
                History
                : 15 November 1999
                : 14 March 2000
                : 12 April 2000
                Categories
                Original Article

                Cell biology
                calcium,membrane fusion,lysosome,endosome,calmodulin
                Cell biology
                calcium, membrane fusion, lysosome, endosome, calmodulin

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