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

Abstract

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.

Syntaxin 7 Complexes with Mouse Vps10p Tail Interactor 1b, Syntaxin 6, Vesicle-associated Membrane Protein (VAMP)8, and VAMP7 in B16 Melanoma Cells

“…Interestingly, in vitro homotypic fusion of yeast vacuoles requires a protein phosphatase I that is activated by calcium and calmodulin (40). Although a functional role has not yet been demonstrated for a protein phosphatase I in lysosomal fusion, there is a requirement for calcium and calmodulin in lysosomal fusion which mirrors the requirement for calcium and calmodulin in homotypic vacuolar fusion (41). Intriguingly, melanosomes are transported to the cell periphery in a manner that is dependent on myosin Va (42) and Rab27a (43).…”

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…Ion dysregulation could in turn have indirect consequences on other processes in OCA2-deficient cells. For example, alterations in intralumenal ion balance or pH can affect cellular membrane fusion events (Peters and Mayer, 1998;Ungermann et al, 1999;Pryor et al, 2000), perhaps explaining the accumulation of melanosomal cargo in vesicular structures in OCA2-deficient melanocytes (Manga et al, 2001). Moreover, disruption of OCA2 transport activity across the melanosomal membrane may indirectly alter ion concentrations or pH in the cytosol; for example, exogenous expression of OCA2 in Saccharomyces cerevisiae led to a depletion of cytoplasmic glutathione due to glutathione transport into the vacuole (Staleva et al, 2002).…”

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

“…Another attractive hypothesis for impaired endolysosomal trafficking is a defect in vesicle fusion or ARTICLE fission processes. It has been postulated before that Ca 2 þ is required for vesicular fusion processes and thereby regulates trafficking of vesicular cargo proteins 30,32,33 . In accordance with this hypothesis, we found that in WT MEFs chelation of cytosolic Ca 2 þ by 1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) mimics the effect on EGF trafficking observed in TPC2 À / À MEFs (Fig.…”