Ca2+/calmodulin signals the completion of docking and triggers a late step of vacuole fusion

Abstract

The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive' as no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Here we show that Ca2+ is released from the vacuolar lumen following completion of the docking step. We have identified calmodulin as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. We propose that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ.

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).…”

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

“…A regulatory role for Ca 2+ has been established in many other fusion reactions. For example, it controls the fusion of secretory vesicles and of vacuoles [31]. We do not observe the obvious differences of free Ca 2+ levels in all the three different ratios of cytosol to vesicles (data not shown).…”

“…Pharmacological studies indicate that calmodulin is required for late-stage vacuole fusion in vitro and in vivo [31]. A large proportion of the bound calmodulin is released from the vacuole at Ca 2+ concentrations below 500 nM, and the Ca 2+ concentration in the cytosol of living yeast cells is 100-150 nM [32], which is within this range.…”

“…Released calmodulin can be recovered from the supernatant. Freshly isolated vacuoles (in the presence of 3 µM Ca 2+ ) carry about 0.15 ng calmodulin per microgram vacuolar protein, corresponding to 20-100 molecules per vacuole [31]. Sullivan et al [33] consider that calcium mobilization is required for nuclear vesicle fusion.…”

Tansley Review No. 108

“…Using an in vitro model for homotypic vacuole fusion in yeast, Peters & Mayer (1998) observed that vesicle fusion was exquisitely sensitive to calmodulin antagonists late in the docking phase and early in the final fusion steps leading to membrane incorporation. Fusion could be blocked at these stages in vivo using temperaturesensitive calmodulin yeast mutants, and the same block in vitro was relieved on adding wild-type calmodulin.…”