The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H+ ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na+/H+ exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca2+ ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca2+ in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).
The role of luminal H+ and Na+ ions in sorting in the endocytic pathway
The acid lumen of endosomes and lysosomes is generated by the activity of the vacuolar H+-ATPase, acting as a proton pump, present in the limiting membrane of the organelles of the endocytic pathway [1,2]. To maintain electroneutrality in the endosome lumen, H+ entry driven by the vacuolar H+-ATPase is accompanied by Cl− entry [3,4]. Mutations in endosomal/lysosomal members of the CLC family of Cl− channels/transporters can lead to disease . Endosomes have a luminal pH of 5.0–6.0, whereas lysosomes are more acidic with a luminal pH of 4.6–5.0 . Late endosomes have a lower pH than early endosomes. The difference is explained by the presence in early endosomes of Na+, K+-ATPase activity that contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport . The pH difference between the lumen of the early endosome and the extracellular environment provides asymmetry to the recycling circuit between the two compartments . Thus, in a classical example, LDL (low-density lipoprotein) molecules are delivered to lysosomal hydrolases for degradation after dissociation from LDL receptors in the acidic lumen of the early endosome, a process that also allows subsequent recycling of the empty receptors to the cell surface. Other ligands, such as EGF (epidermal growth factor), remain bound to their receptors, which are ubiquitinated [7,8] and, after endocytosis, are sorted into luminal vesicles in endosomes. The formation of luminal vesicles and the process of sorting membrane proteins destined for degradation into these vesicles have recently received much attention (reviewed in ). In yeast, genetic screens identified genes encoding 17 soluble Class E Vps (vacuolar protein sorting) proteins and one integral membrane Vps protein required for this process. The soluble Class E proteins function as ESCRT (endosomal sorting complexes required for transport) complexes and associated proteins. The membrane protein is Nhx1p, which shows high homology to the mammalian Na+/H+ exchangers of the NHE (Na+/H+ exchanger) family and is thought to transport sodium ions into the yeast pre-vacuolar (endosomal) compartment in exchange for protons . It has been suggested that the luminal ion concentration and pH are monitored by an unknown transmembrane protein sensor, which transmits this information to the cytosolic face of the membrane to facilitate the binding of cytosolic proteins such as ESCRT proteins . In mammalian cells, the functioning of the ESCRT complexes is less well understood than in yeast, and the mammalian orthologue of Nhx1p has not yet been confirmed among the various members of the NHE family. The site of initial action of the ESCRT proteins in mammalian cells is on early endosomes, but formation of luminal vesicles and sorting into them continues in the late endosomes [MVBs (multivesicular bodies)]. While the ESCRT complexes are implicated in the formation of luminal vesicles as well as in the process of sorting cargo proteins into them, the mechanism of inward budding remains unknown. Experiments with liposomes have suggested that LBPA (lysobisphosphatidic acid), which is normally found in late endocytic organelles, may induce the formation of luminal vesicles. This process is dependent on a pH gradient across the limiting membrane of liposomes similar to that found across endosome membranes and may be regulated by the ESCRTIII-associated protein Alix .
Passage through the endocytic pathway to lysosomes
Two models have been proposed for the process by which late endosomes are formed from early endosomes: a maturation model [12,13] and a model in which endocytic carrier vesicles , formed as a result of the recruitment of cytosolic coat proteins to early endosomes, are required for transfer of cargo to late endosomes. Inhibition of the endosomal proton pump with bafilomycin A1 prevents transfer of endocytosed markers to late endosomes, possibly by inhibition of formation of endocytic carrier vesicles . Recent live-cell imaging studies have shown that large (400–800 nm) vesicles arise from a dynamic early endosome network and undergo conversion such that they lose the small GTPase Rab5 and recruit Rab7, which is enriched on late endosomes . Delivery to lysosomes occurs as a result of kissing events and direct fusion between late endosomes and lysosomes as clearly demonstrated in experiments on NRK (normal rat kidney) fibroblastic cells using live cell confocal microscopy .
Throughout the endocytic pathway, fusion events between organelles are essential to achieve the passage of luminal content as well as membrane proteins through the pathway and to modulate the ratio of the area of the limiting membrane of the organelles to their luminal volume. Homotypic fusion events occur between early endosomes and between late endosomes, but delivery to lysosomes requires heterotypic fusion of lysosomes with late endosomes to form hybrid organelles from which lysosomes are re-formed by a maturation process. The individual fusion steps are mediated by trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes that have been identified and provide specificity to the fusion event [18,19].
The role of luminal Ca2+ in fusion events in the endocytic pathway
There is evidence from cell-free content mixing assays that, for both homotypic and heterotypic fusion events in the endocytic pathway, there is a Ca2+/calmodulin requirement for membrane fusion  with the Ca2+ being released from the lumen of the fusing organelles late in the mechanistic pathway of fusion. Endocytosis can provide a significant contribution to cellular calcium content, since uptake can occur from extracellular fluid. Estimates of free Ca2+ concentration in endocytic organelles vary widely from 3 to 600 μM [21,22], although they are always significantly higher than the resting cytosolic Ca2+ concentration of up to 100 nM.
The first evidence that the release of luminal Ca2+ plays a role in membrane fusion in the endocytic pathway came from cell-free experiments on homotypic vacuole fusion in yeast. It was found that the membrane-impermeable Ca2+ chelating agent BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid] inhibited fusion, whereas EGTA had no effect . A significant difference between BAPTA and EGTA is that at physiological pH, BAPTA exchanges Ca2+ approximately 100 times faster than EGTA, reflecting higher rates of both association and dissociation. BAPTA can thus disperse a cytosolic gradient of Ca2+ concentration, resulting from its mobilization from a sequestered pool, fast enough to suppress a biological response, even though EGTA has no effect. In addition to BAPTA, ionomycin, a Ca2+ ionophore, and Ca2+-ATPase inhibitors all blocked homotypic vacuole fusion. It was also possible to deplete luminal Ca2+ stores by treatment with BAPTA in the presence of ionomycin and show that such Ca2+-depleted vacuoles were unable to fuse. Yet, once luminal Ca2+ was restored, the vacuoles again became fusion-competent. By studying the stage at which vacuolar fusion became resistant to BAPTA, it was shown that release of luminal Ca2+ required for membrane fusion was a post-docking event, i.e. occurred after formation of the trans-SNARE complex. Using cell-free systems, established with subcellular fractions from mammalian cells, complementary data have been obtained for homotypic fusion of early endosomes  and heterotypic fusion of late endosomes with lysosomes . In both systems, fusion was inhibited by BAPTA but not EGTA and was also prevented by pre-incubation with a membrane-permeable ester, EGTA-AM (EGTA-acetoxymethyl ester), which is cleaved within the organelle lumen, resulting in chelation of luminal Ca2+. The inhibitory effects of BAPTA and EGTA-AM were reversed by addition of CaCl2. These results strongly support a role for the release of Ca2+ from the endosome lumen in fusion events in the endocytic pathway. Experiments in which increasing concentrations of CaCl2 were added to fusion assays in the presence of BAPTA suggested that ∼0.5 μM Ca2+ is optimal for fusion. High concentrations of Ca2+, e.g. 1 mM, inhibit both fusion events.
An interesting feature of the differential inhibition of fusion by BAPTA and EGTA is that it provides additional information about the distance between the site of luminal Ca2+ release and the site of bilayer fusion because a chelator with slow binding kinetics will be less effective close to the Ca2+ channel. Given the differential effects of BAPTA and EGTA, it is likely that, in the endocytic pathway, membrane fusion occurs between 20 and 100 nm from the site of Ca2+ release [25–27].
Exactly how the release of luminal Ca2+ causes membrane fusion after trans-SNARE formation is unclear but there is some evidence that it is via a calmodulin-mediated event(s). Direct evidence for this was obtained in experiments on yeast homotypic vacuole fusion where it was also shown that calmodulin is a component of a high-molecular-mass complex on the yeast vacuole which also contains protein phosphatase 1, an essential protein for vacuolar bilayer mixing [23,28]. In mammalian cells, use of calmodulin antagonists has suggested that this protein is important in fusion of endosomes [29,30] and the fusion of late endosomes with lysosomes . The requirement for Ca2+ in cell-free late endosome–lysosome fusion is thought to occur late in the fusion process and the need for a downstream Ca2+ effector is consistent with the observed displacement of the time course of BAPTA inhibition from that which occurred when the reaction was stopped by placing the assay tubes on ice .
Although the results from experiments using BAPTA, EGTA and EGTA-AM make a prima facie case that release of luminal Ca2+ is required for fusion events in the late endocytic pathway, this has been challenged. The BAPTA effects on vacuole fusion have been proposed to occur via effects on the ionic strength of the fusion assay medium, resulting in removal of key peripheral membrane proteins from the vacuoles . At lower BAPTA concentrations where these effects are not observed, inhibition may not be due to Ca2+ chelation because Mg2+ is also able to rescue. Nevertheless, the bulk of published results support a role for release of luminal Ca2+ being at the very least an important regulatory step in fusion in the late endocytic pathway despite the mechanism by which this effect is mediated still being poorly understood .
Luminal Ca2+ and the re-formation of lysosomes
Following late endosome–lysosome fusion, a hybrid organelle is formed. Lysosomes separate as dense organelles following density-gradient centrifugation and are often observed as electron-dense structures by electron microscopy (Figure 1), implying condensed protein/lipid contents. The re-formation of a lysosome from hybrid organelles involves condensation of contents and increase in density in a process that is likely to have analogies to the formation of mature regulated secretory granules in neuroendocrine cells [33,34]. Thus, as for regulated secretory granules, there is evidence that luminal Ca2+ and low pH are required for formation of dense core lysosomes. Treatment of hybrid organelles with either EGTA-AM or bafilomycin A1, an inhibitor of the vacuolar proton pumping ATPase, can prevent lysosome re-formation in a cell-free system .
Lysosomes have condensed contents
Disease and Ca2+ in the late endocytic pathway
If luminal Ca2+ is essential for efficient membrane traffic in the endocytic pathway, one prediction is that diseases that result in an alteration of luminal Ca2+ concentration may show defects in the endocytic pathway. MLIV (mucolipidosis type IV) is an autosomal recessive neurodegenerative lysosomal storage disorder caused by mutations in the gene MCOLN1, which encodes the 580-amino-acid, multispanning, lysosomal membrane protein mucolipin-1. This is a non-specific cation channel modulated by changes in Ca2+ concentration and pH [35,36]. MLIV is characterized by abnormal late endosomal/lysosomal structures which are filled with multilamellar membranous whorls and do not show the characteristic morphology of dense core lysosomes by electron microscopy. It has been suggested that the absence of the mucolipin-1 in MLIV patients, and its orthologue CUP-5 in Caenorhabditis elegans, results in failure to re-form lysosomes from hybrids and thereby causes disease . This may be due to the requirement for intraorganellar Ca2+ for the condensation of content in the re-formation of lysosomes from hybrid organelles. However, loss of mucolipin-1 or CUP-5 may also cause defects in the retrieval of components from endosome–lysosome hybrids, possibly by inhibiting formation of specific transport vesicles or other intermediates. This hypothesis is consistent with the fact that abnormalities in lipid traffic through the late endocytic pathway are clearly shown by the impairment of fluorescently labelled lactosylceramide traffic . Aberrant lactosylceramide trafficking in MLIV cells may be rescued by wild-type mucolipin-1 expression but not by mucolipin-1 mistargeted to the plasma membrane, nor by lysosome-localized mucolipin-1 mutated in its predicted ion pore-selectivity region .
The luminal ionic composition of endocytic organelles clearly plays an important role in their function. How alteration of this composition may be sensed on the cytosolic surface of late endocytic organelles remains unclear but is likely to be very important, for example in regulating the formation of luminal vesicles in MVBs. Release of luminal Ca2+ appears to be necessary for efficient homotypic fusion of endosomes and heterotypic fusion of late endosomes with lysosomes. However, proof that this is the case requires both identification of the channels through which luminal Ca2+ is released and the mechanism by which the released Ca2+ exerts its effects on fusion. Finally, it is also clear that our knowledge of the various ion transporters/channels involved in the regulation of luminal ionic composition is far from complete. With regard to lysosomal Ca2+ alone, in addition to mucolipin-1, the lysosomal NAADP (nicotinic acid–adenine dinucleotide phosphate)-regulated Ca2+ channel  and a ligand-gated P2X4 cation channel  are likely to play a role in regulating the luminal concentration of Ca2+.
Inflammation: A Focus Topic at Life Sciences 2007, held at SECC Glasgow, U.K., 9–12 July 2007. Edited by J. Allen (University College Dublin, Ireland), G. Brown (Cambridge, U.K.), G. Cirino (Napoli, Italy), A. Floto (Cambridge, U.K.), I. Megson (UHI Millennium Institute, U.K.), C. Page (King's College London, U.K.), M. Perretti (Queen Mary, University of London, U.K.) and S. Ward (Bath, U.K.).
endosomal sorting complexes required for transport
mucolipidosis type IV
- NRK cells
normal rat kidney cells
soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor
vacuolar protein sorting
Our experimental work is funded by a Research Grant (G9310915) from the Medical Research Council (MRC) to J.P.L. The Cambridge Institute for Medical Research is supported by a Strategic Award (079895) from the Wellcome Trust.
Present address: Department of Biology (Area 9), University of York, PO Box 373, York YO10 5YW, U.K.