Download The role of calcium and other ions in sorting and delivery in the late

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Biochemical Society Transactions (2007) Volume 35, part 5
The role of calcium and other ions in sorting and
delivery in the late endocytic pathway
J.P. Luzio1 , N.A. Bright and P.R. Pryor2
Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0XY, U.K.
Abstract
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 [5]. Endosomes
have a luminal pH of 5.0–6.0, whereas lysosomes are more
acidic with a luminal pH of 4.6–5.0 [1]. 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 [6].
The pH difference between the lumen of the early endosome
and the extracellular environment provides asymmetry to the
recycling circuit between the two compartments [1]. 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
Key words: calcium, endocytosis, endosome, lysosome, mucolipin, multivesicular body.
Abbreviations used: BAPTA, 1,2-bis-(o-aminophenoxy)ethane-N,N,N ,N -tetra-acetic acid;
EGTA-AM, EGTA-acetoxymethyl ester; ESCRT, endosomal sorting complexes required for transport;
LBPA, lysobisphosphatidic acid; LDL, low-density lipoprotein; MLIV, mucolipidosis type IV; MVB,
multivesicular body; NHE, Na+ /H+ exchanger; NRK cells, normal rat kidney cells; SNARE, soluble
N-ethylmaleimide-sensitive fusion protein-attachment protein receptor; Vps, vacuolar protein
sorting.
1
To whom correspondence should be addressed (email [email protected]).
2
Present address: Department of Biology (Area 9), University of York, PO Box 373, York
YO10 5YW, U.K.
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Authors Journal compilation 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 [9]). 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 [10]. 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 [10]. 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
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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 [11].
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
[14], 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 [15]. 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 [16]. 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 [17].
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 transSNARE (soluble N-ethylmaleimide-sensitive fusion proteinattachment 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 [20] 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)ethaneN,N,N ,N -tetra-acetic acid] inhibited fusion, whereas
EGTA had no effect [23]. 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 cellfree systems, established with subcellular fractions from
mammalian cells, complementary data have been obtained for
homotypic fusion of early endosomes [24] and heterotypic
fusion of late endosomes with lysosomes [20]. In both
systems, fusion was inhibited by BAPTA but not EGTA
and was also prevented by pre-incubation with a membranepermeable 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
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Biochemical Society Transactions (2007) Volume 35, part 5
Figure 1 Lysosomes have condensed contents
(A) Dense core lysosomes in NRK cells were loaded with BSA conjugated
with colloidal gold (black spots) for 4 h followed by a 24 h chase. The
electron-dense content of the organelle lumen can be shown to be
similar to the density of cheddar cheese (B), but not that of cream
cheese (C), by transposing the boxed area directly on to micrographs of
cheese, processed and contrasted in an identical way. Scale bar, 500 nm.
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 [20].
Disease and Ca2+ in the late endocytic
pathway
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 [20]. 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 [20].
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 [31]. 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 [32].
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
C The
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Authors Journal compilation 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 [37]. 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 [38]. 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 lysosomelocalized mucolipin-1 mutated in its predicted ion poreselectivity region [35].
Conclusions
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+
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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 [39] and a
ligand-gated P2X4 cation channel [40] are likely to play a
role in regulating the luminal concentration of Ca2+ .
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.
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Received 20 July 2007
doi:10.1042/BST0351088
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