The role of calcium and other ions in sorting and delivery in the late endocytic pathway.

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. Ca(2+) ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca(2+) 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).

Relationship between endosomes and lysosomes.

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are re-formed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Re-formation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

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.

Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.

Lysosome-endosome fusion and lysosome biogenesis.

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Association of AP1 adaptor complexes with GLUT4 vesicles.

Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes.

Evidence against protein kinase B as a mediator of contraction-induced glucose transport and GLUT4 translocation in rat skeletal muscle.

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4-6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.