Paul R. Pryor

Lysosomes: fusion and function

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans‐SNARE complex consisting of Q‐SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R‐SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell‐free systems, we confirmed this result, but found that the same Q‐SNAREs can combine with an alternative R‐SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co‐immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N‐terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.

Mucolipin‐1 Is a Lysosomal Membrane Protein Required for Intracellular Lactosylceramide Traffic

Mucolipin‐1 is a membrane protein encoded by the gene MCOLN1, mutations in which result in the lysosomal storage disorder mucolipidosis type IV (MLIV). Efficient lysosomal targeting of mucolipin‐1 requires di‐leucine motifs in both the N‐terminal and the C‐terminal cytosolic tails. We have shown that 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 or by lysosome‐localized mucolipin‐1 mutated in its predicted ion pore‐selectivity region. Our data demonstrate that the correct localization of mucolipin‐1 and the integrity of its ion pore are essential for its physiological function in the late endocytic pathway.

Molecular Basis for the Sorting of the SNARE VAMP7 into Endocytic Clathrin- Coated Vesicles by the ArfGAP Hrb

Summary SNAREs provide the specificity and energy for the fusion of vesicles with their target membrane, but how they are sorted into the appropriate vesicles on post-Golgi trafficking pathways is largely unknown. We demonstrate that the clathrin-mediated endocytosis of the SNARE VAMP7 is directly mediated by Hrb, a clathrin adaptor and ArfGAP. Hrb wraps 20 residues of its unstructured C-terminal tail around the folded VAMP7 longin domain, demonstrating that unstructured regions of clathrin adaptors can select cargo. Disrupting this interaction by mutation of the VAMP7 longin domain or depletion of Hrb causes VAMP7 to accumulate on the cells surface. However, the SNARE helix of VAMP7 binds back onto its longin domain, outcompeting Hrb for binding to the same groove and suggesting that Hrb-mediated endocytosis of VAMP7 occurs only when VAMP7 is incorporated into a cis-SNARE complex. These results elucidate the mechanism of retrieval of a postfusion SNARE complex in clathrin-coated vesicles.

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).

Delivery of endocytosed membrane proteins to the lysosome

The delivery of proteins from the plasma membrane to the lysosome for degradation is essential for normal cellular function. There is now a good understanding of the protein complexes involved in sorting proteins at the plasma membrane and into the intralumenal vesicles of the multi-vesicular body. A combination of cell free content mixing assays and live-cell imaging has dissected out the final step in delivery of macromolecules to the lysosome from the multi-vesicular body and provided insights into the molecular mechanisms by which late endosomes and lysosomes exchange lumenal contents. The endocytic pathway has provided a platform with which to understand the autophagic and phagocytic pathways, but the fine details of how traffic through these pathways is regulated remain to be determined.

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.

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.

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from glutamine-contributing SNAREs (Q-SNAREs) embedded in one membrane and an arginine-contributing SNARE (R-SNARE) embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE–binding regulator of SNARE complex formation. Varp colocalizes with and binds to VAMP7, an R-SNARE that is involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7–SNARE motif is trapped between Varp and the VAMP7 longin domain, and hence Varp kinetically inhibits the ability of VAMP7 to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as VAMP7, such as Rab32–GTP.