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1
Haylett, T., and L. Thilo. "Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes." Journal of Cell Biology 103, no. 4 (October 1, 1986): 1249–56. http://dx.doi.org/10.1083/jcb.103.4.1249.
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Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane on self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label in secondary lysosomes increased by first order kinetics (k = [56 min]-1) from less than 0.1% (background level) to a steady-state level of approximately 2.5% of the total label. As analyzed by NaDodSO4 PAGE, labeled molecules of Mr 160-190 kD were depleted and of Mr 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all Mr classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constituents to secondary lysosomes is a limited and selective process, and that only approximately 1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.
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Luzio, J. Paul, Paul R. Pryor, Sally R. Gray, Matthew J. Gratian, Robert C. Piper, and Nicholas A. Bright. "Membrane traffic to and from lysosomes." Biochemical Society Symposia 72 (January 1, 2005): 77–86. http://dx.doi.org/10.1042/bss0720077.
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In the late endocytic pathway, it has been proposed that endocytosed macromolecules are delivered to a proteolytic environment by 'kiss-and-run' events or direct fusion between late endosomes and lysosomes. To test whether the fusion hypothesis accounts for delivery to lysosomes in living cells, we have used confocal microscopy to examine content mixing between lysosomes loaded with rhodamine-dextran and endosomes subsequently loaded with Oregon-Green-dextran. Both kissing and explosive fusion events were recorded. Data from cell-free content-mixing assays have suggested that fusion is initiated by tethering, which leads to formation of a trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein complex and then release of lumenal Ca2+, followed by membrane bilayer fusion. We have shown that the R-SNARE (arginine-containing SNARE) protein VAMP (vesicle-associated membrane protein) 7 is necessary for heterotypic fusion between late endosomes and lysosomes, whereas a different R-SNARE, VAMP 8 is required for homotypic fusion of late endosomes. After fusion of lysosomes with late endosomes, lysosomes are re-formed from the resultant hybrid organelles, a process requiring condensation of content and the removal/recycling of some membrane proteins.
3
Andrews, Norma W. "Lysosomes and the plasma membrane." Journal of Cell Biology 158, no. 3 (July 29, 2002): 389–94. http://dx.doi.org/10.1083/jcb.200205110.
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Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury.
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Green, S. A., K. P. Zimmer, G. Griffiths, and I. Mellman. "Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins." Journal of Cell Biology 105, no. 3 (September 1, 1987): 1227–40. http://dx.doi.org/10.1083/jcb.105.3.1227.
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We have used monospecific antisera to two lysosomal membrane glycoproteins, lgp120 and a similar protein, lgp110, to compare the biosynthesis and intracellular transport of lysosomal membrane components, plasma membrane proteins, and lysosomal enzymes. In J774 cells and NRK cells, newly synthesized lysosomal membrane and plasma membrane proteins (the IgG1/IgG2b Fc receptor or influenza virus hemagglutinin) were transported through the Golgi apparatus (defined by acquisition of resistance to endo-beta-N-acetylglucosaminidase H) with the same kinetics (t1/2 = 11-14 min). In addition, immunoelectron microscopy of normal rat kidney cells showed that lgp120 and vesicular stomatitis virus G-protein were present in the same Golgi cisternae demonstrating that lysosomal and plasma membrane proteins were not sorted either before or during transport through the Golgi apparatus. To define the site at which sorting occurred, we compared the kinetics of transport of lysosomal and plasma membrane proteins and a lysosomal enzyme to their respective destinations. Newly synthesized proteins were detected in dense lysosomes (lgp's and beta-glucuronidase) or on the cell surface (Fc receptor or hemagglutinin) after the same lag period (20-25 min), and accumulated at their final destinations with similar kinetics (t1/2 = 30-45 min), suggesting that these two lgp's are not transported to the plasma membrane before reaching lysosomes. This was further supported by measurements of the transport of membrane-bound endocytic markers from the cell surface to lysosomes, which exhibited additional lag periods of 5-15 min and half-times of 1.5-2 h. The time required for transport of newly synthesized plasma membrane proteins to the cell surface, and for the transport of plasma membrane markers from the cell surface to lysosomes would appear too long to account for the rapid transport of lgp's from the Golgi apparatus to lysosomes. Thus, the observed kinetics suggest that lysosomal membrane proteins are sorted from plasma membrane proteins at a post-Golgi intracellular site, possibly the trans Golgi network, before their delivery to lysosomes.
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Toyomura, Takao, Yoshiko Murata, Akitsugu Yamamoto, Toshihiko Oka, Ge-Hong Sun-Wada, Yoh Wada, and Masamitsu Futai. "From Lysosomes to the Plasma Membrane." Journal of Biological Chemistry 278, no. 24 (April 2, 2003): 22023–30. http://dx.doi.org/10.1074/jbc.m302436200.
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Blott, Emma J., Giovanna Bossi, Richard Clark, Marketa Zvelebil, and Gillian M. Griffiths. "Fas ligand is targeted to secretory lysosomes via a proline-rich domain in its cytoplasmic tail." Journal of Cell Science 114, no. 13 (July 1, 2001): 2405–16. http://dx.doi.org/10.1242/jcs.114.13.2405.
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Fas ligand (FasL) induces apoptosis through its cell surface receptor Fas. T lymphocytes and natural killer cells sort newly synthesised FasL to secretory lysosomes but, in cell types with conventional lysosomes, FasL appears directly on the plasma membrane. Here, we define a proline-rich domain (PRD) in the cytoplasmic tail of FasL that is responsible for sorting FasL to secretory lysosomes. Deletion of this PRD results in cell surface expression of FasL in cells with secretory lysosomes. Positively charged residues flanking the PRD are crucial to the sorting motif and changing the charge of these residues causes mis-sorting to the plasma membrane. In cells with conventional lysosomes, this motif is not recognised and FasL is expressed at the plasma membrane. The FasL PRD is not required for endocytosis in any cell type, as deletion mutants lacking this motif are endocytosed efficiently to the lysosomal compartment. Endogenous FasL cannot internalise extracellular antibody, demonstrating that FasL does not transit the plasma membrane en route to the secretory lysosomes. We propose that an interaction of the PRD of FasL with an SH3-domain-containing protein, enables direct sorting of FasL from the Golgi to secretory lysosomes.
7
Jaiswal, Jyoti K., Norma W. Andrews, and Sanford M. Simon. "Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells." Journal of Cell Biology 159, no. 4 (November 18, 2002): 625–35. http://dx.doi.org/10.1083/jcb.200208154.
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Similar to its role in secretory cells, calcium triggers exocytosis in nonsecretory cells. This calcium-dependent exocytosis is essential for repair of membrane ruptures. Using total internal reflection fluorescence microscopy, we observed that many organelles implicated in this process, including ER, post-Golgi vesicles, late endosomes, early endosomes, and lysosomes, were within 100 nm of the plasma membrane (in the evanescent field). However, an increase in cytosolic calcium led to exocytosis of only the lysosomes. The lysosomes that fused were predominantly predocked at the plasma membrane, indicating that calcium is primarily responsible for fusion and not recruitment of lysosomes to the cell surface.
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WAN, Feng-Yi, Yi-Nan WANG, and Guo-Jiang ZHANG. "The influence of oxidation of membrane thiol groups on lysosomal proton permeability." Biochemical Journal 360, no. 2 (November 26, 2001): 355–62. http://dx.doi.org/10.1042/bj3600355.
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The influence of oxidation of membrane thiol groups on lysosomal proton permeability was studied by measuring lysosomal pH with FITC-conjugated dextran, determining the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Residual membrane thiol groups were measured with 5,5′-dithiobis-(2-nitrobenzoic acid). The lysosomal membrane thiol groups were modified by treatment with diamide and dithiothreitol. SDS/PAGE revealed aggregations of the membrane proteins induced by the treatment of lysosomes with diamide. The cross-linkage of proteins could be abolished by subsequent treatment with dithiothreitol, indicating that the proteins were linked via disulphide bonds. Treating the lysosomes with diamide decreased their membrane thiol groups and caused increases in lysosomal pH, membrane potential and proton leakage, which could be reversed by treatment of the lysosomes with dithiothreitol. This indicates that the lysosomal proton permeability can be increased by oxidation of the membrane thiol groups and restored to the normal level by reduction of the groups. Treatment of the lysosomes with N-ethylmaleimide reduced their membrane thiol groups but did not change the lysosomal pH or their degree of proton leakage. It suggests that protein aggregation may be an important mechanism for the increase in lysosomal proton permeability. The results raise the possibility that the proton permeability of lysosomes in vivo may be affected by the redox states of their membrane thiol groups.
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Rodríguez, Ana, Paul Webster, Javier Ortego, and Norma W. Andrews. "Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells." Journal of Cell Biology 137, no. 1 (April 7, 1997): 93–104. http://dx.doi.org/10.1083/jcb.137.1.93.
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Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca2+, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca2+ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types. Here we demonstrate that elevation in the intracellular free Ca2+ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca2+ ionophore ionomycin or addition of Ca2+containing buffers to streptolysin O–permeabilized cells induced exocytosis of ∼10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca2+ and to be temperature and ATP dependent, similar to Ca2+-regulated secretory mechanisms in specialized cells. These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca2+-regulated exocytosis.
10
Draye, J. P., P. J. Courtoy, J. Quintart, and P. Baudhuin. "A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2109–15. http://dx.doi.org/10.1083/jcb.107.6.2109.
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We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion.
11
Lippincott-Schwartz, J., and D. M. Fambrough. "Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1593–605. http://dx.doi.org/10.1083/jcb.102.5.1593.
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The biochemistry and intracellular transit of an integral membrane glycoprotein of chicken fibroblast lysosomes were studied with monoclonal antibody techniques. The glycoprotein had an apparent molecular weight of 95,000-105,000. Structural analysis involving metabolic labeling with [35S]methionine and cleavage with glycosidases revealed the presence of numerous oligosaccharide chains N-linked to a core polypeptide of apparent molecular weight 48,000. A primary localization of the glycoprotein to lysosomes was demonstrated by the coincidence of antibody binding sites with regions of acridine orange uptake, electron immunocytochemical labeling on the inner surface of lysosome-like vacuolar membranes, and preferential association of the glycoprotein with lysosome-enriched subcellular fractions from Percoll gradients. In addition, small quantities of the glycoprotein were detected on endocytic vesicle and plasma membranes. To study the intracellular pathway of the glycoprotein, we used a monoclonal antibody whose binding to the glycoprotein at the cell surface had no effect on the number or subcellular distribution of antigen molecules. Incubation of chicken fibroblasts with monoclonal antibody at 37 degrees C led to the rapid uptake and subsequent delivery of antibody to lysosomes, where antibody was degraded. This process continued undiminished for many hours on cells continuously exposed to the antibody and was not blocked by the addition of cycloheximide. The rate at which antigen sites were replenished in the plasma membrane of cells prelabeled with antibody (t1/2 = 2 min) was essentially equivalent to the rate of internalization of antibody bound to cell surfaces. These results suggest that there is a continuous and rapid exchange of this glycoprotein between plasma membrane and the membranes of endosomes and/or lysosomes.
12
Oliver, C., R. Dromy, and T. K. Hart. "Density gradient separation of two populations of lysosomes from rat parotid acinar cells." Journal of Histochemistry & Cytochemistry 37, no. 11 (November 1989): 1645–52. http://dx.doi.org/10.1177/37.11.2553801.
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Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.
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Shiki, Yasuko, Yo Ishikawa, Kohji Shirai, Yasushi Saito, and Sho Yoshida. "Effect of Glycyrrhizin on Lysosomes Labilization by Phospholipase A2." American Journal of Chinese Medicine 14, no. 03n04 (January 1986): 131–37. http://dx.doi.org/10.1142/s0192415x86000211.
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The influence of glycyrrhizin on the effect of phospholipase A2 on lysosomes was studied. Treatment of rat liver lysosomes with venom phospholipase A2 caused release of acid phosphatase. This release of acid phosphatase was inhibited by 0.1 mM glycyrrhizin. Glycyrrhizin also inhibited acid phospholiase A2 with pH optimum of 4.5, which is thought to be present in the lysosomal membrane. These results suggests that glycyrrhizin stabilizes lysosomes by inhibiting phospholipase A2 activity in the lysosomal membrane.
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Janvier, Katy, and Juan S. Bonifacino. "Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins." Molecular Biology of the Cell 16, no. 9 (September 2005): 4231–42. http://dx.doi.org/10.1091/mbc.e05-03-0213.
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The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.
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Williams, M. A., and M. Fukuda. "Accumulation of membrane glycoproteins in lysosomes requires a tyrosine residue at a particular position in the cytoplasmic tail." Journal of Cell Biology 111, no. 3 (September 1, 1990): 955–66. http://dx.doi.org/10.1083/jcb.111.3.955.
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Human lysosome membrane glycoprotein h-lamp-1 is a highly N-glycosylated protein found predominantly in lysosomes, with low levels present at the cell surface. The signal required for delivery of h-lamp-1 to lysosomes was investigated by analyzing the intracellular distribution of h-lamp-1 with altered amino acid sequences expressed from mutated cDNA clones. A cytoplasmic tail tyrosine residue found conserved in chicken, rodent, and human deduced amino acid sequences was discovered to be necessary for efficient lysosomal transport of h-lamp-1 in COS-1 cells. In addition, the position of the tyrosine residue relative to the membrane and carboxyl terminus also determined lysosomal expression. Supplanting the wild-type h-lamp-1 cytoplasmic tail onto a cell surface reporter glycoprotein was sufficient to cause redistribution of the chimera to lysosomes. A similar chimeric protein replacing the cytoplasmic tyrosine residue with an alanine was not expressed in lysosomes. Altered proteins that were not transported to lysosomes were found to accumulate at the cell surface, and unlike wild-type lysosomal membrane glycoproteins, were unable to undergo endocytosis. These data indicate that lysosomal membrane glycoproteins are sorted to lysosomes by a cytoplasmic signal containing tyrosine in a specific position, and the sorting signal may be recognized both in the trans-Golgi network and at the cell surface.
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Madden, E. A., and B. Storrie. "Effect of acidotropic amines on the accumulation of newly synthesized membrane and luminal proteins in Chinese-hamster ovary (CHO) cell lysosomes." Biochemical Journal 258, no. 3 (March 15, 1989): 843–51. http://dx.doi.org/10.1042/bj2580843.
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Lysosomes were isolated by sequential gradient centrifugation [Madden, Wirt & Storrie (1987) Arch. Biochem. Biophys. 257, 27-38] from control or acidotropic-amine-treated Chinese-hamster ovary (CHO) cells. By marker-enzyme analysis, the preparation from chloroquine or NH4Cl-treated cells was about 25-fold enriched for lysosomes compared with the postnuclear supernatant and contained little or no marker activities for the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, mitochondria, cytosol and peroxisomes. The yield of amine-treated lysosomes was about 60% relative to the postnuclear supernatant. Electron microscopy and cytochemistry demonstrated that the amine-treated preparation was highly purified. Cytochemical analyses after a short-term pulse of horseradish peroxidase revealed that endosomal contamination of the lysosomal preparation was less than 1%. Lysosomal polypeptides were biosynthetically labelled with [35S]methionine and identified by SDS/polyacrylamide-gel electrophoresis. As expected, the bulk accumulation of luminal proteins into lysosomes was decreased. The bulk accumulation of membrane proteins was increased by acidotropic amine treatment. There were also several qualitative differences in each lysosomal compartment, with new species observed and other species absent. These data suggest that a low pH is not necessary for the normal accumulation of the bulk of membrane proteins in lysosomes and that membrane trafficking from Golgi apparatus to lysosomes occurs at a high rate in acidotropic-amine-treated CHO cells.
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Bagshaw, Richard D., Don J. Mahuran, and John W. Callahan. "Lysosomal Membrane Proteomics and Biogenesis of Lysosomes." Molecular Neurobiology 32, no. 1 (2005): 027–42. http://dx.doi.org/10.1385/mn:32:1:027.
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Deng, Y. P., and B. Storrie. "Animal cell lysosomes rapidly exchange membrane proteins." Proceedings of the National Academy of Sciences 85, no. 11 (June 1, 1988): 3860–64. http://dx.doi.org/10.1073/pnas.85.11.3860.
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Goo, Marisa S., Laura Sancho, Natalia Slepak, Daniela Boassa, Thomas J. Deerinck, Mark H. Ellisman, Brenda L. Bloodgood, and Gentry N. Patrick. "Activity-dependent trafficking of lysosomes in dendrites and dendritic spines." Journal of Cell Biology 216, no. 8 (June 19, 2017): 2499–513. http://dx.doi.org/10.1083/jcb.201704068.
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In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins.
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Jonas, A. J., and H. Jobe. "N-acetyl-d-glucosamine countertransport in lysosomal membrane vesicles." Biochemical Journal 268, no. 1 (May 15, 1990): 41–45. http://dx.doi.org/10.1042/bj2680041.
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Countertransport of GlcNAc was examined in membrane vesicles prepared from rat liver lysosomes which had been lysed by exposure to 5 mM-methionine methyl ester. These vesicles have a random orientation, have intact ATP-dependent acidification and are largely free of soluble hydrolases. Vesicular volume (24.69 +/- 4.51 microliters/mg of protein) was greater than that of lysosomes (3.02 +/- 0.56 microliters/mg of protein), corresponding to a doubling of diameter. Characteristics of GlcNAc transport in vesicles (Km = 1.3 mM) were similar to those observed in intact lysosomes (Km = 4.4 mM). Sulphation or phosphorylation of the substrate resulted in loss of recognition by the carrier. Hydroxyl group orientation at multiple positions did not appear to be critical, whereas orientation of the acetyl group appeared to have a fundamental role in recognition by the carrier. Based on these criteria, phenyl isothiocyanate-GlcNAc (PITC-GlcNAc) was identified as a possible substrate for transport. Under mild conditions, PITC-GlcNAc reversibly inhibited GlcNAc countertransport in lysosomes and vesicles. This and other modified substrates may be of value in identification of the GlcNAc/GalNAc lysosomal transporter. Lysosomal membrane vesicle preparation is a technique that should be useful for the study of other lysosomal transport systems.
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Harter, C., and I. Mellman. "Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane." Journal of Cell Biology 117, no. 2 (April 15, 1992): 311–25. http://dx.doi.org/10.1083/jcb.117.2.311.
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We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail.
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Iveson, G. P., S. J. Bird, and J. B. Lloyd. "Passive diffusion of non-electrolytes across the lysosome membrane." Biochemical Journal 261, no. 2 (July 15, 1989): 451–56. http://dx.doi.org/10.1042/bj2610451.
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An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo.
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Mathews, P. M., J. B. Martinie, and D. M. Fambrough. "The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes." Journal of Cell Biology 118, no. 5 (September 1, 1992): 1027–40. http://dx.doi.org/10.1083/jcb.118.5.1027.
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A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.
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Luzio, J. Paul, Michael D. J. Parkinson, Sally R. Gray, and Nicholas A. Bright. "The delivery of endocytosed cargo to lysosomes." Biochemical Society Transactions 37, no. 5 (September 21, 2009): 1019–21. http://dx.doi.org/10.1042/bst0371019.
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In mammalian cells, endocytosed cargo that is internalized through clathrin-coated pits/vesicles passes through early endosomes and then to late endosomes, before delivery to lysosomes for degradation by proteases. Late endosomes are MVBs (multivesicular bodies) with ubiquitinated membrane proteins destined for lysosomal degradation being sorted into their luminal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery. Cargo is delivered from late endosomes to lysosomes by kissing and direct fusion. These processes have been studied in live cell experiments and a cell-free system. Late endosome–lysosome fusion is preceded by tethering that probably requires mammalian orthologues of the yeast HOPS (homotypic fusion and vacuole protein sorting) complex. Heterotypic late endosome–lysosome membrane fusion is mediated by a trans-SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) complex comprising Syntaxin7, Vti1b, Syntaxin8 and VAMP7 (vesicle-associated membrane protein 7). This differs from the trans-SNARE complex required for homotypic late endosome fusion in which VAMP8 replaces VAMP7. VAMP7 is also required for lysosome fusion with the plasma membrane and its retrieval from the plasma membrane to lysosomes is mediated by its folded N-terminal longin domain. Co-ordinated interaction of the ESCRT, HOPS and SNARE complexes is required for cargo delivery to lysosomes.
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Traub, L. M., S. I. Bannykh, J. E. Rodel, M. Aridor, W. E. Balch, and S. Kornfeld. "AP-2-containing clathrin coats assemble on mature lysosomes." Journal of Cell Biology 135, no. 6 (December 15, 1996): 1801–14. http://dx.doi.org/10.1083/jcb.135.6.1801.
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Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.
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Rupar, C. Anthony, Susan Albo, and Jeffery D. Whitehall. "Rat liver lysosome membranes are enriched in α-tocopherol." Biochemistry and Cell Biology 70, no. 6 (June 1, 1992): 486–88. http://dx.doi.org/10.1139/o92-075.
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The subcellular distribution of α-tocopherol has been studied in rat liver. Lysosomal membranes were found to be considerably enriched in α-tocopherol with 6300 pmol/mg membrane protein, whereas mitochondrial membranes and microsomes contained 530 and 200 pmol/mg membrane protein, respectively. The 37-fold higher specific content of α-tocopherol in lysosomal membranes relative to homogenate indicates that lysosomes could be a target of cellular pathology in vitamin E deficiency states.Key words: α-tocopherol, lysosomes, mitochondria, membrane.
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Ono, Koh, Sung O. Kim, and Jiahuai Han. "Susceptibility of Lysosomes to Rupture Is a Determinant for Plasma Membrane Disruption in Tumor Necrosis Factor Alpha-Induced Cell Death." Molecular and Cellular Biology 23, no. 2 (January 15, 2003): 665–76. http://dx.doi.org/10.1128/mcb.23.2.665-676.2003.
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ABSTRACT Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-α)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-α-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-α in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.
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Luzio, J. P., B. M. Mullock, P. R. Pryor, M. R. Lindsay, D. E. James, and R. C. Piper. "Relationship between endosomes and lysosomes." Biochemical Society Transactions 29, no. 4 (August 1, 2001): 476–80. http://dx.doi.org/10.1042/bst0290476.
Full textAbstract:
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 reformed. 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. Reformation 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.
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Elstak, Edo D., Maaike Neeft, Nadine T. Nehme, Jarno Voortman, Marc Cheung, Monireh Goodarzifard, Hans C. Gerritsen, et al. "The munc13-4–rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane." Blood 118, no. 6 (August 11, 2011): 1570–78. http://dx.doi.org/10.1182/blood-2011-02-339523.
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Abstract Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4–rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4–rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.
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Mundy, Dorothy I., Wei Ping Li, Katherine Luby-Phelps, and Richard G. W. Anderson. "Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content." Molecular Biology of the Cell 23, no. 5 (March 2012): 864–80. http://dx.doi.org/10.1091/mbc.e11-07-0598.
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Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking.
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Trivedi, Purvi C., Jordan J. Bartlett, and Thomas Pulinilkunnil. "Lysosomal Biology and Function: Modern View of Cellular Debris Bin." Cells 9, no. 5 (May 4, 2020): 1131. http://dx.doi.org/10.3390/cells9051131.
Full textAbstract:
Lysosomes are the main proteolytic compartments of mammalian cells comprising of a battery of hydrolases. Lysosomes dispose and recycle extracellular or intracellular macromolecules by fusing with endosomes or autophagosomes through specific waste clearance processes such as chaperone-mediated autophagy or microautophagy. The proteolytic end product is transported out of lysosomes via transporters or vesicular membrane trafficking. Recent studies have demonstrated lysosomes as a signaling node which sense, adapt and respond to changes in substrate metabolism to maintain cellular function. Lysosomal dysfunction not only influence pathways mediating membrane trafficking that culminate in the lysosome but also govern metabolic and signaling processes regulating protein sorting and targeting. In this review, we describe the current knowledge of lysosome in influencing sorting and nutrient signaling. We further present a mechanistic overview of intra-lysosomal processes, along with extra-lysosomal processes, governing lysosomal fusion and fission, exocytosis, positioning and membrane contact site formation. This review compiles existing knowledge in the field of lysosomal biology by describing various lysosomal events necessary to maintain cellular homeostasis facilitating development of therapies maintaining lysosomal function.
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Van Dyke, R. W. "Acidification of rat liver lysosomes: quantitation and comparison with endosomes." American Journal of Physiology-Cell Physiology 265, no. 4 (October 1, 1993): C901—C917. http://dx.doi.org/10.1152/ajpcell.1993.265.4.c901.
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Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.
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Rodríguez, A., E. Samoff, M. G. Rioult, A. Chung, and N. W. Andrews. "Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport." Journal of Cell Biology 134, no. 2 (July 15, 1996): 349–62. http://dx.doi.org/10.1083/jcb.134.2.349.
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Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the parasitophorous vacuole. Here we directly demonstrate directional migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L6E9 myoblasts exposed to T. cruzi trypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periphery, in the direction of the parasite attachment site, but only when their original position was less than 11-12 microns from the invasion site. Lysosomes more distant from the invasion area exhibited only the short multi-directional saltatory movements previously described for lysosomes, regardless of their proximity to the cell margins. Specific depletion of peripheral lysosomes was obtained by microinjection of NRK cells with antibodies against the cytoplasmic domain of lgp 120, a treatment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and L6E9 cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent redistribution of lysosomes to the host cell periphery, and reduced trypomastigote entry. Our results therefore demonstrate that during T. cruzi invasion of host cells lysosomes are mobilized from the immediately surrounding area, and that availability of lysosomes at the cell periphery and microtubule/kinesin-mediated transport are requirements for parasite entry.
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Nicot, Anne-Sophie, Hanna Fares, Bernard Payrastre, Andrew D. Chisholm, Michel Labouesse, and Jocelyn Laporte. "The Phosphoinositide Kinase PIKfyve/Fab1p Regulates Terminal Lysosome Maturation in Caenorhabditis elegans." Molecular Biology of the Cell 17, no. 7 (July 2006): 3062–74. http://dx.doi.org/10.1091/mbc.e05-12-1120.
Full textAbstract:
Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7–positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.
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Jadot, M., S. Wattiaux-De Coninck, and R. Wattiaux. "The permeability of lysosomes to sugars. Effect of diethylstilbestrol on the osmotic activation of lysosomes induced by glucose." Biochemical Journal 262, no. 3 (September 15, 1989): 981–84. http://dx.doi.org/10.1042/bj2620981.
Full textAbstract:
We have investigated the effect on the osmotic activation of rat liver lysosomes, by glucose penetration, of different substances known to inhibit the glucose transport through the plasma membrane. Diethylstilbestrol is the most efficient, particularly when purified lysosomes are used. It has no effect on osmotic activation induced by hypo-osmotic sucrose or by iso-osmotic KCl. It is proposed that diethylstilbestrol reacts with specific sites involved in the glucose translocation through the lysosomal membrane. These sites could not be identified by binding experiments, presumably owing to the considerable unspecific binding of the compound to the membrane.
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Luzio, J. Paul, Viviane Poupon, Margaret R. Lindsay, Barbara M. Mullock, Robert C. Piper, and Paul R. Pryor. "Membrane dynamics and the biogenesis of lysosomes (Review)." Molecular Membrane Biology 20, no. 2 (January 2003): 141–54. http://dx.doi.org/10.1080/0968768031000089546.
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Sandoval, Ignacio V., and Oddmund Bakke. "Targeting of membrane proteins to endosomes and lysosomes." Trends in Cell Biology 4, no. 8 (August 1994): 292–97. http://dx.doi.org/10.1016/0962-8924(94)90220-8.
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Harding, C. V., and H. J. Geuze. "Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment." Journal of Immunology 151, no. 8 (October 15, 1993): 3988–98. http://dx.doi.org/10.4049/jimmunol.151.8.3988.
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Abstract Exogenous protein Ag are processed within endocytic compartments to produce peptides that bind to class II MHC (MHC-II) molecules for presentation to T cells. We have now identified a subcellular compartment in which immunogenic peptides bind to MHC-II as a subset of high density lysosomes. Immunoelectron microscopy of whole cells and dense Percoll gradient subcellular fractions showed early tubulovesicular lysosomes with high levels of MHC-II. Typical mature lysosomes contained less MHC-II. Pulse-chase biosynthetic labeling of macrophages followed by immunoprecipitation of MHC-II from dense lysosomal fractions showed that MHC-II molecules targeted efficiently to lysosomes after biosynthesis. Moreover, lysosomal MHC-II molecules were rapidly loaded with immunogenic peptide (as detected by T cells) soon after exposure of macrophages to Ag and before similar expression of peptide-MHC-II complexes on the plasma membrane; this loading was blocked at 18 degrees C. We propose that nascent MHC-II molecules target to early tubulovesicular lysosomes and bind immunogenic peptides therein; the resulting peptide-MHC-II complexes are then transported to the plasma membrane.
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Dolat, Lee, and Elias T. Spiliotis. "Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion." Journal of Cell Biology 214, no. 5 (August 22, 2016): 517–27. http://dx.doi.org/10.1083/jcb.201603030.
Full textAbstract:
Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate–dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes.
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Moseley, R. H., and R. W. Van Dyke. "Organic cation transport by rat liver lysosomes." American Journal of Physiology-Gastrointestinal and Liver Physiology 268, no. 3 (March 1, 1995): G480—G486. http://dx.doi.org/10.1152/ajpgi.1995.268.3.g480.
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Hepatic organic cation transport has been characterized in rat liver plasma membrane vesicles, using the quaternary amine tetraethylammonium (TEA) as a model substrate. Sinusoidal TEA uptake is stimulated by an inside-negative membrane potential; TEA transport across the canalicular membrane is mediated by electroneutral organic cation-H+ exchange. Substrates for these transport processes include procainamide ethobromide (PAEB) and vecuronium, cationic drugs that undergo biliary excretion. Given the apparent absence of sinusoidal transport mechanisms able to generate high hepatocyte-to-blood organic cation concentration ratios, intracellular transport of organic cations may involve sequestration and concentration within acidified organelles. Therefore, the characteristics of TEA uptake were examined in isolated rat liver lysosomes that are acidified by a well-described H(+)-adenosinetriphosphatase (ATPase). Lysosomal uptake of [14C]TEA was a time- and ATP-dependent process, reaching steady state after 30-60 min. Steady-state [14C]TEA uptake was significantly reduced by omission of ATP and by addition of monensin, conditions that alter lysosomal pH and membrane potential gradients, and by the H(+)-ATPase inhibitors, N-ethylmaleimide and bafilomycin A. ATP-dependent lysosomal [14C]TEA uptake was significantly inhibited by PAEB, vecuronium, and other organic cationic substrates of canalicular TEA/H+ exchange. These findings demonstrate that rat liver lysosomes sequester certain organic cationic drugs, most likely via organic cation/H+ exchange driven by H(+)-ATPase. Canalicular organic cation/H+ exchange may reflect, in part, the exocytic insertion of this transporter from an intracellular compartment to this membrane domain.
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Boonen, Marielle, Isabelle Hamer, Muriel Boussac, Anne-Françoise Delsaute, Bruno Flamion, Jérôme Garin, and Michel Jadot. "Intracellular localization of p40, a protein identified in a preparation of lysosomal membranes." Biochemical Journal 395, no. 1 (March 15, 2006): 39–47. http://dx.doi.org/10.1042/bj20051647.
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Unlike lysosomal soluble proteins, few lysosomal membrane proteins have been identified. Rat liver lysosomes were purified by centrifugation on a Nycodenz density gradient. The most hydrophobic proteins were extracted from the lysosome membrane preparation and were identified by MS. We focused our attention on a protein of approx. 40 kDa, p40, which contains seven to ten putative transmembrane domains and four lysosomal consensus sorting motifs in its sequence. Knowing that preparations of lysosomes obtained by centrifugation always contain contaminant membranes, we combined biochemical and morphological methods to analyse the subcellular localization of p40. The results of subcellular fractionation of mouse liver homogenates validate the lysosomal residence of p40. In particular, a density shift of lysosomes induced by Triton WR-1339 similarly affected the distributions of p40 and β-galactosidase, a lysosomal marker protein. We confirmed by fluorescence microscopy on eukaryotic cells transfected with p40 or p40–GFP (green fluorescent protein) constructs that p40 is localized in lysosomes. A first molecular characterization of p40 in transfected Cos-7 cells revealed that it is an unglycosylated protein tightly associated with membranes. Taken together, our results strongly support the hypothesis that p40 is an authentic lysosomal membrane protein.
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Rao, Swathi K., Chau Huynh, Veronique Proux-Gillardeaux, Thierry Galli, and Norma W. Andrews. "Identification of SNAREs Involved in Synaptotagmin VII-regulated Lysosomal Exocytosis." Journal of Biological Chemistry 279, no. 19 (March 1, 2004): 20471–79. http://dx.doi.org/10.1074/jbc.m400798200.
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Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C2A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.
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Bonifacino, J. S., L. Yuan, and I. V. Sandoval. "Internalization and recycling to serotonin-containing granules of the 80K integral membrane protein exposed on the surface of secreting rat basophilic leukaemia cells." Journal of Cell Science 92, no. 4 (April 1, 1989): 701–12. http://dx.doi.org/10.1242/jcs.92.4.701.
Full textAbstract:
The 80K (80 × 10(3) Mr) integral membrane protein, first described in the secretory granules of rat basophilic leukaemia (RBL) cells, is also localized to lysosomes in these cells. The protein displays the same distribution in natural killer lymphocytes (RNK-7), wherein it codistributes with cytolysin in secretory granules. In contrast, the protein is absent from the endocrine and exocrine secretory granules of rat pancreatic acinar and pituitary cells, respectively, where it is confined to lysosomes. The protein colocalizes with lysosomal integral membrane proteins in all the cells studied, indicating that is largely restricted to secretory granules with lysosomal properties (LSG) and lysosomes. The protein expressed on the surface of secreting RBL cells is internalized by endocytosis via coated pits, and found in coated vesicles, endosomes, multivesicular bodies and Golgi system, before being recycled to LSG and partly delivered to lysosomes. The recycled protein is re-expressed on the surface of cells stimulated to secrete a second time.
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Bonet-Ponce, Luis, Alexandra Beilina, Chad D. Williamson, Eric Lindberg, Jillian H. Kluss, Sara Saez-Atienzar, Natalie Landeck, et al. "LRRK2 mediates tubulation and vesicle sorting from lysosomes." Science Advances 6, no. 46 (November 2020): eabb2454. http://dx.doi.org/10.1126/sciadv.abb2454.
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Genetic variation around the LRRK2 gene affects risk of both familial and sporadic Parkinson’s disease (PD). However, the biological functions of LRRK2 remain incompletely understood. Here, we report that LRRK2 is recruited to lysosomes after exposure of cells to the lysosome membrane–rupturing agent LLOME. Using an unbiased proteomic screen, we identified the motor adaptor protein JIP4 as an LRRK2 partner at the lysosomal membrane. LRRK2 can recruit JIP4 to lysosomes in a kinase-dependent manner via the phosphorylation of RAB35 and RAB10. Using super-resolution live-cell imaging microscopy and FIB-SEM, we demonstrate that JIP4 promotes the formation of LAMP1-negative tubules that release membranous content from lysosomes. Thus, we describe a new process orchestrated by LRRK2, which we name LYTL (LYsosomal Tubulation/sorting driven by LRRK2), by which lysosomal tubulation is used to release vesicles from lysosomes. Given the central role of the lysosome in PD, LYTL is likely to be disease relevant.
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Flannery, Andrew R., Cecilia Czibener, and Norma W. Andrews. "Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes." Journal of Cell Biology 191, no. 3 (November 1, 2010): 599–613. http://dx.doi.org/10.1083/jcb.201003021.
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Syt VII is a Ca2+ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca2+-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes.
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Advani, Raj J., Bin Yang, Rytis Prekeris, Kelly C. Lee, Judith Klumperman, and Richard H. Scheller. "Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes." Journal of Cell Biology 146, no. 4 (August 23, 1999): 765–76. http://dx.doi.org/10.1083/jcb.146.4.765.
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A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome.
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Stahl-Meyer, Jonathan, Lya Katrine Kauffeldt Holland, Bin Liu, Kenji Maeda, and Marja Jäättelä. "Lysosomal Changes in Mitosis." Cells 11, no. 5 (March 3, 2022): 875. http://dx.doi.org/10.3390/cells11050875.
Full textAbstract:
The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.
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Encarnação, Marisa, Lília Espada, Cristina Escrevente, Denisa Mateus, José Ramalho, Xavier Michelet, Inês Santarino, et al. "A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair." Journal of Cell Biology 213, no. 6 (June 20, 2016): 631–40. http://dx.doi.org/10.1083/jcb.201511093.
Full textAbstract:
Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis.
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Nabi, I. R., A. Le Bivic, D. Fambrough, and E. Rodriguez-Boulan. "An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes." Journal of Cell Biology 115, no. 6 (December 15, 1991): 1573–84. http://dx.doi.org/10.1083/jcb.115.6.1573.
Full textAbstract:
Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.
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Raposo, Graça, Marie-Neige Cordonnier, Danièle Tenza, Bernadette Menichi, Antoine Dürrbach, Daniel Louvard, and Evelyne Coudrier. "Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells." Molecular Biology of the Cell 10, no. 5 (May 1999): 1477–94. http://dx.doi.org/10.1091/mbc.10.5.1477.
Full textAbstract:
Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.