ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate

Membrane vesicles were isolated from purified liver lysosomes of rats treated with Triton WR-1339. In order to preserve ATP-dependent acidification activity, proteolysis of membranes was minimized by adding protease inhibitors and by centrifuging to form dilute bands of vesicles rather than highly concentrated pellets. The membrane vesicle fraction represented about 20% of the total lysosomal protein, 80% of the ATPase activity, and 3% of the solute proteins as marked by N-acetylglucosaminidase. About one-half of the membranes were oriented right side out. The space unavailable to [14C]sucrose corresponded to 3 microliters/mg of membrane protein which indicates that the membranes form vesicles about one-tenth the size of lysosomes. Uptake of either [14C]methylamine or [14C]chloroquine by lysosomal membrane vesicles was ATP-dependent, indicating acidification of the intravesicle space. The acidification activity was inhibited when either 1.5 microM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, 100 microM dicyclohexylcarbodiimide, or millimolar concentrations of such permeant weak bases as ammonium sulfate and dansyl cadaverine were added. Acidification of lysosomal vesicles by ATP occurred electroneutrally. This acidification activity was not dependent on added salts but was inhibited by the anion transport inhibitors pyridoxal phosphate and diisothiocyanostilbene disulfonic acid, thus suggesting co-transport of protons and anions. Results which indicate that phosphate is the transported anion included (a) ATP-dependent uptake of [32P]phosphate by lysosomal membrane vesicles and (b) stimulation of ATP-dependent acidification of these vesicles by added phosphate. These observations provide further evidence that maintenance of the acid intralysosomal pH necessary for activation of lysosomal hydrolases is due to an ATP-driven proton pump located in the lysosomal membrane.     

Enhancement of lysosomal proton permeability induced by photooxidation of membrane thiol groups

Effects of photooxidation of membrane thiol groups on lysosomal proton permeability were studied by measuring intralysosomal pH with fluorescein isothiocyanate-dextran and monitoring proton leakage with p-nitrophenol. Methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linking of the membrane proteins, which was established by the measurement of residual membrane thiol groups with 5,5'-dithio-bis(2-nitrobenzoic acid) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The cross-linking of proteins could be abolished by subsequent treatment of the photodamaged lysosomes with dithiothreitol, indicating that the proteins were linked via disulfide bonds. In addition, the photodamage of lysosomes raised the intralysosomal pH and caused leakage of the lysosomal protons, which could also be reversed by subsequent dithiothreitol treatment. This indicates that lysosomal proton permeability can be increased by photooxidation of the membrane thiol groups and recovered to the normal level by reduction of the groups.     

The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells.

Following entry into non-phagocytic HeLa cells, the facultative pathogen Salmonella typhimurium survives and replicates within a membrane-bound vacuole. Preceding the initiation of intracellular replication there is a lag phase, during which the bacteria modulate their environment. This phase is characterized by the rapid recycling of early endosomal proteins present on the nascent vacuole followed by the acquisition of a subset of lysosomal proteins. To gain a better understanding of the mechanism of intracellular survival, we have followed the biogenesis of the S. typhimurium-containing vacuole (SCV) in HeLa cells expressing different mutant forms of the small GTPase rab7. We demonstrate that the SCV recruits pre-existing lysosomal glycoproteins (Lgps) in a rab7-dependent manner, without directly interacting with lysosomes. We also show the transient accumulation, in the vicinity of the SCV, of novel rab7- and Lgp-containing vesicles containing very low amounts of cathepsin D. The size of these vesicles is dependent on rab7 activity, suggesting a role for rab7 in their homotypic fusion. Taken together, these results indicate that rab7 regulates SCV biogenesis during the phase characterized by the rapid acquisition of lysosomal proteins. We propose that SCV maturation involves its interaction with rab7/Lgp-containing vesicles which are possible intermediate cargo components of the late endocytic pathway.     

Effect of ionophores on intralysosomal pH.

1. The effect of ionophores on the intralysosomal pH (as estimated from the distribution of a weak acid or base), on the distribution of 42K+ across the lysosomal membrane, and on the intralysosomal degradation of 125I-labelled bovine serum albumin has been studied. 2. Nigericin and X537A equilibrate both 42K+ and H+ across the lysosomal membrane. Gramicidin equilibrates H+ across the lysosomal membrane, this equilibration being more effective in a NaCl than in a KCl medium. Thus all three ionophores exhibit the same ion specificity as in other membranes. 3. The effect of the exchange-diffusion ionophores cannot be imitated by the combination of valinomycin with an uncoupler. Valinomycin by itself also has no effect. 4. X537A and nigericin inhibit the intralysosomal degradation of 125I-labelled albumin only when potassium is present. In a sucrose-containing medium no effect is found. Similar results were obtained with gramicidin. 5. These data suggest that the lysosomal membrane is impermeable to monovalent cations at 25 or 37 degrees C, and that the transport of protons is organised in such a way that electroneutrality is maintained.     

Membrane lipids and proteins as modulators of urothelial endocytic vesicles pathways

The increased studies on urinary bladder umbrella cells as an important factor for maintaining the permeability barrier have suggested new pathways for the discoidal/fusiform endocytic vesicles which is one of the main features of the umbrella cells. The biological role of these vesicles was defined, for many years, as a membrane reservoir for the umbrella cell apical plasma membrane which are subject to an increased tension during the filling phase of the micturition cycle and, therefore, the vesicles are fused with the apical membrane. Upon voiding, the added membrane is reinserted via a non-clathrin or caveolin-dependant endocytosis thereby restoring the vesicle cytoplasmic pool. However, in the last decade, new evidence appeared indicating alternative pathways of the endocytic vesicles different than the cycling process of exocytosis/endocytosis. The purpose of this review is to analyze the molecular modulators, such as membrane lipids and proteins, in the permeability of endocytic vesicles, the sorting of endocytosed material to lysosomal degradation pathway and recycling of both membrane and fluid phases.     

Mechanism of cholesterol transfer from the Niemann-Pick type C2 protein to model membranes supports a role in lysosomal cholesterol transport

Cells acquire cholesterol either by de novo synthesis in the endoplasmic reticulum or by internalization of cholesterol-containing lipoproteins, particularly low density lipoprotein (LDL), via receptor-mediated endocytosis. The inherited disorder Niemann-Pick type C (NPC), in which abnormal LDL-cholesterol trafficking from the endo/lysosomal compartment leads to substantial cholesterol and glycolipid accumulation in lysosomes, is caused by defects in either of two genes that encode for proteins designated as NPC1 and NPC2. NPC2 is a small intralysosomal protein that has been characterized biochemically as a cholesterol binding protein. We determined the rate and mechanism by which NPC2 delivers cholesterol to model phospholipid membranes. A fluorescence dequenching assay was used to monitor the kinetics of cholesterol transfer from the protein to membranes. The endogenous tryptophan fluorescence of the NPC2 was quenched upon binding of cholesterol, and the subsequent addition of acceptor vesicles resulted in dequenching of the tryptophan signal, enabling the monitoring of cholesterol transfer to membranes. The rates of cholesterol transfer were evaluated as a function of acceptor vesicle concentration, acceptor vesicle phospholipid headgroup composition, and aqueous phase properties. The results suggest that NPC2 rapidly transports cholesterol to phospholipid vesicles via a collisional mechanism which involves a direct interaction with the acceptor membrane. Transfer of cholesterol to membranes is faster in an acidic environment and is greatly enhanced by the presence of the unique lysosomal/late endosomal phospholipid lyso-bisphosphatidic acid (LBPA) (also known as bismonoacylglycerol phosphate). Finally, we found that the rate of transfer of cholesterol from vesicles to NPC2 was dramatically increased by the presence of lyso-bisphosphatidic acid in the donor vesicles. These results support a role for the NPC2 protein in the egress of LDL derived cholesterol out of the endosomal/lysosomal compartment.     

Multiple pathways of exocytosis, endocytosis, and membrane recycling: validation of a Golgi route

A number of pathways for intracellular membrane traffic have been detected in various cell types. The major established routes are: 1) the lysosomal pathway, which is the major route utilized in phagocytic and cultured cells; 2) the transcellular route, which represents the major type of traffic in nonfenestrated, capillary endothelial cells and which also appears to be the preferred route for the transport of immunoglobulins (intact) across cells; 3) the exocytosis pathway, utilized in secretory cells for discharge of secretory products, and which is also believed to be used for delivery of intrinsic membrane glycoproteins; 4) the plasmalemma to Golgi route, also highly developed in secretory cells, which is believed to be utilized for the recycling of secretory granule membranes; and 5) the biosynthetic pathways for transport of secretory products, lysosomal enzymes, and membrane proteins from the endoplasmic reticulum to the Golgi complex and for transport of lysosomal enzymes from the Golgi complex to lysosomes. It has become clear that cells repeatedly reutilize or recycle the membranes used in these various transport operations. Clathrin-coated vesicles have been found to be involved in transport along all these routes, which suggests that there are multiple populations of coated vesicles with different transport functions in every cell. It has become clear that the Golgi complex is the site where the membrane and product traffic converges and is sorted and directed to its correct destinations. The validation of a transport route from the cell surface to the Golgi complex raises the possibility that bound ligands and membrane constituents could be modified or repaired in transit during recycling through the Golgi complex, which is a biosynthetic compartment.     

Saposin D solubilizes anionic phospholipid-containing membranes.

Saposin (Sap) D is a late endosomal/lysosomal small protein, generated together with three other similar proteins, Sap A, B, and C, from the common precursor, prosaposin. Although the functions of saposins such as Sap B and C are well known (Sap B promotes the hydrolysis of sulfatides and Sap C that of glucosylceramide), neither the physiological function nor the mechanism of action of Sap D are yet fully understood. We previously found that a dramatic increase of Sap D superficial hydrophobicity, occurring at the low pH values characteristic of the late endosomal/lysosomal environment, triggers the interaction of the saposin with anionic phospholipid-containing vesicles. We have presently found that, upon lipid binding, Sap D solubilizes the membranes, as shown by the clearance of the vesicles turbidity. The results of gel filtration, density gradient centrifugation, and negative staining electron microscopy demonstrate that this effect is due to the transformation of large vesicles to smaller particles. The solubilizing effect of Sap D is highly dependent on pH, the lipid/saposin ratio, and the presence of anionic phospholipids; small variations in each of these conditions markedly influences the activity of Sap D. The present study documents the interaction of Sap D with membranes as a complex process. Anionic phospholipids attract Sap D from the medium; when the concentration of the saposin on the lipid surface reaches a critical value, the membrane breaks down into recombinant small particles enriched in anionic phospholipids. Our results suggest that the role played by Sap D is more general than promoting sphingolipid degradation, e.g. the saposin might also be a key mediator of the solubilization of intralysosomal/late endosomal anionic phospholipid-containing membranes.     

Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Autophagy, heterophagy, microautophagy and crinophagy as the means for intracellular degradation

It is generally accepted that the lysosomal compartment plays an important role in the degradation of cellular components. In this communication we discuss various experimental models which have been used to study mechanisms of intralysosomal degradation and also discuss the evidence obtained in support of the following proposals: 1. The autophagosomes can be isolated into high purity and are the subcellular locus of induced protein degradation. 2. Different membrane components such as proteins and lipids are degraded at different rates inside the lysosomes. Intralysosomal hydrolysis is not the rate limiting step in degradation. 3. Lysosomes take up soluble material in vitro by invagination and pinching off of their membranes (microautophagy). 4. Secretory vesicles can degrade their secretory contents by fusing with the lysosomes.     

pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [14C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H+ greater than Cs+ greater than Rb+ greater than K greater than Na+ greater than Li+ greater than Mg2+, Ca2+. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondiffusible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Microautophagy in mammalian cells: Revisiting a 40-year-old conundrum

The term microautophagy was first used in 1966 by de Duve and Wattiaux1 and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen (“multivesicular lysosomes”). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.     

Microautophagy in mammalian cells: revisiting a 40-year-old conundrum

The term microautophagy was first used in 1966 by de Duve and Wattiaux and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen ("multivesicular lysosomes"). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.     

Immunological discrimination of intralysosomal, cytosolic, and two membrane sialidases present in rat tissues

Cytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma membrane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.     

Exosome Secretion: The Art of Reutilizing Nonrecycled Proteins?

Multivesicular bodies contain membrane vesicles which either undergo lysosomal digestion or are released in the extracellular environment as exosomes. Evidence is accumulating that supports a physiological role for exosomes in, for example, antigen presentation or removal of transferrin receptor during reticulocyte development. Here, inspired by observations on exosomal release from reticulocytes, we discuss the potential involvement of the so-called ESCRT mechanism in the entrapment of both lysosomal and exosomal cargo within the intralumenal vesicles of multivesicular bodies. We propose that this mechanism operates at different sites in the endocytic itinerary in different cells, thereby providing a tool for directional sorting. We also explore the possibility that the efficiency of sorting of molecules into exosomes increases when the recycling kinetics of molecules decreases, exosomal sorting being favored by intermolecular interactions occurring within lipid domains, or with protein webs, that slow lateral mobility. These considerations are mirrored in the context of current knowledge on the mechanism of protein sorting for degradation in lysosomes, and the hijacking of such mechanisms by some retroviruses for particle budding.     

Synaptophysin and synapsin I as tools for the study of the exo-endocytotic cycle

Synaptophysin, an integral protein of the synaptic vesicle membrane, and synapsin I, a phosphoprotein associated with the cytoplasmic side of synaptic vesicles, represent useful markers that allow to follow the movements of the vesicle membrane during recycling. The use of antibodies against these proteins to label nerve terminals during experimental treatments which stimulate secretion has provided evidence that during the exo-endocytotic cycle synaptic vesicles transiently fuse with the axolemma, from which they are specifically recovered. When recycling is blocked, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and to diffusion of the vesicle components in the plane of the membrane.     

Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles.

In several cell types, specific membrane proteins are retained intracellularly and rapidly redistributed to the surface in response to stimulation. In fat and muscle, the GLUT4 glucose transporter is dynamically retained because it is rapidly internalized and slowly recycled to the plasma membrane. Insulin increases the recycling of GLUT4, resulting in a net translocation to the surface. We have shown that fibroblasts also have an insulin-regulated recycling mechanism. Here we show that GLUT4 is retained within the transferrin receptor-containing general endosomal recycling compartment in Chinese hamster ovary (CHO) cells rather than being segregated to a specialized, GLUT4-recycling compartment. With the use of total internal reflection microscopy, we demonstrate that the TR and GLUT4 are transported from the pericentriolar recycling compartment in separate vesicles. These data provide the first functional evidence for the formation of distinct classes of vesicles from the recycling compartment. We propose that GLUT4 is dynamically retained within the endosomal recycling compartment in CHO cells because it is concentrated in vesicles that form more slowly than those that transport TR. In 3T3-L1 adipocytes, cells that naturally express GLUT4, we find that GLUT4 is partially segregated to a separate compartment that is inaccessible to the TR. We present a model for the formation of this specialized compartment in fat cells, based on the general mechanism described in CHO cells, which may explain the increased retention of GLUT4 and its insulin-induced translocation in fat cells.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

Dynamics of the apical plasma membrane recycling system during cell division

The members of the family of Rab11 small GTPases are critical regulators of the plasma membrane vesicle recycling system. While previous studies have determined that the Golgi apparatus disperses during mitosis and reorganizes after cytokinesis, the fate of the recycling system during the cell cycle is more obscure. We have now studied in MDCK cells the fate during mitosis of an apical recycling system cargo, the polymeric IgA receptor (pIgAR), and regulators of the recycling system, Rab11a and its interacting proteins myosin Vb, Rab11-FIP1, Rab11-FIP2 and pp75/Rip11. Rab11a, pIgAR and myosin Vb containing vesicles dispersed into diffuse puncta in the cytosol during prophase and then became clustered near the spindle poles after metaphase, increasing in intensity throughout telophase. A similar pattern was observed for Rab11-FIP1 and Rab11-FIP2. However, Rab11-FIP1 lost colocalization with other recycling system markers during late prophase, relocating to the pericentriolar material. During telophase, Rab11-FIP1 returned to recycling system vesicles. Western blot analysis indicated that both Rab11a and pIgAR remained associated with membrane vesicles throughout the cell cycle. This behavior of the Rab11a-containing apical recycling endosome system during division was distinct from that of the Golgi apparatus. These results indicate that critical components of the apical recycling system remain associated on vesicles throughout the cell cycle and may provide a means for rapid re-establishment of plasma membrane components after mitosis.