The study of endocytosis has traditionally focused on the contents of endocytic vacuoles, i.e., extracellular fluid, dissolved solutes, and macromolecules or particles which specifically or nonspecifically bind to the plasma membrane (PM). Substances that are endocytosed include important nutrients, toxins, effector molecules (growth factors, hormones, antibodies), enzymes, and pathogens. This article centers on the properties and dynamics of the endocytic vacuole membrane. In many instances we will stress observations on cultured mouse macrophages with which we are most familier. We will emphasize four points: (a) Movement of vesicles is rapid such that endocytosed membrane and contents move from one cellular compartment to another in seconds to minutes. (b) Vesicular movement requires the interiorization and flow of large amounts of PM) (c) In many instances, internalized PM must recycle or return intact to the cell surface. (d) During recycling, contents and membrane components can be sorted from one another; e.g., endocytosed contents can accumulate within the cell while the container (membrane) can move into and out of the cell after one or more fusion events with other endocytic vacuoles, lysosomes (Ly), or Golgi apparatus. While it has been difficult to obtain direct evidence, the literature is replete with examples in which rapid membrane flow and recycling readily explains the data. Two of the more striking examples derive from studies of pinocytosis in cultured ceils. Fibroblasts, for instance, interiorize the equivalent of 50% of their surface area and 5-10% of their cell volume during each hour of pinocytic activity. Yet, the overall dimensions of the cells and the vacuolar system remain constant throughout hours, even days, of endocytic activity. Since it is unlikely that internalized PM is rapidly degraded, it was proposed that

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Endocytosis and the recycling of plasma membrane.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

Bone resorption depends on the formation, by osteoclasts, of an acidic extracellular compartment wherein matrix is degraded. The mechanism by which osteoclasts transport protons into that resorptive microenvironment was identified by means of adenosine triphosphate-dependent weak base accumulation in isolated osteoclast membrane vesicles, which exhibited substrate and inhibition properties characteristic of the vacuolar, electrogenic H+-transporting adenosine triphosphatase (H+-ATPase). Identify of the proton pump was confirmed by immunoblot of osteoclast membrane proteins probed with antibody to vacuolar H+-ATPase isolated from bovine kidney. The osteoclast's H+-ATPase was immunocytochemically localized to the cell-bone attachment site. Immunoelectron microscopy showed that the H+-ATPase was present in the ruffled membrane, the resorptive organ of the cell.

https://science.sciencemag.org/content/sci/245/4920/855.full.pdf

Osteoclastic bone resorption by a polarized vacuolar proton pump

Lysosomes, the terminal organelles on the endocytic pathway, digest macromolecules and make their components available to the cell as nutrients. Hydrolytic enzymes specific to a wide range of targets reside within the lysosome; these enzymes are activated by the highly acidic pH (between 4.5 and 5.0) in the organelles' interior. Lysosomes generate and maintain their pH gradients by using the activity of a proton-pumping V-type ATPase, which uses metabolic energy in the form of ATP to pump protons into the lysosome lumen. Because this activity separates electric charge and generates a transmembrane voltage, another ion must move to dissipate this voltage for net pumping to occur. This so-called counterion may be either a cation (moving out of the lysosome) or an anion (moving into the lysosome). Recent data support the involvement of ClC-7, a Cl(-)/H(+) antiporter, in this process, although many open questions remain as to this transporter's involvement. Although functional results also point to a cation transporter, its molecular identity remains uncertain. Both the V-ATPase and the counterion transporter are likely to be important players in the mechanisms determining the steady-state pH of the lysosome interior. Exciting new results suggest that lysosomal pH may be dynamically regulated in some cell types.

/pdf/lysosomal-acidification-mechanisms-53xx1ofvg0.pdf

Lysosomal Acidification Mechanisms

In a living cell membrane-bound compartments are continuously either separated or united through fusion reactions, and literally thousands of such reactions take place every minute. The formation of membrane vesicles from pre-existing membranes, and their fusion with specific acceptor membranes, constitute a prerequisite for the transport of most impermeant molecules and macromolecules into the cell by endocytosis, out of the cell by exocytosis, and between the cellular organelles (Palade, 1975; Silverstein, 1978; Evered & Collins, 1982). Less frequent, but equally crucial, are fusion events in fertilization, cell division, polykaryon formation, enucleation, etc. (for reviews see Poste & Nicholson, 1978). Although a great deal is known about the properties and consequences of individual forms of membrane fusion in cellular systems, and about fusion in artificial lipid membranes, the molecular basis for the reactions remain largely unclear.

Membrane fusion proteins of enveloped animal viruses.

Fluorescein isothiocyanate-conjugated dextran was introduced preferentially into hepatic lysosomes by intraperitoneal injection into rats. The pH in isolated lysosomes, measured by fluorescein fluorescence, was approximately 5 and gradually increased in KCl (to 7.0) at 25 degrees C. In the presence of Mg2+, ATP caused acidification of lysosomes that was reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Mn2+, Co2+, and Fe2+ could replace Mg2+ but Ca2+ could not. Cu2+, Zn2+, and Cd2+ were inhibitory. A membrane-permeant anion, in practice chloride, was required for this acidification. ATP analogues, including 5'-adenylyl imidodiphosphate, could not be substituted for ATP. ATP-driven acidification was sensitive to N-ethylmaleimide and quercetin but insensitive to oligomycin, ouabain, and vanadate. There were some differences between "normal" lysosomes and tritosomes; the acidification was resistant to azide and N,N'-dicyclohexylcarbodiimide in normal lysosomes but sensitive to these reagents in tritosomes. These results provide evidence for the presence of an electrogenic proton pump driven by MgATP (H+-ATPase) on lysosomes.

https://www.pnas.org/content/pnas/79/9/2758.full.pdf

Yoshinori Moriyama

Papers

Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution.