Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity

Abstract

SummaryThe endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle.

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

“…Recent work has shown that after ended hydrolytic digestion, endolysosomes form into small non-acidic storage lysosomes containing inactive lysosomal hydrolases [13]. These storage lysosomes can then fuse to LEs and existing endolysosomes to recycle their content of lysosomal constituents and regenerate lysosomal compartments (see model in Fig.…”

“…These processes generate hybrid endo- and autolysosomal compartments, in which degradation catalyzed by lysosomal enzymes takes place. Following the completion of digestion, endolysosomes condense to reform small lysosomes that function as storage organelles for resident hydrolases [13]. …”

Triggered recruitment of ESCRT machinery promotes endolysosomal repair

“…We thus sought a way to follow ESCRT dynamics during both the onset and resolution of endolysosomal injury. Briefly exposing cells to lysosomotropic rupturing agents induces transient membrane disruption (9, 11, 27, 36) and accordingly, cells treated with LLOME for less than a minute acquired ESCRT puncta shortly after adding drug but not at later times (fig. S20).…”

“…GPN (glycyl-L-phenylalanine 2-naphthylamide) is an alternative rupturing agent that accumulates in endolysosomes like LLOME (11, 27). However, GPN is processed into metabolites thought to promote osmotic rupture (27, 37) potentially similar to damage caused by materials that normally transit the endolysosomal network.…”

“…We next compared the timing of ESCRT recruitment with loss and recovery of endolysosomal function, using the Magic Red fluorogenic indicator of lysosomal protease activity (11). Magic Red fluorescence was lost after brief exposure to GPN as above, but returned after washing GPN away (Fig.…”