Lysosomes are the major digestive compartments with many hydrolytic enzymes and acidic lumenal pH. For efficient degradation of macromolecules, lysosomes repeatedly undergo dynamic fusion events, which include both homotypic fusion and heterotypic fusion with late endosomes, phagosomes and autophagosomes; yet cells are able to maintain an approximately constant number and size of lysosomes. Clearly, lysosomal fusion/scission events must be tightly regulated for appropriate lysosomal biogenesis and function; however, the molecular mechanism of their regulation is poorly understood. Arl8 is the first small GTPase that has been shown to be localized primarily to lysosomes in mammalian cells. We conducted the first genetic analysis of an
arl-8-deletion mutant using C. elegans, and found that ARL-8 is an essential GTPase for lysosome biogenesis in the macrophage-like coelomocytes.
arl-8 mutants display the unique combination of lysosomal phenotype that are unprecedented in previous studies. First, loss-of-function of ARL-8 leads to supernumerary lysosomes that are smaller than wild type. Second, the defects of these aberrant lysosomes are not limited to their morphology: Distributions of V-ATPase subunits among lysosomes are disorganized, and most lysosomes are defective for their luminal acidification in
arl-8 mutant coelomocytes. The localization of lysosomal aspartic protease ASP-1 is also compromised in
arl-8 mutants: While ASP-1 is distributed among virtually all lysosomes in wild-type, it localizes to a small population of lysosomes in
arl-8 mutants.
arl-8 mutant coelomocytes can endocytose macromolecules; however, the endocytosed macromolecules accumulate in a lysosomal fraction with relatively low levels of ASP-1. These results imply that loss of
arl-8 leads to the generation of numerous small lysosomes, where lysosomal enzymes and endocytosed macromolecules accumulate in distinct lysosome subpopulations. Furthermore, we found that
arl-8 acts upstream to
cup-5, which is the orthologue of human mucolipin-1 implicated in mucolipidosis type IV. Previous studies have shown that
cup-5 mutant coelomocytes display enlarged lysosome-like vacuoles probably due to defects in the maturation/scission process of lysosomes. Interestingly, loss of
arl-8 strongly suppresses formation of enlarged lysosome-like vacuoles in
cup-5 mutants. Collectively, our findings suggest that ARL-8 acts as an essential node that tightly links appropriate maintenance of number and size of lysosomes with their intrinsic functions.