Rab7: A Key to Lysosome Biogenesis

Abstract

The molecular machinery behind lysosome biogenesis and the maintenance of the perinuclear aggregate of late endocytic structures is not well understood. A likely candidate for being part of this machinery is the small GTPase Rab7, but it is unclear whether this protein is associated with lysosomes or plays any role in the regulation of the perinuclear lysosome compartment. Previously, Rab7 has mainly been implicated in transport from early to late endosomes. We have now used a new approach to analyze the role of Rab7: transient expression of Enhanced Green Fluorescent Protein (EGFP)-tagged Rab7 wt and mutant proteins in HeLa cells. EGFP-Rab7 wt was associated with late endocytic structures, mainly lysosomes, which aggregated and fused in the perinuclear region. The size of the individual lysosomes as well as the degree of perinuclear aggregation increased with the expression levels of EGFP-Rab7 wt and, more dramatically, the active EGFP-Rab7Q67L mutant. In contrast, upon expression of the dominant-negative mutants EGFP-Rab7T22N and EGFP-Rab7N125I, which localized mainly to the cytosol, the perinuclear lysosome aggregate disappeared and lysosomes, identified by colocalization of cathepsin D and lysosome-associated membrane protein-1, became dispersed throughout the cytoplasm, they were inaccessible to endocytosed molecules such as low-density lipoprotein, and their acidity was strongly reduced, as determined by decreased accumulation of the acidotropic probe LysoTracker Red. In contrast, early endosomes associated with Rab5 and the transferrin receptor, late endosomes enriched in the cation-independent mannose 6-phosphate receptor, and the trans-Golgi network, identified by its enrichment in TGN-38, were unchanged. These data demonstrate for the first time that Rab7, controlling aggregation and fusion of late endocytic structures/lysosomes, is essential for maintenance of the perinuclear lysosome compartment. INTRODUCTIONThe "kiss-and-run" model for lysosome biogenesis proposes that maintenance of the lysosomal compartment depends on continuous fusions of late endocytic structures accompanied by fission events (Storrie and Desjardins, 1996). This model implies that, in addition to heterotypic fusions between late endosomes and lysosomes in the perinuclear region, there could also be continuous exchange within the lysosomal vesicle population (homotypic fusion). Indeed, evidence for fusions and bidirectional traffic of soluble material between lysosomes and late endosomes has been reported (Jahraus et al., 1994;Mullock et al., 1994;van Deurs et al., 1995;Futter et al., 1996;Storrie and Desjardins, 1996;Bright et al., 1997;Mullock et al., 1998).Several different molecules are part of the machinery responsible for vesicle docking and fusion, Rab GTPases and SNAREs being among the best studied (Rothman and Warren, 1994;Denesvre and Malhotra, 1996;Pfeffer, 1996Pfeffer, , 1999Olkkonen and Stenmark, 1997;Mayer, 1999;Waters and Pfeffer, 1999). Rab proteins are important regulators of membrane traffic on the biosynthetic ...

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