The small GTPase ARL8 associates with lysosomes and recruits several effectors that mediate coupling to kinesins for anterograde transport, as well as tethering for eventual fusion with other organelles. Herein we report the identification of the "RUN- and FYVE-domain-containing" proteins RUFY3 and RUFY4 as novel ARL8 effectors that couple lysosomes to dynein-dynactin for retrograde transport. Using various biochemical approaches, we find that RUFY3/4 interact with both GTP-bound ARL8 and

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... -dynactin. In addition, we show that RUFY3/4 are both necessary and sufficient for concentration of lysosomes in the juxtanuclear area of the cell. RUFY3/4 also promote retrograde transport of lysosomes in the axon of hippocampal neurons. The function of RUFY3/4 in retrograde transport is required for juxtanuclear redistribution of lysosomes upon serum starvation or cytoplasmic alkalinization, and may underlie the reported roles of RUFY3/4 in axon development/degeneration, cancer and immunity. These studies thus establish RUFY3/4 as novel ARL8-dependent, dynein-dynactin adaptors, and highlight the role of ARL8 in the regulation of both anterograde and retrograde lysosome transport.