Analysis of native Ist1 dynamics reveals multiple pools of ESCRT-III on endosomes.

Protein trafficking within the endosomal system involves several distinct membrane remodeling events, including those with opposing orientations that lead to the production of intraluminal vesicles (ILVs) and recycling tubules. Components of the endosomal sorting complex required for transport (ESCRT) machinery have been implicated in both pathways, although few studies have directly examined their native dynamics in mammalian cells. Here, we demonstrate that the endogenous ESCRT-III subunit Ist1 exists in at least two different pools on endosomes. High-speed, live-cell imaging further showed that one pool of Ist1 forms transiently on endosomes, while the other is relatively stable. However, upon growth factor stimulation, the stable pool of Ist1 becomes more mobile, and the transient pool accumulates more rapidly on endosomes. Our data indicate that ESCRT-III dynamics are distinct from that of other ESCRT complexes and additionally suggest an intrinsic amount of time is required for ESCRT-mediated ILV biogenesis, irrespective of environmental conditions.