Ofir Gershony 1 Ofer Shapira 2 Mehtap Abu-Qarn 1 Inbar Segal 1 Dikla Nachmias 1 Leah Gheber 2 Natalie Elia 1
1Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
2Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel

The AAA ATPase VPS4 functions at the very last step of ESCRT mediated membrane fission. During these processes, it’s involved in the membrane scission reaction through disassembly of ESCRT-III helical filaments. VPS4 is the only enzyme in the ESCRT machinery and one of the most conserved ESCRT components in evolution. Here, we present a novel function of human VPS4A as a microtubule (MT) binding protein. Using an in vitro microscopic assay, in which rhodamine-labeled MT are incubated with a GFP tagged protein, we characterize VPS4 binding to MT. We show that VPS4A-GFP, overexpressed in HEK293T cell lysates, binds MT and demonstrate that this interaction is specific using recombinant VPS4A-GFP. Additionally, we find that the addition of nucleotides reduce the MT binding ability of VPS4A. These results were further validated using a MT binding protein spin-down assay. This MT binding property has not been attributed, as of yet, to any ESCRT component. Phylogenetic analysis reveals that VPS4 is closely related to the MT severing proteins spastin and katanin, which may hint at a possible similarity in function. A VPS4A deletion mutant, in which a sequence that is based on spastin`s MT binding domain has been removed, still binds strongly to MT, suggesting that VPS4A binds MT through a different domain than spastin. This novel function of VPS4 as a MT binding protein could provide new insights on its role in the mechanism of the ESCRT machinery and on the regulation of many ESCRT-mediated cellular events.

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