Vfa1 Binds to the N-terminal Microtubule-interacting and Trafficking (MIT) Domain of Vps4 and Stimulates Its ATPase Activity

Abstract

Background: Vfa1 was recently identified as a Vps4-binding protein, yet its function was unknown. Results: Vfa1 potently stimulates Vps4 ATPase activity, and Vps4-Vfa1 interaction consists of a binding mechanism previously seen in other Vps4 regulator structures. Conclusion: Biochemical and biophysical studies show that Vfa1 is a novel regulator of Vps4 in the multivesicular body pathway. Significance: Vps4-Vfa1 interaction further expands the complexity of Vps4 regulation.

A Novel Mechanism of Regulating the ATPase VPS4 by Its Cofactor LIP5 and the Endosomal Sorting Complex Required for Transport (ESCRT)-III Protein CHMP5

“…Vta1 is a protein cofactor in yeast that functions as a Vps4 activator (41,50). The basal activity of Vta1 has been attributed to the structural features near the C terminus, whereas binding of the ESCRT-III proteins Did2 and Vps60 to the N terminus provides an additional level of stimulation (42).…”

“…Malachite Green ATPase Assay-The Malachite Green assay was used to measure the ATPase activity of VPS4B as described previously with modifications (50). Proteins used in the assay were dialyzed in the ATPase buffer (100 mM Tris-HCl, pH 7.4, 20 mM KCl, 6 mM MgCl 2 ) and stored at Ϫ80°C.…”

Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines

“…MIM1 sequences are found in CHMP1-3 94 and IST1 87 , and have been visualized in multiple MIT domain complexes (Figure 3B) to reveal a short amphipathic helix binding in the groove between MIT helices 2 and 3 94; 97; 98; 99 . In contrast, MIM2 sequences, which have been identified in CHMP4, CHMP6 and IST1 (IST1 has both MIM1 and MIM2 sequences), bind to Vps4 MIT domains in an extended conformation that lines the groove formed by helices 1 and 3 26; 53; 100 . An MIM2 sequence that can bind the Vps4 MIT domain has also been characterized in the S. cerevisiae protein Vfa1 100 , although it is not clear whether Vfa1 is a previously unrecognized ESCRT-III subunit.…”

Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains

“…All proteins were purified using a protocol as previously described (52). Briefly, bacterial cells were grown to mid-log phase in LB media at 37°C and induced with 0.2 mM isopropyl ␤-D-1-thiogalactopyranoside for an additional 16 -20 h at 16°C.…”

“…IST1 fragments were injected at 0.5 mM against 0.05 mM Spartin 8 -101 , 0.5 mM against 0.05 mM LIP5 , and 0.85 mM against 0.09 mM VPS4B . GST Pulldown Analysis-GST pulldown analysis was performed following standard protocols in a phosphate-buffered saline solution supplemented with 1 mM DTT and 0.1% (v/v) Tween 20 (52). GST alone, GST-tagged IST1 290 -335 and its mutants, and GST-tagged CHMP2A…”